CA2109461A1

CA2109461A1 - Indirect fluorescent assay of blood samples

Info

Publication number: CA2109461A1
Application number: CA002109461A
Authority: CA
Inventors: Robert A. Levine; Stephen C. Wardlaw; Leon W. M. M. Terstappen; Diether J. Recktenwald; Thomas Mercolino
Original assignee: Robert A. Levine; Stephen C. Wardlaw; Leon W. M. M. Terstappen; Diether J. Recktenwald; Thomas Mercolino; Becton, Dickinson And Company
Current assignee: Becton Dickinson and Co
Priority date: 1992-10-30
Filing date: 1993-10-28
Publication date: 1994-05-01
Also published as: JP2679945B2; TW297094B; EP0595641B1; US5635362A; AU4870993A; JPH06281651A; DE69329726D1; US5460979A; NO933919D0; US5776710A; ES2152243T3; FI934804A; EP0595641A2; FI934804A0; EP0595641A3; CN1088310A; US5759794A; AU668212B2; US5342790A; DE69329726T2

Abstract

ABSTRACT OF THE DISCLOSURE

A patient's health is diagnosed by centrifuging blood samples in a transparent tube, which tube contains one or more groups of particles such as lyposomes or plasticbeads of different densities for each group. Each group of density-defined particles carries antigens or antibodies which are specific to a complement antigen or antibody which may be in the blood sample being tested, and which are indicative of the patient's health. A label-tagged antibody which is specific to all bound antibody/antigen couples is added to the blood sample so as to form labeled antibody+antigen-antibody complexes (AAAC) in the blood sample. Upon centrifugation, the complexed particles will settle out in different areas in the tube according to the respective density of the particles, and the degree of label emission of the particle layers can enable qualitative or quantitative analyses of the blood sample to be made. Unbound labeled antibodies will be washed away from the complexed layers by the washing action of the descending blood cells during the centrifugation step. Unbound labeled antibodies will thus not interfere with the analysis.

Description

2 ~

Indirect Fluorescent Assay of Blood Samples Technical Field This inven~ion relates to the one-step simultaneous determination of the presence or absence of either partner component of one or more active couples of binding biologic particles, and where desired, their quantification in a whole blood, blood plasma, or serum sample. ~ :

Background Art Analyses of blood samples for the presence or absence of antibodies or antigens are used in the diagnosis of diseases, such as HiV infection, hepatitis, Lyme disease, prenatal profiles inclucling TORCH (an acronym for: "Toxoplasmosis, Rubella, Cytomegalovirus, Herpes") profiles, as well as other infectious disease profiles.
Presently, such serologic diagnoses are often performed by standard indirect fluorescent immunoassay. In a standard indirect fluorescent immunoassay, an antigen, which is the coupling partner for the antibedy to be det0cted, is first affixed to a solid support medium such as a glass slide, a paper membrane or the like. A
sample of serum from tho patient is then allowed to incubate in contact with the affixed antigen for a period of time sufficient for the partner antibody, if present, to become attached to the affixed antigen. The support su~face is then washed to remove all u~bound an~ibodies. A reagent consisting of a !abelled antibody to human immune (antibody) globulins is next brought into contact with the support surface and incubated for a time sufficient to cause linkage of the labelled material and any traces of the patient's antibodies which may have bound to the ~ixed antigen. The excess reagent is then washed off and the support sur~ace is examined to deterrnine if any label is present. Examination of the prepared sample is done visually, or by by spectrophotometry or fluorometry. It will be appreciated ~hat the aforesaid procedure requir0s multiple specimen handling steps, inclucling washing, and analysis techniques, and is thus labor ineensive and time-consuming. The aforesaid proceclure can detect the presence or absenc~ of only one antigen-specHic antibody per test, but cannot differentiate b~twean specific IgG or IgM without further testing, nor can it detect ~ 2 ~

multiple antigens and/or antibodies simultaneously.

