CA1202236A - Immunoassay methods employing patterns for the detection of soluble and cell surface antigens - Google Patents
Immunoassay methods employing patterns for the detection of soluble and cell surface antigensInfo
- Publication number
- CA1202236A CA1202236A CA000442166A CA442166A CA1202236A CA 1202236 A CA1202236 A CA 1202236A CA 000442166 A CA000442166 A CA 000442166A CA 442166 A CA442166 A CA 442166A CA 1202236 A CA1202236 A CA 1202236A
- Authority
- CA
- Canada
- Prior art keywords
- antigens
- detecting
- surface means
- antibodies
- providing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N21/8483—Investigating reagent band
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/80—Fluorescent dyes, e.g. rhodamine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/804—Radioisotope, e.g. radioimmunoassay
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/809—Multifield plates or multicontainer arrays
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/823—Immunogenic carrier or carrier per se
Abstract
IMMUNOASSAY METHODS EMPLOYING PATTERNS FOR THE DETECTION
OF SOLUBLE AND CELL SURFACE ANTIGENS
Abstract Methods for determining the presence of antigens or antibodies in an aqueous sample or presence of antigens on the surface of cells. A preferred embodiment employs fluorescent antigens which compete with the sample antigens for antibody binding sites. The antibodies are deposited on a support surface means in alternating patterns. The surface means and fluorescence detector are translocated with respect to each other and a signal generated by the detection of the repeating pattern of fluorescence. The signal is analyzed by means of a gated integrator responsive to a gate track control means also located on the surface means. Immunoassay methods having increased sensitivity are thereby obtained.
OF SOLUBLE AND CELL SURFACE ANTIGENS
Abstract Methods for determining the presence of antigens or antibodies in an aqueous sample or presence of antigens on the surface of cells. A preferred embodiment employs fluorescent antigens which compete with the sample antigens for antibody binding sites. The antibodies are deposited on a support surface means in alternating patterns. The surface means and fluorescence detector are translocated with respect to each other and a signal generated by the detection of the repeating pattern of fluorescence. The signal is analyzed by means of a gated integrator responsive to a gate track control means also located on the surface means. Immunoassay methods having increased sensitivity are thereby obtained.
Description
22~
IMMUNOASSAY METHODS EMPLOYING PATTERNS FOR THE DETECTION
OF SOLUBLE AND CELL SURFACE AI~TI~ENS
.
Field of the Invention -This invention relates generally to immunoassay methods useful for detecting soluble and cell surface antigens and more specifically, relates to methods employing patterns of immunological reactions formed on solid phase surfaces.
Background of the Invention The detection of specified antiyens and/or their specific binding partners, antibodies, has in recent years become of utmost importance in both the research and clinical environment. The detection of antigens and antibodies can often be related to various disease states and consequent-ly is of extreme usefulness in diagnosis as well as gain-ing basic understandings concerning the genesis of disease including cancer as well as the effectiveness of therapies therefor.
Consequently, i~proved methods for detecting antigens - found in aqueous samples, i.e. soluble antigens, as well as antigens found on the surface of tissues and cells are constantly sought. Typically, immunoassay methods may be characterized by their speed/facility of employment and by their sensitivity.
It is therefore an object of the present invention to provide new and novel methods which are susceptible to use in auto~ated and semi-automated instruments. It is another object to provide new immunoassay methods having a desirably high level of sensitivity to the antigens to be detected. It is a still further object to provide methods which incorporate a noise reduction technique for the generation of signals haviny superior signal-to-noise ratios.
Summary of the Invention In accordance with the objects of the present invention, methods are provided which permit the detection of antiyens solubilized within a sarnple solution or located on the surface of cells by employing surfaces having appropriate antibodies (i.e., antibodies specific for the particular antigen to be detected) bound thereto in particularized patterns. These patterns, expediently produced by alternating areas with the presence and absence of antibodies, per~it the generation of signals responsive to the presence of label having superior signal-to-noise ratios than those generally provided by conventional techniques.
Immunological reactions between antigens and antibodies will occur substantially only in or on those surface areas having antibodies attached thereto or deposited thereon but not in those areas having an absence of antibodies.
Accessory labeled antigens are made to compete with anti-gens from the sample for antibody binding sites. By effecting translocation of the surface vis-a-vis a detec-tor capable of detecting the label, a signal representing the difference in measurable levels of label between areas having antibodies and areas having no antibodies may be obtained. This repetitive signal can be expediently analyzed electronically and/or mathematicaily pursuant to well-known conventional methods t~ determine the quantity of antigens originally present in the sample.
Alternately, the so-called sandwich techniques may be employed whereby antigens or haptens, immunologically similar to the antigens to be detected are deposited on . , , CRL-l
IMMUNOASSAY METHODS EMPLOYING PATTERNS FOR THE DETECTION
OF SOLUBLE AND CELL SURFACE AI~TI~ENS
.
Field of the Invention -This invention relates generally to immunoassay methods useful for detecting soluble and cell surface antigens and more specifically, relates to methods employing patterns of immunological reactions formed on solid phase surfaces.
Background of the Invention The detection of specified antiyens and/or their specific binding partners, antibodies, has in recent years become of utmost importance in both the research and clinical environment. The detection of antigens and antibodies can often be related to various disease states and consequent-ly is of extreme usefulness in diagnosis as well as gain-ing basic understandings concerning the genesis of disease including cancer as well as the effectiveness of therapies therefor.
Consequently, i~proved methods for detecting antigens - found in aqueous samples, i.e. soluble antigens, as well as antigens found on the surface of tissues and cells are constantly sought. Typically, immunoassay methods may be characterized by their speed/facility of employment and by their sensitivity.
It is therefore an object of the present invention to provide new and novel methods which are susceptible to use in auto~ated and semi-automated instruments. It is another object to provide new immunoassay methods having a desirably high level of sensitivity to the antigens to be detected. It is a still further object to provide methods which incorporate a noise reduction technique for the generation of signals haviny superior signal-to-noise ratios.
Summary of the Invention In accordance with the objects of the present invention, methods are provided which permit the detection of antiyens solubilized within a sarnple solution or located on the surface of cells by employing surfaces having appropriate antibodies (i.e., antibodies specific for the particular antigen to be detected) bound thereto in particularized patterns. These patterns, expediently produced by alternating areas with the presence and absence of antibodies, per~it the generation of signals responsive to the presence of label having superior signal-to-noise ratios than those generally provided by conventional techniques.
Immunological reactions between antigens and antibodies will occur substantially only in or on those surface areas having antibodies attached thereto or deposited thereon but not in those areas having an absence of antibodies.
Accessory labeled antigens are made to compete with anti-gens from the sample for antibody binding sites. By effecting translocation of the surface vis-a-vis a detec-tor capable of detecting the label, a signal representing the difference in measurable levels of label between areas having antibodies and areas having no antibodies may be obtained. This repetitive signal can be expediently analyzed electronically and/or mathematicaily pursuant to well-known conventional methods t~ determine the quantity of antigens originally present in the sample.
Alternately, the so-called sandwich techniques may be employed whereby antigens or haptens, immunologically similar to the antigens to be detected are deposited on . , , CRL-l
2~
the surface, antibodies reacted therewith followed by addition of the sample containing the antigens to be detected. Finally, these exposed antigens are detected by the application of labeled antibodies. Detection of the label and subsequent signal handling would otherwise be identical to the competitive type procedures described.
Brief Description of the Drawings Further understanding of the principles and scope of the present invention may be had by reference to the drawings wherein: -Figure 1 diagra~matically depicts the operation of the present invention in a preferred embodiment;
Figure la illustrates the periodic, photomultiplier tubedetected signal;
Figure 2 shows the alternating pattern of deposition on a tape type surface; and Figure 3 illustrates an alternative e~bodiment of the present invention.
Detailed Description and Best Mode The principles of flow cytometry in combination with various labeling techniques such as those employing fluorescence can be applied to the determination and quan-tification of antigens present in a solution or on the surface of a cell. The use of fluorochromes or fluorescent dyes as a label lends itself particularly well to the present invention, however, other types of labels may be equally expedient. For the sake of simplicity, the discussion will be limited to the use of fluorescent dycs"
CRL-l ~0~
however, it is understood, that one skilled in the art may employ other labels such as metal particles (colloidal gold), enzymes, radioisotopes or the like with equal facility.
A typical problem encountered with conventional im~uno-assay techniques is that of obtaining a sufficiently high signal-to-noise ratio in order to acquire the sensitivity desired. Typically, background fluorescence operates to -10 mask the desired signal particularly when the signal is generated by a weak immunological reaction commonly occasioned by low antigen concentration~ The present invention provides novel ways of circumventing this problem by utilizing the noise reduction technique of producing a periodic signal which is analyzed with the aid of a periodic reference signal~ This is accomplished by reducing the antigen~antibody immunological reactions to specified and circumscribed areas on a surface such as that provided by a tape strip. Further, the invention is equally applicable to both competitive--inhibition type assays and sandwich type assays. In the former assay typer antibody to the desired antigen is deposited either on the surface or within the tape itself but in either ~ case, in alternating specified locations so that a particular repeating pattern is produced while in the latter, antigen is deposited instead of antibody and an antibody-antigen-antibody bridge produced. Since the first type of assay is conceptually easier to understand, it will be fully described first.
A surface such as tha~ provided by tape 10 is depicted in Figure 2. Areas 11 have the antibody attached thereto while areas 12 are characterized by the absence oE
antibody. Areas 11 and 12 are li~ited to section 15 of tape 10 in contrast to a gate-track control section 16 described later. As may be readily appreciated, the sllape :
CRL-l 2~Gi of the areas may be varied greatly without departure from the objectives and principles of the present invention.
The tape, with antibodies attached, is then advantageously incubated with a known quantity of fluorescinated antigens which are as equally reactive ~or at least at a compara-tively known level) to the antibody deposited on the tape as are the antigens to be determined within the sample.
Thus, the fluorescinated antigens and the sample antigens -10 compete for availahle antibody binding sites. Unbound antigens are then ideally removed, advantageously accomplished by any standard washing technique such as by passing the tape through a bath or a spray.
With reference to Figure 1, the tape is thereafter passed through an instrument capable of illurninating the tape by use of such standard optical devices as a light source l;
lenses 2, 11; filter 3; aperture 4; and mirrors 5; and measuring resultant bulk fluorescence again acco~plished with such well-known arrangements as lens 11; mirrors 5;
aperture 6; lens 7i filter 8 and photomultiplier tube 9.
As the tape passes the focused light source, the alternat-ing areas containing antibody and areas containing no antibody will be illuminated.
Those areas containing antibody can be expected to have some proportion of labeled antigen bound thereto. That amount will be dependent upon the level of competition fOL^
antibody binding sites generated by the antigens in the sample. Thus, in areas having no antibodies, and c~nse-quently no fluorescent antigens bound thereto, a very low or nonexistent signal of fluorescence will be measured.
If manufactured perfectly, these areas would provide virtually no signal but in practicality, the representa-tive signal have some elevated, albeit noisy level (V! lnFigure lA idealized in the figure as a pe~fectly Colls'~ t CRL-l 2~
level) representative of the background. In contrast, in those areas having antibody and consequently some amount of fluorescinated antigen bound thereto, a higher level signal V2 will be detected by the photomultiplier tube S (PMT). Analysis of the signal will be keyed by the gate control track and associated circuit depicted in Figure 1 and discussed hereaEter~ Due to the relative transloca~
tion of the siæe limited area vis-a-vis the detector (iOe., it may be advantageous to move the detector rather than the tape), this PrlT derived signal will be periodic in nature and by capitalizing on this characteristic, a superior signal-to-noise signal may be obtained. The difference between the high level and low level of fluorescence will be inversely related to the amount of antigen present in the sample or serum and r~y be enhanced by a switched or gated su~ming technique as described below.
Figure la graphically shows the type o signal resultant fro~ the PMT of the present invention. It is to be understood that the relative magnitudes of Vl and V2 are not drawn to scale but rather, have been dra~n to emphasize the periodic nature of the wavefor~, their difference rom ground (OV) and Eurther illustrate ideal square-wave signals. In reality the detected siynal will reflect physical and electronic imperfections of the system such as noise, imperfect area boundaries on the surface and the like.
Nonetheless the diEference in levels, i.e., V2 ~ V1, 7here V2 equals the level of fluorescence measurec in areas of the tape containing antibody and Vl is the level of fluorescence measured in areas containing no antibody, may be advantageously enhanced using a gated integrator.
CRL-l A gated integrator is a summing device that operates as follows: signals are integrated positively or negatively at time intervals that are synchronized with a gate track (described below). The diference V2-Vl in each signal is summed (integrated) with respect to the previous difference signals. Thus, the desired signal componen~ V2 will add coherently to the previous desired signal compo-nents, prior V2s, because these desired components are integrated positively while background signals Vls are integrated negatively. This switching of positive or negative integration is accomplished by gating the PMT
signal to the positive or negative ports of a standard integrator in response to a signal derived from the gate track reader. The gate track reader signal is fed to a gate and an inverter to a gate as depicted in Figure 1 The gates control the input of the PMT signal to the integrator. Thus, judicious timing of the physical and electronic aspects of this invention will permit summing the difference in PMT signals in order to add desired signal components minus the background signals.
With additional reference to the figures, the gate control track 16 ma~ be advantageuusly located proximal to the regions containing antibody and indicates the beginnin and end of the regions containing antibody. Additionally, such a gate control track may be ad~antageously employed to permit coating different areas of the tape, disk or other surface with various types of antibodies or r,lultiple controls. Thus, the control track could code for the presence of various test materials, antibodies, and/or controls as well as different tests~
The advantage to many areas particularly in the advanta-geous stripe-type format as opposed to a limited number of areas, i.e., one sample and one reference location, is that nonspecific binding or other spatial inhomogeneities CRL-l for binding antigen to the substrate may have relatively large spatial periods that are equal to or greater than the dimensions of the sample and reference areas. By using many stripes at a higher spatial frequency than these background inhomogeneities, the resulting, slowly varying background detection signal can be rejected for the most part by the use of the aforedescribed periodic gating or gated integrator.
In the case of the alternative sandwich type assay, antigens having immunologically similar reactivity to the antigens to be detected, i.e., they may be haptens as that term is com~only understood in the art, are deposited on the surface instead of antibodies. Parenthetically, it is appropriate to note that in this format, the invention may be used to detect the presence of antibodies in a fluid sample and thus will find yet further advantageous and important utility in the diagnostic arena.
Then, through a series of immunlogical reactions, a sandwich of alternating antibody-antigen levels is created. The surface antigens are reacted with an excess of antibody ~o that all antigenically reactive sites are covered. The sample with antigens is reacted therewith followed by reaction with labeled antibody. The labeled antibody will thus only attach to those antigenic sites supplied by the sample antigens and consequently, the level of detectable label can be related to the quantity and presence of antigen in the sample. The detectlon an~-analysis of the PMT signal (i.e., the detectable l~bel)in all other respects identical to that described above.
Still another and simpler sandwich technique involves the deposition of a first antibody on the surface specific for the sample antigens to be detected. Ater contacting ~he sample with the attached first antibody and removing CRL-]
~L21~;~23~
unreacted materials, a second labeled antibody is allowed to react with the antigen but at a different epitopic site than that for which the first antibody is specific. The subsequent detection and analysis are as previously described.
Similarly, the principles of the present invention may be used to determine the presence of selected cell surface antigens or presence of cells containing such surface antigens. This may be accomplished by contacting the cells with a surface having attached thereto in specified areas the antibody specific for the cell surface antigen to be detected~ By virtue of the standard imlDunological reaction, the cells having the desired cell surface antigens are attached to those areas of the tape containing the antibody.