Disclosure of the Invention Copending USSN 071770,875, filed October 4, 1991, discloses a method an:l paraphenalia for performing differential erythrocyte counts by forming densimetrically distributed bands of microbeads having different band specific gravities, hereinafter referred to as density-markers. This invention relates to a method and paraphenalia for rapidly and easily determining the presence or absence of either partner component of one or more active couples of binding bio!ogic particles in whole blood, sera or plasma samples. Examples of such detectable couples are: TSH/Anti TSH
complex; T4/Anti T4 complex; Rubella antibody/Anti Rubella antibody; HIV
antibody/HlV antigen; all of which afe where the TSH, the T4, the Rubella antibody, and the HIV an~ibody are the target analytes. The method is perFormed in a centrifug tube by merely centrifuging the blood sample containing the several reagents in the .
tube, anc~ observing the results of the centrifugation step. The determination can be made without axposing the physician or technici~rl to the blood sample.

Red cells, when centrifuged in a tube con~aining ,a whole bloed sample will form a continual density gradient layar in the bottom of the tube, with the most dense red cells settling on the bottom of the red cell layer. When the blood sample is centrifuged in a tube containing ehe groups of different specific gravity beads referred to above, or different specific gravity liposomes, the keads or liposomes will form spaced, distinctly visible marker rings in the packed red cell layer. The oentrifuge tube may also contain a cylindrical plastic insert which may be fixed to the bottom of the tube or may be freely movable in the tube, and which, if freely movable, has a specific gravity such that it will sink through the red cell layer in the centrifuged blood sample. The insert restricts the available space in the tube which the red cells can oceupy, and therefore increases ~he distance between the marker rings which form in the centrifuged red cell layer, and displaces the beads or liposomes to the periphery of the tube where they may be seen and easily detected without their signal being extinguished by the red cells.

In perForming the method of this invention, th~ beads or liposomes will be coupled with an antigen or antibody, or another biologically active substance whose complemen~, or - 2 ~

binding partners, (which may be designated as "the target analytes") may be present in the pa~ient's blood. Examples of biologically active complementary couples include:
enzymes and their substrates; nucleotides and ~heir complemen~ary nucleotides;
naturally occurring protein binders, such as thyroid binding globulin (TGB) and thyroxine; the "intrinsic factor", and vitamin B-12; and specific antibodies which will selectively couple with RNA-DNA hybrids, as described by Stollar and Rashtchian, in their article "Immunochemical Approaches to Gene Pro~e Assays", Analytical Biochemistry 1987; 161, 387-394.

Each density-marker group, of which there may be only on~, will be bound with a coupling particle, which is specific to a target analyte, which analyte may be present in ~he blood or other biological specimen sample. The sample is added to the tube so as to allow the density-marker/coupling particle group or groups to intermix with the sample sufficiently to cause any target analytes present in the sample to couple with their complement partners on the density-markers.

Wh~n bound couples are created using ~he method of this invention, after the coupling step is completed, a labeled or tagged "anti-antigan-antibody-complex" antibody (AAAC antibody) which is specific to (and which may be in the tube prior to addition o~
the sample thereto) all couples on the density-markers. This AAAC antibody may be dry coated on the interior of the tube, for example, or may be present in liquid form in an evacua~ed tube such as describcd in US Patent No. 5,086,784, granted Februa~
11, 1992 to Robert A. Levine and Stephen C. Wardlaw.

Antibodies of this typ~ have been made by the Irnmunocytometry System Division of Becton Dickinson and Company, of San Jose, California. Instead of being specific to all antigen/antibody couples, the tagged AAAC antibody may be speciFic for immune globulin subgroups (IgG or IgM antibody-antigen complexes). Likewise, if the desired analy~e is an RNA or DNA, then the tube will contain a labeled antibody that is specific to or will bind to the RNA-DNA pair, as described in Stollar and Rashtchian.

The label may be a liposome encapsulated colorant, or a fluorescent colorant; or may be a radioactive energy emitter. The label must be detectable and pre~rably : :- .. , ' ' :

r~ 2 ~

quantifiable. The tagged antibody binds to all of the density-markers which havecouples formed thereon. The sample is centrifuged to densimetrioally separate the density-markers into spaced apart-bands or rings in the tube. The different density-marker bands are then ~xamined in the tube to determine which bands, if any, have a detectable quantity of the label, and to measure the quantity of the label, if appropriate. ~:.
The label most likely to be used would be a fluorescent molecule such as FITC.