Alternatively, antigens may be advantageously employed to coat specific areas of the tape (antigens are often more stable and thus easily manipulated than antibodies) and the sandwich technique or antibody bridge used to attach the cells containing the antigens of interest with the antigens present on the tape. In either case, the cells are then advantageously stained employing techniques conventional in the art. For instance, the cells may be stained with fluorescein diacetate which, in free .solution is not fluorescent since the fluorescein ~fluorescent portion) is quenched by the presence of the acetate moiety. Such a stain is typically adsorbed into tl~ cc11 where it~ in the presence of cellular esterase whicn cleaves off the acetate rnoiety, becomes fluorescent~
In yet another embodiment, the present invention may be employed to determine the presence of serum antibodies specific for cellular antigens such as the red blood cel1 typing antigens (A, B, A3, O, D). Such a test woul-l be CRL-l ~2~2~
accomplished in the following manner. Cells containing the blood type-specific antigens are applied to specified areas of the tape or other surface which areas are coded by the gate control track. The tape treated in this manner is then passed through the sample serum under con-ditions appropriately adjusted to allow an immunological reaction between the cells present on the tape and the antibodies present in the serum. ~he tape is then washed of unreacted materials such as by spraying or by subse-~10 quent baths, and passed through a fluorescinated Coombsserum. Subsequently, the tape is read by a device such as that described above and fluorescence detected whereupon, with the aid of the gate control track, the blood type of an individual or animal may be determined.
Various alternatives to the tape may be advantageously employed and include, for example, such surfaces as a disk having radii defined sectors which alternately contain and do not contain antibody. Clearly, in such an embodiment, the preferred motion (or effective translocation with respect the detector) would be rotary as opposed to the linear type of motion associated with the tape. Still yet another alternative would include the preparation of a surface such as the photoslide which may be completely illuminated and the detector moved to accomplish piecemeal interrogation or detection of specified areas. It is to be understood that with appropriate alterations, all of these alternative embodiments may be substituted for tlle tape in the prior discussion.
Additionally, alternatives to the use of cells may he advantageously employed in various of the above embodi-ments and would include, for instance, the use of latex particles. Such particles may be preferable as the~ are typically more capable of withstanding harsh mecharic^
and~or chemical treatment than are cells. Also, la -~
, .
CRL-l ~oæ~
particles may prove more useful for optimizing coating uniformity and edge clefinition.
Other principles of light based flow cytometry apparatus, such as those obtainable from the assignee hereof, may be employed in conjunction with th~e methods and principles of the present invention. For instance and with reference to Figure 3, alternatives to the detection of bulk fluores-cence include the detection of light reflected from the -10 tape (detectors 20 and 23), light transmitted or absorbed by the tape ~detector 36), intrinsic fluorescence versus bound fluorescence and the detection of multiple fluores-cent colors (detectors 20, 233. For example, one color may be employed specifically for the antibody and a second color tagged to a nonspecific antigen. Other embodiments contemplate the use of a tape 10 having a reference channel in addition to the gate control track and antibody areas~ Such a reference signal may be particularly expedient for expanding the number or variety of tests which can be performed simultanteously on the surface.
The placement of dichroic filters 28, ~2, 25 and 34 as well as the detectors 20, 23, 34 themselves (along with apertures 21, 24, 35) for the particular detection of transmitted signal, scattered signal and fluorescence may be adjusted pursuant to techniques well-known in the art.
Further, it is to be understood that all antigen-antibody reactions discussed throuyhout the foregoing discussion depend upon appropriate conditions conducive for ir--uno-logical reactions~ It is also assumed that the sel ctio.of antigens, haptens or ligands and the selection of the ligand binding partner, i.e., antibody or reactive por-tions thereof r as all those terms are commonly employed, is in accordance with their reactivity, specificity and affinity so that immunological reactions will in fact occur. Thus, the terms antigens and antibodies shall b~
CRL-l understood to apply generically to this invention thus encompassing all possible immunological variations a.s conventionally understood in the immunoassay art.
Generally then, from the discussion, drawings as well as the disclosure, it may be readily apparent that one skilled in the art can derive various modifications of the present invention without departing from the spirit and scope thereofO
t ,~
the surface, antibodies reacted therewith followed by addition of the sample containing the antigens to be detected. Finally, these exposed antigens are detected by the application of labeled antibodies. Detection of the label and subsequent signal handling would otherwise be identical to the competitive type procedures described.
Brief Description of the Drawings Further understanding of the principles and scope of the present invention may be had by reference to the drawings wherein: -Figure 1 diagra~matically depicts the operation of the present invention in a preferred embodiment;
Figure la illustrates the periodic, photomultiplier tubedetected signal;
Figure 2 shows the alternating pattern of deposition on a tape type surface; and Figure 3 illustrates an alternative e~bodiment of the present invention.
Detailed Description and Best Mode The principles of flow cytometry in combination with various labeling techniques such as those employing fluorescence can be applied to the determination and quan-tification of antigens present in a solution or on the surface of a cell. The use of fluorochromes or fluorescent dyes as a label lends itself particularly well to the present invention, however, other types of labels may be equally expedient. For the sake of simplicity, the discussion will be limited to the use of fluorescent dycs"
CRL-l ~0~
however, it is understood, that one skilled in the art may employ other labels such as metal particles (colloidal gold), enzymes, radioisotopes or the like with equal facility.
A typical problem encountered with conventional im~uno-assay techniques is that of obtaining a sufficiently high signal-to-noise ratio in order to acquire the sensitivity desired. Typically, background fluorescence operates to -10 mask the desired signal particularly when the signal is generated by a weak immunological reaction commonly occasioned by low antigen concentration~ The present invention provides novel ways of circumventing this problem by utilizing the noise reduction technique of producing a periodic signal which is analyzed with the aid of a periodic reference signal~ This is accomplished by reducing the antigen~antibody immunological reactions to specified and circumscribed areas on a surface such as that provided by a tape strip. Further, the invention is equally applicable to both competitive--inhibition type assays and sandwich type assays. In the former assay typer antibody to the desired antigen is deposited either on the surface or within the tape itself but in either ~ case, in alternating specified locations so that a particular repeating pattern is produced while in the latter, antigen is deposited instead of antibody and an antibody-antigen-antibody bridge produced. Since the first type of assay is conceptually easier to understand, it will be fully described first.
A surface such as tha~ provided by tape 10 is depicted in Figure 2. Areas 11 have the antibody attached thereto while areas 12 are characterized by the absence oE
antibody. Areas 11 and 12 are li~ited to section 15 of tape 10 in contrast to a gate-track control section 16 described later. As may be readily appreciated, the sllape :
CRL-l 2~Gi of the areas may be varied greatly without departure from the objectives and principles of the present invention.
The tape, with antibodies attached, is then advantageously incubated with a known quantity of fluorescinated antigens which are as equally reactive ~or at least at a compara-tively known level) to the antibody deposited on the tape as are the antigens to be determined within the sample.
Thus, the fluorescinated antigens and the sample antigens -10 compete for availahle antibody binding sites. Unbound antigens are then ideally removed, advantageously accomplished by any standard washing technique such as by passing the tape through a bath or a spray.
With reference to Figure 1, the tape is thereafter passed through an instrument capable of illurninating the tape by use of such standard optical devices as a light source l;
lenses 2, 11; filter 3; aperture 4; and mirrors 5; and measuring resultant bulk fluorescence again acco~plished with such well-known arrangements as lens 11; mirrors 5;
aperture 6; lens 7i filter 8 and photomultiplier tube 9.
As the tape passes the focused light source, the alternat-ing areas containing antibody and areas containing no antibody will be illuminated.
Those areas containing antibody can be expected to have some proportion of labeled antigen bound thereto. That amount will be dependent upon the level of competition fOL^
antibody binding sites generated by the antigens in the sample. Thus, in areas having no antibodies, and c~nse-quently no fluorescent antigens bound thereto, a very low or nonexistent signal of fluorescence will be measured.
If manufactured perfectly, these areas would provide virtually no signal but in practicality, the representa-tive signal have some elevated, albeit noisy level (V! lnFigure lA idealized in the figure as a pe~fectly Colls'~ t CRL-l 2~
level) representative of the background. In contrast, in those areas having antibody and consequently some amount of fluorescinated antigen bound thereto, a higher level signal V2 will be detected by the photomultiplier tube S (PMT). Analysis of the signal will be keyed by the gate control track and associated circuit depicted in Figure 1 and discussed hereaEter~ Due to the relative transloca~
tion of the siæe limited area vis-a-vis the detector (iOe., it may be advantageous to move the detector rather than the tape), this PrlT derived signal will be periodic in nature and by capitalizing on this characteristic, a superior signal-to-noise signal may be obtained. The difference between the high level and low level of fluorescence will be inversely related to the amount of antigen present in the sample or serum and r~y be enhanced by a switched or gated su~ming technique as described below.
Figure la graphically shows the type o signal resultant fro~ the PMT of the present invention. It is to be understood that the relative magnitudes of Vl and V2 are not drawn to scale but rather, have been dra~n to emphasize the periodic nature of the wavefor~, their difference rom ground (OV) and Eurther illustrate ideal square-wave signals. In reality the detected siynal will reflect physical and electronic imperfections of the system such as noise, imperfect area boundaries on the surface and the like.
Nonetheless the diEference in levels, i.e., V2 ~ V1, 7here V2 equals the level of fluorescence measurec in areas of the tape containing antibody and Vl is the level of fluorescence measured in areas containing no antibody, may be advantageously enhanced using a gated integrator.
CRL-l A gated integrator is a summing device that operates as follows: signals are integrated positively or negatively at time intervals that are synchronized with a gate track (described below). The diference V2-Vl in each signal is summed (integrated) with respect to the previous difference signals. Thus, the desired signal componen~ V2 will add coherently to the previous desired signal compo-nents, prior V2s, because these desired components are integrated positively while background signals Vls are integrated negatively. This switching of positive or negative integration is accomplished by gating the PMT
signal to the positive or negative ports of a standard integrator in response to a signal derived from the gate track reader. The gate track reader signal is fed to a gate and an inverter to a gate as depicted in Figure 1 The gates control the input of the PMT signal to the integrator. Thus, judicious timing of the physical and electronic aspects of this invention will permit summing the difference in PMT signals in order to add desired signal components minus the background signals.
With additional reference to the figures, the gate control track 16 ma~ be advantageuusly located proximal to the regions containing antibody and indicates the beginnin and end of the regions containing antibody. Additionally, such a gate control track may be ad~antageously employed to permit coating different areas of the tape, disk or other surface with various types of antibodies or r,lultiple controls. Thus, the control track could code for the presence of various test materials, antibodies, and/or controls as well as different tests~
The advantage to many areas particularly in the advanta-geous stripe-type format as opposed to a limited number of areas, i.e., one sample and one reference location, is that nonspecific binding or other spatial inhomogeneities CRL-l for binding antigen to the substrate may have relatively large spatial periods that are equal to or greater than the dimensions of the sample and reference areas. By using many stripes at a higher spatial frequency than these background inhomogeneities, the resulting, slowly varying background detection signal can be rejected for the most part by the use of the aforedescribed periodic gating or gated integrator.
In the case of the alternative sandwich type assay, antigens having immunologically similar reactivity to the antigens to be detected, i.e., they may be haptens as that term is com~only understood in the art, are deposited on the surface instead of antibodies. Parenthetically, it is appropriate to note that in this format, the invention may be used to detect the presence of antibodies in a fluid sample and thus will find yet further advantageous and important utility in the diagnostic arena.
Then, through a series of immunlogical reactions, a sandwich of alternating antibody-antigen levels is created. The surface antigens are reacted with an excess of antibody ~o that all antigenically reactive sites are covered. The sample with antigens is reacted therewith followed by reaction with labeled antibody. The labeled antibody will thus only attach to those antigenic sites supplied by the sample antigens and consequently, the level of detectable label can be related to the quantity and presence of antigen in the sample. The detectlon an~-analysis of the PMT signal (i.e., the detectable l~bel)in all other respects identical to that described above.
Still another and simpler sandwich technique involves the deposition of a first antibody on the surface specific for the sample antigens to be detected. Ater contacting ~he sample with the attached first antibody and removing CRL-]
~L21~;~23~
unreacted materials, a second labeled antibody is allowed to react with the antigen but at a different epitopic site than that for which the first antibody is specific. The subsequent detection and analysis are as previously described.
Similarly, the principles of the present invention may be used to determine the presence of selected cell surface antigens or presence of cells containing such surface antigens. This may be accomplished by contacting the cells with a surface having attached thereto in specified areas the antibody specific for the cell surface antigen to be detected~ By virtue of the standard imlDunological reaction, the cells having the desired cell surface antigens are attached to those areas of the tape containing the antibody.
Alternatively, antigens may be advantageously employed to coat specific areas of the tape (antigens are often more stable and thus easily manipulated than antibodies) and the sandwich technique or antibody bridge used to attach the cells containing the antigens of interest with the antigens present on the tape. In either case, the cells are then advantageously stained employing techniques conventional in the art. For instance, the cells may be stained with fluorescein diacetate which, in free .solution is not fluorescent since the fluorescein ~fluorescent portion) is quenched by the presence of the acetate moiety. Such a stain is typically adsorbed into tl~ cc11 where it~ in the presence of cellular esterase whicn cleaves off the acetate rnoiety, becomes fluorescent~
In yet another embodiment, the present invention may be employed to determine the presence of serum antibodies specific for cellular antigens such as the red blood cel1 typing antigens (A, B, A3, O, D). Such a test woul-l be CRL-l ~2~2~
accomplished in the following manner. Cells containing the blood type-specific antigens are applied to specified areas of the tape or other surface which areas are coded by the gate control track. The tape treated in this manner is then passed through the sample serum under con-ditions appropriately adjusted to allow an immunological reaction between the cells present on the tape and the antibodies present in the serum. ~he tape is then washed of unreacted materials such as by spraying or by subse-~10 quent baths, and passed through a fluorescinated Coombsserum. Subsequently, the tape is read by a device such as that described above and fluorescence detected whereupon, with the aid of the gate control track, the blood type of an individual or animal may be determined.
Various alternatives to the tape may be advantageously employed and include, for example, such surfaces as a disk having radii defined sectors which alternately contain and do not contain antibody. Clearly, in such an embodiment, the preferred motion (or effective translocation with respect the detector) would be rotary as opposed to the linear type of motion associated with the tape. Still yet another alternative would include the preparation of a surface such as the photoslide which may be completely illuminated and the detector moved to accomplish piecemeal interrogation or detection of specified areas. It is to be understood that with appropriate alterations, all of these alternative embodiments may be substituted for tlle tape in the prior discussion.
Additionally, alternatives to the use of cells may he advantageously employed in various of the above embodi-ments and would include, for instance, the use of latex particles. Such particles may be preferable as the~ are typically more capable of withstanding harsh mecharic^
and~or chemical treatment than are cells. Also, la -~
, .
CRL-l ~oæ~
particles may prove more useful for optimizing coating uniformity and edge clefinition.
Other principles of light based flow cytometry apparatus, such as those obtainable from the assignee hereof, may be employed in conjunction with th~e methods and principles of the present invention. For instance and with reference to Figure 3, alternatives to the detection of bulk fluores-cence include the detection of light reflected from the -10 tape (detectors 20 and 23), light transmitted or absorbed by the tape ~detector 36), intrinsic fluorescence versus bound fluorescence and the detection of multiple fluores-cent colors (detectors 20, 233. For example, one color may be employed specifically for the antibody and a second color tagged to a nonspecific antigen. Other embodiments contemplate the use of a tape 10 having a reference channel in addition to the gate control track and antibody areas~ Such a reference signal may be particularly expedient for expanding the number or variety of tests which can be performed simultanteously on the surface.
The placement of dichroic filters 28, ~2, 25 and 34 as well as the detectors 20, 23, 34 themselves (along with apertures 21, 24, 35) for the particular detection of transmitted signal, scattered signal and fluorescence may be adjusted pursuant to techniques well-known in the art.
Further, it is to be understood that all antigen-antibody reactions discussed throuyhout the foregoing discussion depend upon appropriate conditions conducive for ir--uno-logical reactions~ It is also assumed that the sel ctio.of antigens, haptens or ligands and the selection of the ligand binding partner, i.e., antibody or reactive por-tions thereof r as all those terms are commonly employed, is in accordance with their reactivity, specificity and affinity so that immunological reactions will in fact occur. Thus, the terms antigens and antibodies shall b~
CRL-l understood to apply generically to this invention thus encompassing all possible immunological variations a.s conventionally understood in the immunoassay art.