If desired, the different density beads can hav~ different intrinsic colors, so that each (if thore are mor~ than one band) differently colored band wiil designate a diff~rent target analyte. If differently colored density-markers are used, the colors of the labeled bands in the tube will indicate which bound analytes are in the samplel and which analytes are not, in the event that bands of density-markers placed in the tube do not d~monstrate any iabel associated therewith. If colored density-markers are not used, then the position of the labeled bands in the tube will ir~icate which analytes are in ~ :
the sample, and which are not. This information, of course, permits diagnosis of the health of the sample donor.

It is ~herefore an object of this invention to provide~ an improved technique for analyzing a biological specimen sample to determine the presence or absence of certain target analytes therein.

It is a furth~r object to provide an imprQved ~echnique of the character described wherein the analysis is performed densimeerically in a transparent specimen tube.

It is an additional object of this invention to provide an improved technique of the character described wherein the analysis is performed by using beads of different specific graviti~s coupled to antibodies and/or antigens so that muitiple assays may be performed in one tube at one time.

It is another object of this invention to provide an improved t~hnique of the charaGter described wherein ~he analysis is peflormed by forming highlighted antibody/antigcn couple bands in the sampl~.

2 ~

These and other objects and advantages will b0come more readily apparent from th~
following detailed description of a preferred embodiment of the invention when taken in conjunction with the accompanying drawing in which:

Description of the Drawing FIG. 1 is side elevational view of a centrifuge tube adapted to perform the procedure of this invention;

FIG. 2 is a view of the tube of FIG. 1 showing a centrifuged whole blood sample therein, and with the red blood cell layer being blown up or increased in size to particularly point out the nature of the invention; and FIG. 3 is an axial sectional view of a second embodiment of a centrifuge tube adapted for use in performing the invention.

De~ailed Description of the Best Mode Referriny now to FIGS. 1 and 2, there is shown in Fl(3. 1, a tube 2, which may be a glass capill~ry tube, or other transparsnt tube, and which may contain a float or insert 4 made of a plastic, which has a specitic gravity that causes the insert 4 to settle through the red blood celis to the bottom 6 of the tube 2 when the latter is centrifuged with the blood sample therein The fraternal groups of antibody and/or antigen-coupled plastic beads of different specific gravities may be disposed in a clump 5 in the tube 2. A plastic cap 10 closes th bottom 6 of the tube 2. The specific gravity each group of beads will be greater than the specific gravi~y of the ligh~est of the red cells, ie, the youngest reticulocytes.

The blood sample is drawn into the tube 2 and, after a suitable inbubation period, is cen~rifuged ther~in along with the insert 4 and beads 5. The bead clump 5 disperses in the blood sample during th~ incubation p~riod, and then settles into distinct band which form lines in the red cell layer as shown in FIG. 2 during the c~ntrifugation step, while the float 4 settles into an~ through the red cells R. The tube 2 will also contain ~he tagged AAAC~ antibodies d~scribsd abova.

~:

, ., , . ~ : . : . . : .
.. . ~ , : . ~ :

" , . ~ . ..

2 ~

The white cells W layar out in bands above a red oellhNhite cell interface 1. The density-marker bead bands B layer out densimetrically in the the red cell layer.Examination of the bands B will indicate which of the bands B have been tagged since the fluorophore tags will be detectable only in tagged bands.

FIG. 3 shows an alternative form of centrifuge tube which can be used to practice the invention. The tube 12 has a compound funnel-shaped bore with an enlarged open end part 14 and a restricted closed end part 16. The bore is sized so as to cause the red cells R in the centri~uged blood sample to settle into the restricted part 16 of the bore, with the white cells and plasma staying for the most part in the enlarged part 14 of the bore. The tagged density-marker bands B disperse in the centrifuged red cell layer. The tube 12 is formed from a transparent glass or plastic material. It will be noted that the embodiment shown in FIG. 3 does not use a float component. :

It will be noted from FIGS. 2 and 3 that the density- markèr bands are sufficiently spaced apart that each can be assayed for fluorescence, or other ~nergy emissions, and can even be ~uantified as set forth hereinaft~r, without interference from any other bands B. When a blood sample is assayed, the nature of the red blood cells, ie, the fact that thay pack when centrifuged in a manner which axcludes the plasma, ensures that virtually all of the non-bound labeled AAAC antibodies in the tube will ~nd up in the plasma layer, and will not intorfere with the procedure.