Generally then, from the discussion, drawings as well as the disclosure, it may be readily apparent that one skilled in the art can derive various modifications of the present invention without departing from the spirit and scope thereofO
t ,~
Claims (64)
1. A method for determining the presence of antigens in an aqueous sample comprising the steps of:
(a) providing surface means having first antibodies, specific for the antigens to be determined, associated there-with in a specified pattern of areas, each area alternating with the presence and absence of said first antibodies;
(b) further providing an aqueous solution containing a known quantity of labeled antigens having substantially the same reactivity with said first antibodies as the anti-gens to be determined;
(c) contacting the aqueous sample and the aqueous solution with said surface means under conditions appropriate for permitting an immunological reaction to occur whereby said labeled and sample antigens compete for binding on said first antibodies;
(d) illuminating at least one first area having first antibodies associated therewith and detecting the effect of said illumination on said label associated with said area by detection means for providing a first signal responsive to the presence of said labeled antigens;
(e) translocating said surface means with respect to said detector means and further illuminating at least one second area having an absence of first antibodies associated therewith and detecting the effect of said illumination on said label associated with said area whereby a second signal responsive to the presence of said labeled antigens is provided; and (f) analyzing said first and second signals whereby the presence of antigens in said aqueous sample may be determined based on the relative absence or presence of bound labeled antigen in said first area in comparison to said second area.
(a) providing surface means having first antibodies, specific for the antigens to be determined, associated there-with in a specified pattern of areas, each area alternating with the presence and absence of said first antibodies;
(b) further providing an aqueous solution containing a known quantity of labeled antigens having substantially the same reactivity with said first antibodies as the anti-gens to be determined;
(c) contacting the aqueous sample and the aqueous solution with said surface means under conditions appropriate for permitting an immunological reaction to occur whereby said labeled and sample antigens compete for binding on said first antibodies;
(d) illuminating at least one first area having first antibodies associated therewith and detecting the effect of said illumination on said label associated with said area by detection means for providing a first signal responsive to the presence of said labeled antigens;
(e) translocating said surface means with respect to said detector means and further illuminating at least one second area having an absence of first antibodies associated therewith and detecting the effect of said illumination on said label associated with said area whereby a second signal responsive to the presence of said labeled antigens is provided; and (f) analyzing said first and second signals whereby the presence of antigens in said aqueous sample may be determined based on the relative absence or presence of bound labeled antigen in said first area in comparison to said second area.
2. The method as provided in claim 1 wherein said providing step comprises providing surface means further comprising gate track control means, corresponding to the patterned areas of antibody presence and absence on said surface means, and said analyzing step further comprising detecting said gate track control means for enabling the analyzing step by associating said first and second signals with said first and second areas respectively.
3. The method as provided in claim 2 wherein the analyzing step comprises processing said first and second signals with gated integrator means whereby detecting said gate track control means enables said gated integrator means to positively integrate said first signals and negatively integrate said second signals from said detection means whereby the presence of said antigen in said aqueous sample may be determined.
4. The method as provided in claim 3 wherein said illuminating step comprises illuminating a fluorescent dye label at a light frequency which causes the label to fluoresce, and the step of detecting the effect of said illumination comprises detecting bulk fluorescence as-sociated with said label.
5. The method as provided in claim 4 further compris-ing the step of removing said antigens to be determined and said labeled antigens which have not reacted immunologically with said first antibodies on said surface means by washing said surface means.
6. The method as provided in claim 3 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence.
7. The method as provided in claim 1 wherein said provid-ing step comprises providing a generally rectangular tape adapted for substantially linear motion.
8. A method for determining within a sample the presence of cells having a specified surface antigen, comprising the steps of:
(a) providing surface means having a pattern of areas alternating with the presence and absence of first antibodies associated therewith and specific for the surface antigens to be determined;
(b) contacting said sample containing the cells with said surface means under conditions appropriate to allow an immunological reaction to occur;
c) staining the cells which have immunologically reacted with a detectable label;
(d) illuminating said surface means and detecting the effect of said illumination whereby a first signal responsive to the presence of said label in an area having antibodies associated therewith is generated;
(e) further illuminating said surface means and further detecting the effect of said illumination whereby a second signal responsive to the presence of said label in an area having an absence of antibodies associated therewith is generated; and (f) analyzing said first and second signals and deter-mining the presence of cells having a specific surface antigen based on the association of said label with said area having antibodies specific for said cellular surface antigen.
(a) providing surface means having a pattern of areas alternating with the presence and absence of first antibodies associated therewith and specific for the surface antigens to be determined;
(b) contacting said sample containing the cells with said surface means under conditions appropriate to allow an immunological reaction to occur;
c) staining the cells which have immunologically reacted with a detectable label;
(d) illuminating said surface means and detecting the effect of said illumination whereby a first signal responsive to the presence of said label in an area having antibodies associated therewith is generated;
(e) further illuminating said surface means and further detecting the effect of said illumination whereby a second signal responsive to the presence of said label in an area having an absence of antibodies associated therewith is generated; and (f) analyzing said first and second signals and deter-mining the presence of cells having a specific surface antigen based on the association of said label with said area having antibodies specific for said cellular surface antigen.
9. The method as provided in claim 8 wherein the step of staining the cells is accomplished by applying to the cells an effective solution of fluorescein diacetate.
10. The method as provided in claim 8 further comprising the step of removing unbound cells prior to the detecting steps.
11. The method as provided in claim 8 wherein said analyzing step is enabled by additionally detecting gate track control means associated with said surface means and corresponding to the pattern of antibody presence and absence on said surface means whereby said first and second signals are identified with areas of presence and absence of said first antibodies respectively.
12. The method as provided in claim 10 wherein said analyzing step is enabled by additionally detecting gate track control means associated with said surface means and corresponding to the pattern of antibody presence and absence on said surface means whereby said first and second signals are identified with areas of presence and absence of said first antibodies respectively.
13. The method as provided in claim 11 wherein based on detecting said gate track control means said analyzing step comprises processing said first and second signals with gated integrator means whereby the presence of cells containing the specified surface antigen may be determined.
14. The method as provided in claim 12 wherein based on detecting said gate track control means said analyzing step comprises processing said first and second signals with gated integrator means whereby the presence of cells contain-ing the specified surface antigen may be determined.
15. The method as provided in claim 8 further compris-ing detecting gate track control means associated with said surface means and corresponding to the pattern of antibody presence and absence on said surface means for controlling gated integrator means whereby said first detected signals are integrated positively and said second detected signals are integrated negatively and the presence of cells contain-ing the specified surface antigen may be determined.
16. A method for determining the presence of cells hav-ing a specified surface antigen, said cells present within a sample, comprising the steps of:
(a) providing surface means having second antigens applied thereto in a specified pattern of areas alter-nating with the presence and absence of said second antigens;
(b) providing an antibody-complex specific for the specified cell surface antigen and for the second antigen whereby, after allowing an immunological reaction between said antibody-complex, said cell, and said second antigen, an antibody bridge between the surface means and the cells may be formed;
(c) permitting under appropriate conditions, said immunological reaction to occur;
(d) staining the cells with a detectable label;
(e) illuminating an area of said surface means having second antigens present and detecting the effect of said illumination on said labeled cells whereby a first signal responsive to the presence of label is generated;
(f) illuminating an area of said surface means having second antigens absent and detecting the effect of said illumination on said labeled cells whereby a second signal responsive to the presence of label is generated; and (g) analyzing said first and second signals whereby the presence of cells having a specified surface antigen may be determined.
(a) providing surface means having second antigens applied thereto in a specified pattern of areas alter-nating with the presence and absence of said second antigens;
(b) providing an antibody-complex specific for the specified cell surface antigen and for the second antigen whereby, after allowing an immunological reaction between said antibody-complex, said cell, and said second antigen, an antibody bridge between the surface means and the cells may be formed;
(c) permitting under appropriate conditions, said immunological reaction to occur;
(d) staining the cells with a detectable label;
(e) illuminating an area of said surface means having second antigens present and detecting the effect of said illumination on said labeled cells whereby a first signal responsive to the presence of label is generated;
(f) illuminating an area of said surface means having second antigens absent and detecting the effect of said illumination on said labeled cells whereby a second signal responsive to the presence of label is generated; and (g) analyzing said first and second signals whereby the presence of cells having a specified surface antigen may be determined.
17. The method as provided in claim 16 wherein the step of staining the cells is accomplished by applying to the cells a solution of fluorescein diacetate.
18. The method as provided in claim 16 further com-prising the step of removing unbound cells prior to the label detecting steps.
19. The method as provided in claim 16 wherein said providing step comprises providing surface means having gate track control means corresponding to the patterned areas of antigen presence and absence on said surface means, and said analyzing step further comprises detecting said gate track control means for enabling the analyzing step by identifying said first and second signals with said areas having a presence and absence of second antigens respectively.
20. The method as provided in claim 18 wherein said providing step comprises providing surface means having gate track control means corresponding to the patterned areas of antigen presence and absence on said surface means, and said analyzing step further comprises detecting said gate track control means for enabling the analyzing step by identifying said first and second signals with said areas having a presence and absence of second antigens respectively.
21. The method as provided in claim 19 wherein said analyzing step comprises processing said first and second signals with gated integrator means in response to said gate track control means whereby said first signals are integrated positively and said second signals are integrated negatively and the presence of cells containing the specified surface antigen may be determined.
22. The method as provided in claim 20 wherein said analyzing step comprises processing said first and second signals with gated integrator means in response to said gate track control means whereby said first signals are integrated positively and said second signals are integrated negatively and the presence of cells containing the specified surface antigen may be determined.
23. A method for determining the presence of antigens in an aqueous sample comprising the steps of:
(a) providing first antibodies and surface means having second antigens with substantially the same immunological reactivity with said first antibodies as the antigens to be determined, said second antigens associated with said surface in a specified pattern of areas, each area of presence alternating with an area of absence of said second antigens;
(b) immunologically reacting substantially all said second antigens with said first antibodies and removing substantially all unbound first antibodies;
(c) contacting the aqueous sample with said surface means under conditions appropriate for permitting an immunological reaction to occur whereby said antigens to be determined may be bound to said first antibodies;
(d) further providing an aqueous solution containing second labeled antibodies immunologically reactive with said antigens to be determined under conditions appropriate for permitting an immunological reaction to occur whereby said second labeled antibodies may be bound to said antigens to be determined if present;
(e) illuminating an area of said surface means having second antigens present and detecting the presence of said label whereby a first signal is generated;
(f) illuminating an area of said surface means having an absence of second antigens and detecting the presence of said label whereby a second signal is generated; and (g) analyzing said first and second signals whereby the presence of antigens in said aqueous sample may be determined.
(a) providing first antibodies and surface means having second antigens with substantially the same immunological reactivity with said first antibodies as the antigens to be determined, said second antigens associated with said surface in a specified pattern of areas, each area of presence alternating with an area of absence of said second antigens;
(b) immunologically reacting substantially all said second antigens with said first antibodies and removing substantially all unbound first antibodies;
(c) contacting the aqueous sample with said surface means under conditions appropriate for permitting an immunological reaction to occur whereby said antigens to be determined may be bound to said first antibodies;
(d) further providing an aqueous solution containing second labeled antibodies immunologically reactive with said antigens to be determined under conditions appropriate for permitting an immunological reaction to occur whereby said second labeled antibodies may be bound to said antigens to be determined if present;
(e) illuminating an area of said surface means having second antigens present and detecting the presence of said label whereby a first signal is generated;
(f) illuminating an area of said surface means having an absence of second antigens and detecting the presence of said label whereby a second signal is generated; and (g) analyzing said first and second signals whereby the presence of antigens in said aqueous sample may be determined.
24. The method as provided in claim 23 further com-prising the step of detecting gate track control means associated with said surface means and corresponding to the pattern of second antigen presence and absence on said surface means and for enabling the analyzing step.
25. The method as provided in claim 24 wherein the analyzing step comprises processing said first and second signals with gated integrator means based on said gate track control means whereby said first signals are integrated positively and said second signals are integrated negatively whereby the presence of said antigen in said aqueous sample may be determined.
26. The method as provided in claim 25 wherein the further providing step comprises providing second antibodies labeled with a fluorescent dye, said illuminating steps comprise illuminating at a frequency of light which causes the label to fluoresce, and the step of detecting the effect of said illumination comprises detecting bulk fluorescence associated with said label.
27. The method as provided in claim 26 further com-prising the step of removing, prior to said illuminating step, substantially all unbound second labeled antibodies which have not reacted immunologically by washing said sur-face means.
28. The method as provided in claim 26 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence.
29. The method as provided in claim 23 wherein said providing step comprises providing surface means compris-ing a generally rectangular tape adapted for substantially linear motion.
30. A method for determining the presence of antigens in an aqueous sample comprising the steps of:
(a) providing surface means having first antibodies specific for a first epitopic site on said antigens to be determined, and which are associated with said surface in a specified pattern of areas, each area alternating with the presence and absence of said first antibodies;
(b) contacting the aqueous sample with said surface means under conditions appropriate for permitting an immuno-logical reaction to occur whereby said antigens to be deter-mined may be bound to said first antibodies;
(c) further providing an aqueous solution containing second labeled antibodies immunologically reactive with a second epitopic site on said antigens to be determined under conditions appropriate for permitting an immunological reaction to occur whereby said second labeled antibodies may be bound to said antigens to be determined if present;
(d) illuminating an area having said first antibodies present and detecting the effect of said illumination on said label whereby a first signal responsive to the presence of said labeled antibodies is provided;
(e) illuminating an area having an absence of said first antibodies and detecting the effect of said illumination on said label whereby a second signal responsive to the presence of said labeled antibodies is provided; and (f) analyzing said first and second signals whereby the presence of antigens in said aqueous sample may be determined.
(a) providing surface means having first antibodies specific for a first epitopic site on said antigens to be determined, and which are associated with said surface in a specified pattern of areas, each area alternating with the presence and absence of said first antibodies;
(b) contacting the aqueous sample with said surface means under conditions appropriate for permitting an immuno-logical reaction to occur whereby said antigens to be deter-mined may be bound to said first antibodies;
(c) further providing an aqueous solution containing second labeled antibodies immunologically reactive with a second epitopic site on said antigens to be determined under conditions appropriate for permitting an immunological reaction to occur whereby said second labeled antibodies may be bound to said antigens to be determined if present;
(d) illuminating an area having said first antibodies present and detecting the effect of said illumination on said label whereby a first signal responsive to the presence of said labeled antibodies is provided;
(e) illuminating an area having an absence of said first antibodies and detecting the effect of said illumination on said label whereby a second signal responsive to the presence of said labeled antibodies is provided; and (f) analyzing said first and second signals whereby the presence of antigens in said aqueous sample may be determined.
31. The method as provided in claim 30 further comprising detecting gate track control means associated with said surface means and corresponding to the pattern of said first antibody presence and absence on said surface means for enabling the analyzing step.
32. The method as provided in claim 30 wherein the analyzing step comprises processing said first and second signals with gated integrator means said integrator being gated in response to the detection of said gate track con-trol means whereby the presence of said antigen in said aqueous sample may be determined.
33. The method as provided in claim 32 wherein the further providing step comprises providing second antibodies labled with a fluorescent dye, said illuminating steps comprise illuminating at a frequency of light which causes the label to fluoresce, and the step of detecting the effect of said illumination comprises detecting bulk fluorescence associated with said label.
34. The method as provided in claim 33 further com-prising the step of removing, prior to said illuminating steps, substantially all unbound second labeled antibodies which have not reacted immunologically by washing said surface means.
35. The method as provided in claim 33 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence.
36. The method as provided in claim 30 wherein said providing step comprises providing surface means comprising a generally rectangular tape adapted for substantially linear motion.
37. The method as provided in claim 4 further compris-ing the step of removing said antigens to be determined and said labeled antigens which have not reacted immunologically with said first antibodies on said surface means by spraying said surface means.
38. The method as provided in claim 4 further compris-ing the step of removing said antigens to be determined and said labeled antigens which have not reacted immunologically with said first antibodies on said surface means by washing and spraying said surface means.