A general example of the use of the invention to quantify a target analyte in a sample is as follows. The physician will identify from the literature an approxirnate range of how many molecules or units of a target analyte can be expected to b~ found in aknown volume sample of the biologic fluid being assayed~ For example, assums that a patient infected or exposed to Lyme Disease will be expected to have ~0 Lyme analyte units per milliliter of blood at the rnost. The physician wiil add at least 100 density-marker/antigen/antibody coupl~d units to the blood sample per milliliter being sampled, and will also add at least 100 labeled AAAC antiboclies per milliliter of sample to the container. Since there are an excess of bonding sites and tagging particles in the sample as compared to the maximum number of analyte units expected to be found in the sample, the degree or intensity of label emission from the Lyme 6 .

r~
~ - 2 ~

bead band will be proportional to the nurnber o~ Lyme analytes which are actually present in the sample. A quantification of the Lyme analyte in the blood can thus be approximated by measuring the emission intensity. The key to the quantification procedure is to provide a functional excess of binding sites and tagged antibodies in the sample as compared to the maximum number of analyte units which can be expected to be found in the sample. One may still be able to quantitate the analyte even if the bound-AAAC antibody units are present in molar amounts less than theanalyte, provided that there exists a mathematical relationship between the amount of analyte present and the amount of analyte eventually bound to the density marker-AAAC antibody couples.

In c~ses where a laboratory wishes to utilize serum or plasma samples, or where rnore predictable density gradients are required, such as might be the case if many densities need to be separated, then the narrow~d portion of the tube shown in FIG. 3 could be prefilled with a stable material, such as geiled Ficoll, having the required densi~y gradient. This density gradient material, in addition to separating the resident bands, will serve to wash the unbound AAAC antibodies away from the bound layers duringthe centrifugation step.

It will be appreciated that the invention has been described in connection with blood diagnosis, but the invention is also applicable to àiagnose other biological fluids for the presence or absence of highly specific complement couples found in such oth~r biological fluids. As with the analysis of plasma, when a biological fluid other than whole blood is assayed, the centrifugation step should be performed in the density gradient fluid, such as Ficoll gel, as noted above, which will not mix with the aqueous phase of tha biological fluid, and will allow densimetric separa~ion of the bands in the density gradient fluid, wi~h concurrent washing by the gradient fluid of the densi~
markers, to ensure separation of all non-bound label frorn the bands. This eliminates non-bound label interferenGe with quantification of the labeled bands. The inherent washing of non-bound label from labeled cells when whole blood is being tested, and when a non-cellular fluid is being tested in gelled Ficoll, which washing occurs during .
the centrifugation step, ~liminates the separate washing steps required by the prior art, and prevents unbound label from interf~ring with the accuracy of the procedure. This 7 . :

2 ~

inherent washing is an important con~ributor to the operability of this invention.

Since many changes and variations of the disclosed embodiments of the invention may be made without departing from the inventive concept, it is not intended to limit the invention otherwise than as required by the appended claims.

What isclaimed is: : :

; , . . ' . .

:~

Claims

1. A method for detecting a suspect target analyte in a biologic fluid sample in a transparent tube, said method comprising the steps of:
a) adding a group of density markers to the sample, which density markers have apredetermined specific gravity, each density marker in said group being coupled with a binding material to form density marker couples which are specific to the suspect target analyte;
b) adding labeled couple-binding antibodies to the sample;
c) incubating the density marker/labeled couple-binding antibody sample mixture;d) densimetrically aggregating the density markers into a distinct band; and e) determining if the band exhibits the presence of the labeled couple-binding antibodies, and therefore the presence of the target analyte.

2. A method for detecting one or more different target analytes in a biologic fluid sample in a transparent tube, said method comprising the steps of:
a) adding density markers to the sample, there being one group of density markers for each target analyte suspected to be in the sample, each group of density markershaving a different specific gravity from each other group of density markers, and each density marker in each group thereof being coupled with a binding material which is specific to one of the target analytes, whereby each of the different density marker/binding material couple groups is specific to a different one of the suspected target analytes;
b) adding labeled couple-binding antibodies to the sample;
c) incubating the density marker/labeled couple-binding antibody sample mixture;d) densimetrically aggregating the density markers into one or more distinct bands;
and e) determining which, if any of the bands exhibit the presence of a labeled couple-binding antibody, and therefore the presence of a target analyte.

3. The method of Claim 2 further comprising the step of displacing unbound labeled couple-binding antibodies from the density markers during the step of densimetrically separating

4. The method of Claim 3 wherein the fluid sample is whole blood, and wherein the density-markers have a specific gravity that is greater than the specific gravity of the lightest of the red cells.

5. The method of Claim 4 wherein said step of displacing is performed by the red cells in the whole blood sample.

6. The method of Claim 2 wherein the fluid sample is an aqueous base biological sample.

7. The method of Claim 6 further comprising the step of displacing unbound labeled couple-binding antibodies from the density markers during the step of densimetrically separating.

8. The method of Claim 7 wherein said step of displacing is performed by providing a sample-immiscible density gradient material in the tube, into which density gradient mater the density-markers will settle.

9. The method of Claim 2 wherein the density-markers are densimetrically separated into a portion of the tube having an internal sample-occupying portion which is less in cross sectional area than the cross sectional area of the remainder of the tube.

10. The method of Claim 9 wherein said portion of the tube is formed by positioning an axial insert in the bore of the tube.

11. The method of Claim 9 wherein said portion of the tube is formed by a localized constriction of the tube bore.

12. A method for detecting a suspect target analyte in a blood sample in a transparent tube, said method comprising the steps of:
a) adding a group of density markers to the sample, which density markers have aspecific gravity which ensures that said density markers will settle into the red cell layer of the blood upon centrifugation of the sample in the tube, each density marker in said group being coupled with a binding material to form density marker couples which are specific to the suspect target analyte;
b) adding labeled couple-binding antibodies to the sample;

c) incubating the density marker/labeled couple-binding antibody sample mixture;d) centrifuging the sample so as to aggregate the density markers into a distinct band in the red cell layer; and e) determining if the band exhibits the presence of the labeled couple-binding antibodies, and therefore the presence of the target analyte.

13. A method for detecting one or more different target analytes in a blood sample in a transparent tube, said method comprising the steps of:
a) adding density markers to the sample, there being one group of density markers for each target analyte suspected to be in the sample, each group of density markershaving a different specific gravity from each other group of density markers, with all of the density markers having a specific gravity that will ensure that said density markers will settle into the red cell layer of the blood upon centrifugation of the sample in the tube, and each density marker in each group thereof being coupled with a bindingmaterial which is specific to one of the target analytes, whereby each of the different density marker/binding material couple groups is specific to a different one of the suspected target analytes;
b) adding labeled couple-binding antibodies to the sample;
c) incubating the density marker/mabeled couple-binding antibody sample mixture;d) centrifugating the sample so as to aggregate the density markers into one or more distinct bands; and e) determining which, if any of the bands exhibit the presence of a labeled couple binding antibody, and therefore the presence of a target analyte.

14. Paraphenalia for use in centrifugally analysing a biological fluid sample for the presence or absence of a target analyte, said paraphenalia including:
a) a transparent tube having a bore for receiving the fluid sample;
b) means for forming a local constriction in said tube bore; and c) a quantity of antibody and/or antigen-coupled formed bodies in said tube.

15. The paraphenalia of Claim 14 wherein said formed bodies include several different fraternal groups of formed bodies, each group having a specific gravity that is different from the specific gravity of the bodies in each other group.

16. The paraphenalia of Claim 14 wherein the sample to be tested is whole blood,and said formed bodies have a specific gravity which is greater than the specific gravity of the lightest of the red blood cells in the sample of whole blood.