39. The method as provided in claim 3 wherein the step of detecting the effect of said illumination comprises detecting light reflected from said surface means.
40. The method as provided in claim 3 wherein the step of detecting the effect of said illumination com-prises detecting light transmitted through said surface means.
41. The method as provided in claim 3 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence and intrinsic fluorescence of said surface means for use as a reference whereby the true level of bulk fluorescence may be assessed.
42. The method as provided in claim 3 wherein the step of detecting the effect of said illumination comprises a first fluorescent dye specifically attached to said anti-bodies and a second fluorescent dye nonspecifically attached to said labeled antigens.
43. The method as provided in claim 1 wherein said providing step comprises providing a disk adapted for rotary motion.
44. The method as provided in claim 1 wherein said providing step comprises providing a photoslide adapted for substantially complete illumination.
45. The method as provided in claim 44 further com-prising detecting the effect of illumination by sequentially detecting portions substantially smaller than the total area of said photoslide.
46. The method as provided in claim 26 further compris-ing the step of removing, prior to said illuminating step, substantially all unbound second labeled antibodies which have not reacted immunologically by spraying said surface means.
47. The method as provided in claim 26 further compris-ing the step of removing, prior to said illuminating step, substantially all unbound second labeled antibodies which have not reacted immunologically by washing and spraying said surface means.
48. The method as provided in claim 26 wherein the step of detecting the effect of said illumination comprises detecting light reflected from said surface means.
49. The method as provided in claim 26 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence and intrinsic fluorescence of said surface means for use as a reference whereby the true level of bulk fluorescence may be assessed.
50. The method as provided in claim 26 wherein the step of detecting the effect of said illumination comprises a combination of detecting light scatter, bulk fluorescence, and intrinsic fluorescence of said surface means.
51. The method as provided in claim 23 wherein said providing step comprises providing surface means comprising a disk adapted for rotary motion.
52. The method as provided in claim 23 wherein said providing step comprises providing surface means comprising a photoslide, and said detecting the effect of illumination comprises sequentially detecting portions of said photoslide substantially smaller than the total area of said photoslide.
53. The method as provided in claim 33 further compris-ing the step of removing, prior to said illuminating steps, substantially all unbound second labeled antibodies which have not reacted immunologically by spraying said surface means.
54. The method as provided in claim 33 further comprising the step of removing, prior to said illuminating steps, sub-stantially all unbound second labeled antibodies which have not reacted immunologically by washing and spraying said surface means.
55. The method as provided in claim 33 wherein the step of detecting the effect of said illumination comprises detecting light reflected from said surface means.
56. The method as provided in claim 33 wherein the step of detecting the effect of said illumination comprises detecting light transmitted through said surface means.
57. The method as provided in claim 33 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence and intrinsic fluorescence of said surface means for use as a reference whereby the true level of bulk fluorescence may be assessed.
58. The method as provided in claim 33 wherein the step of detecting the effect of said illumination comprises detecting bulk fluorescence reflected from said surface means.
59. The method as provided in claim 30 wherein said providing step comprises providing surface means comprising a disk adapted for rotary motion.
60. The method as provided in claim 1 wherein the providing step comprises providing surface means having first antibodies specific for the antigen to be determined, said first antibodies attached to latex particles and said particles associated with said surface means in a specified pattern of areas, each area alternating with the presence and absence of said first antibodies.
61. The method as provided in claim 8 wherein the providing step comprises providing surface means having first antibodies specific for the antigens to be determined, said first antibodies attached to latex particles and said particles associated with said surface means in a specific pattern of areas, each area alternating with the presence and absence of said first antibodies.
62. The method as provided in claim 16 wherein the providing step comprises providing surface means having second antigens, said second antigens attached to latex particles and said particles associated with said surface means in a specific pattern of areas, each area alternat-ing with the presence and absence of said second antigen.
63. The method as provided in claim 23 wherein the providing step comprises providing surface means having second antigens, said second antigens attached to latex particles and said particles associated with said sur-face means in a specified pattern of areas, each area alternating with the presence and absence of said second antigens.
64. The method as provided in claim 30 wherein the providing step comprises providing surface means having first antibodies, said first antibodies attached to latex particles and said particles associated with said surface means in a specified pattern of areas, each area alternat-ing with the presence and absence of said first antibodies.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US455,765 | 1983-01-05 | ||
| US06/455,765 US4487839A (en) | 1983-01-05 | 1983-01-05 | Immunoassay methods employing patterns for the detection of soluble and cell surface antigens |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1202236A true CA1202236A (en) | 1986-03-25 |
Family
ID=23810202
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000442166A Expired CA1202236A (en) | 1983-01-05 | 1983-11-29 | Immunoassay methods employing patterns for the detection of soluble and cell surface antigens |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4487839A (en) |
| EP (1) | EP0117019B1 (en) |
| JP (1) | JPS59125063A (en) |
| AT (1) | ATE57577T1 (en) |
| CA (1) | CA1202236A (en) |
| DE (1) | DE3483397D1 (en) |
Families Citing this family (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4621063A (en) * | 1982-10-12 | 1986-11-04 | The Center For Immunological Studies | Methods for the detection and quantitation of immunological substances |
| US4591570A (en) * | 1983-02-02 | 1986-05-27 | Centocor, Inc. | Matrix of antibody-coated spots for determination of antigens |
| GB8331514D0 (en) * | 1983-11-25 | 1984-01-04 | Janssen Pharmaceutica Nv | Visualization method |
| US4647544A (en) * | 1984-06-25 | 1987-03-03 | Nicoli David F | Immunoassay using optical interference detection |
| USRE33581E (en) * | 1984-06-25 | 1991-04-30 | Immunoassay using optical interference detection | |
| US4791069A (en) * | 1984-09-21 | 1988-12-13 | Ortho Diagnostic Systems Inc. | Methods for attaching ligands or anti-ligands to a solid phase |
| FR2576418B1 (en) * | 1985-01-24 | 1988-04-01 | Ambroise Thomas Pierre | NEW SUPPORT FOR IN VITRO ORGANIC TESTS AND APPLICATIONS OF THIS SUPPORT |
| DE3686474T2 (en) * | 1985-10-11 | 1993-02-11 | Abbott Lab | DIAGNOSTIC SAMPLE. |
| US4741999A (en) * | 1986-02-26 | 1988-05-03 | The Research Foundation Of State University Of New York | Monoclonal antibodies useful in the identification of microorganisms causing periodontal disease |
| US5759774A (en) * | 1988-05-18 | 1998-06-02 | Cobe Laboratories, Inc. | Method of detecting circulating antibody types using dried or lyophilized cells |
| AU2684488A (en) * | 1988-06-27 | 1990-01-04 | Carter-Wallace, Inc. | Test device and method for colored particle immunoassay |
| US5089387A (en) * | 1988-07-07 | 1992-02-18 | Adeza Biomedical Corporation | Dna probe diffraction assay and reagents |
| GB8902803D0 (en) * | 1989-02-08 | 1989-03-30 | Plessey Co Plc | A method for detecting optical phase changes during biosensor operation,biosensing apparatus and a biosensor adapted for use in the same |
| WO1992006379A1 (en) * | 1990-10-09 | 1992-04-16 | Idemitsu Petrochemical Co., Ltd. | Method of immunological quantitative analysis |
| DE69132843T2 (en) * | 1990-12-06 | 2002-09-12 | Affymetrix Inc N D Ges D Staat | Identification of nucleic acids in samples |
| US5196350A (en) * | 1991-05-29 | 1993-03-23 | Omnigene, Inc. | Ligand assay using interference modulation |
| US5441894A (en) * | 1993-04-30 | 1995-08-15 | Abbott Laboratories | Device containing a light absorbing element for automated chemiluminescent immunoassays |
| US5413939A (en) * | 1993-06-29 | 1995-05-09 | First Medical, Inc. | Solid-phase binding assay system for interferometrically measuring analytes bound to an active receptor |
| US5770389A (en) * | 1993-09-27 | 1998-06-23 | Abbott Laboratories | Apparatus and method for determining the quanity of an analyte in a biological sample by means of transmission photometry |
| US20010051350A1 (en) | 1995-05-02 | 2001-12-13 | Albert Nazareth | Diagnostic detection device and method |
| US20030153023A1 (en) * | 1999-05-13 | 2003-08-14 | Starzl Timothy W. | Enumeration method of analyte detection |
| EP1204842A4 (en) * | 1999-08-19 | 2003-04-02 | Univ California | Apparatus and method for visually identifying micro-forces with a palette of cantilever array blocks |
| US20030154149A1 (en) * | 2002-02-13 | 2003-08-14 | Dilip Gajendragadkar | System and method of creating and executing a restricted stock sale plan |
| US7867754B1 (en) | 2002-08-01 | 2011-01-11 | Purdue Research Foundation | Microarrays for analyte detection |
| US7445938B2 (en) * | 2003-01-24 | 2008-11-04 | General Dynamics Advanced Information Systems, Inc. | System and method for detecting presence of analytes using gratings |
| US7027163B2 (en) * | 2003-01-24 | 2006-04-11 | General Dynamics Advanced Information Systems, Inc. | Grating sensor |
| JP2004309416A (en) * | 2003-04-10 | 2004-11-04 | Sony Corp | Sensor unit and sensing method, sensor unit and sensing method for biosubstance, sensor unit and sensing method for secrete, and feeling sensor unit and sensing method |
| AU2004273783A1 (en) | 2003-07-12 | 2005-03-31 | Accelr8 Technology Corporation | Sensitive and rapid biodetection |
| US20120077206A1 (en) | 2003-07-12 | 2012-03-29 | Accelr8 Technology Corporation | Rapid Microbial Detection and Antimicrobial Susceptibility Testing |
| US7341841B2 (en) * | 2003-07-12 | 2008-03-11 | Accelr8 Technology Corporation | Rapid microbial detection and antimicrobial susceptibility testing |
| US20150285793A1 (en) * | 2008-10-17 | 2015-10-08 | Medmira, Inc. | Downward or vertical flow diagnostic device and assay |
| ES2551922T3 (en) | 2011-03-07 | 2015-11-24 | Accelerate Diagnostics, Inc. | Rapid cell purification systems |
| US10254204B2 (en) | 2011-03-07 | 2019-04-09 | Accelerate Diagnostics, Inc. | Membrane-assisted purification |
| US9677109B2 (en) | 2013-03-15 | 2017-06-13 | Accelerate Diagnostics, Inc. | Rapid determination of microbial growth and antimicrobial susceptibility |
| KR20170132856A (en) | 2015-03-30 | 2017-12-04 | 액셀러레이트 다이어그노스틱스, 아이엔씨. | Instruments and systems for rapid microbiological identification and antimicrobial susceptibility testing |
| US10253355B2 (en) | 2015-03-30 | 2019-04-09 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing |
| CN113250762A (en) * | 2021-04-21 | 2021-08-13 | 广西电网有限责任公司电力科学研究院 | Low-pressure cylinder shaft seal steam flow testing method for high-medium pressure cylinder combined steam turbine |
| CN113250761A (en) * | 2021-04-21 | 2021-08-13 | 广西电网有限责任公司电力科学研究院 | Low-pressure cylinder shaft seal steam flow testing system of high-medium pressure cylinder-combined steam turbine |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3941876A (en) * | 1973-04-25 | 1976-03-02 | Gte New Ventures Corporation | In vitro method for determining allergic hypersensitivity |
| US4031197A (en) * | 1973-04-25 | 1977-06-21 | Gte New Ventures Corporation | In vitro method for determining allergic hypersensitivity |
| US4011308A (en) * | 1974-01-04 | 1977-03-08 | General Electric Company | Method for surface immunological detection of biological particles by the use of tagged antibodies |
| GB1506017A (en) * | 1974-03-04 | 1978-04-05 | Int Diagnostic Tech | Fluorometric system and method for the detection of biologically derived substances |
| DE2539193C3 (en) * | 1975-09-03 | 1979-04-19 | Siemens Ag, 1000 Berlin Und 8000 Muenchen | Process for the production of a planar conductor track system for integrated semiconductor circuits |
| DE2832491A1 (en) * | 1978-07-24 | 1980-02-07 | Merck Patent Gmbh | MEANS AND METHOD FOR STAINING CELL SWABS |
| GB2065298B (en) * | 1979-12-12 | 1985-02-20 | Wei Kung Wang | Method and apparatus for detecting biological particles by induced signal |
| US4303611A (en) * | 1980-08-11 | 1981-12-01 | Eastman Kodak Company | Analyzer apparatus featuring a simplified incubator |
| JPS57182651A (en) * | 1981-05-07 | 1982-11-10 | Olympus Optical Co Ltd | Detection apparatus of particle agglomeration pattern |
| AU596946B2 (en) * | 1984-07-10 | 1990-05-24 | F. Hoffmann-La Roche Ltd | Methods of assay |
-
1983
- 1983-01-05 US US06/455,765 patent/US4487839A/en not_active Expired - Lifetime
- 1983-11-29 CA CA000442166A patent/CA1202236A/en not_active Expired
- 1983-12-22 JP JP58241106A patent/JPS59125063A/en active Granted
-
1984
- 1984-01-04 EP EP84300031A patent/EP0117019B1/en not_active Expired - Lifetime
- 1984-01-04 DE DE8484300031T patent/DE3483397D1/en not_active Expired - Lifetime
- 1984-01-04 AT AT8484300031T patent/ATE57577T1/en active
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59125063A (en) | 1984-07-19 |
| DE3483397D1 (en) | 1990-11-22 |
| ATE57577T1 (en) | 1990-11-15 |
| EP0117019B1 (en) | 1992-04-29 |
| JPH0473107B2 (en) | 1992-11-19 |
| EP0117019A1 (en) | 1984-08-29 |
| US4487839A (en) | 1984-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1202236A (en) | Immunoassay methods employing patterns for the detection of soluble and cell surface antigens | |
| US5219763A (en) | Agglutination method for the determination of multiple ligands | |
| US7556932B2 (en) | Particle based homogeneous assays using capillary electrophoresis with laser-induced fluorescence detection | |
| EP0118894A2 (en) | Particle reagent size distribution measurements for immunoassay | |
| JPH0754324B2 (en) | Test agent for measuring antigen and / or antibody in liquid sample | |
| CA2269162A1 (en) | Method for the determination of analyte concentration in a lateral flow sandwich immunoassay exhibiting high-dose hook effect | |
| EP1448990B1 (en) | Particle-based ligand assay with extended dynamic range | |
| US4554257A (en) | Assaying immunoreactive and like substances by measurement of aggregate classes | |
| JP2005510706A5 (en) | ||
| Tu et al. | Ultrasensitive heterogeneous immunoassay using photothermal deflection spectroscopy | |
| CA2076349A1 (en) | Solid-phase interferometric immunoassay system | |
| US4251360A (en) | Method and apparatus for the detection of a specific binding protein | |
| ES2125982T3 (en) | TURBIDIMETRIC IMMUNOLOGICAL TEST OF INITIAL SPEED. | |
| ATE403866T1 (en) | METHOD FOR DETECTING ANTIBODIES IN LIQUID SAMPLES | |
| EP0222781A1 (en) | Microtiter - surface - flocculation assay for antigen or antibody screening | |
| WO1998022825A3 (en) | A whole blood/mitogen assay for the early detection of a subject with cancer and kit | |
| Wang et al. | A simplified solid-phase immunofluorescence assay for measurement of serum immunoglobulins | |
| CA1245551A (en) | Assay of immunoreactive and like substances | |
| CA1197186A (en) | Method of enumerating serologically selected cell populations | |
| Ligler et al. | Array biosensor for simultaneous detection of multiple analytes | |
| KR20040064054A (en) | Strips and fluoresence scanner for the measurement of high sensitivity c-reactive protein in serum and whole blood | |
| Maggio et al. | Immunology: current and future technology assessment | |
| JP3375739B2 (en) | Highly sensitive immunoassay method | |
| Paraf et al. | Immunoassays | |
| Ho et al. | Large-area waveguide sensor for multiple analytes detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |