Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS3853467 A
Publication typeGrant
Publication date10 Dec 1974
Filing date15 Aug 1973
Priority date15 Aug 1973
Publication numberUS 3853467 A, US 3853467A, US-A-3853467, US3853467 A, US3853467A
InventorsI Giaever
Original AssigneeGen Electric
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Method and apparatus for immunological detection of biological particles
US 3853467 A
Abstract
Method and apparatus for the detection of biological particles such as viruses, bacteria, and other cells by detection of the occurrence of an immunological reaction between the particle to be detected and its antibody. In one embodiment a substrate having an etchable metal surface is coated with a layer of antibodies to the biological particle to be detected. The antibody coated substrate is exposed to a fluid suspected of containing the biological particles to be detected. After such exposure, the structure is coated with a layer of a non-etachable metal of sufficient thickness to cover the antibody layer but of insufficient thickness to completely coat the biological particles and form a continuous film. The structure is then exposed to an etchant. After etching, positions at which biological particles had been immunologically bonded to their antibodies are observable as voids in the structure.
Images(1)
Previous page
Next page
Description  (OCR text may contain errors)

I United States Patent Giaever Dec. 10, 1974 METHOD AND APPARATUS FOR G. M. Edelman et al., Proc. Nat. Acad. Sci., 68, (9),

IMMUNOLOGICAL DETECTION OF 2153-2157 P 1971 BIOLOGICAL PARTICLES Primary Examiner-Morris O. Wolk [75] Inventor. Ivar Giaever, Schenectady, NY. Assistant Examiner sidney Maramz [73] Assignee: General Electric Company, Attorney, Agent, or Firm-Paul I. Edelson; Joseph T.

Schenectady, NY. Cohen; Jerome C. Squillaro [22] Filed: Aug. 15, 1973 [57] ABSTRACT [21] Appl' 388406 Method and apparatus for the detection of biological particles such as viruses, bacteria, and other cells by [52] US. Cl. 23/230 B, 23/253 R, 23/253 TP, detection of the occurrence of an immunological reac- 117/71 R, 195/1035 R, 424/12, 156/13 tion between the particle to be detected and its anti- [51] Int. Cl. G01n 21/04, GOln 31/06, body. In one embodiment a substrate having an etch- GOln 33/16 able metal surface is coated with a layer of antibodies [58] Field of Search 23/230 B, 253 R, 253 TP; to the biological particle to be detected. The antibody 195/1035 R; 424/12; 117/71 R coated substrate is exposed to a fluid suspected of containing the biological particles to be detected. [56] References Cited After such exposure, the structure is coated with a UNITED STATES PATENTS layer of a non-etachable metal of sufficient thickness 3,250,596 5/1966 Grafe 23/230 B to cover the antibody layer of i i thick 3,492,396 1/1970 Dalton 424/12 to completely Coat the blologlcal Partlcles and 3,646,346 2/1972 Catt 424/12 x form a Continuous The Structure is then exposed 3,770,380 11/1973 Smith 424/12 x o n tchant. After etching, positions at which biolog- OTHER "PUBLIC ATIONS Chemical Abstracts, 63:3532d (1965). Chemical Abstracts, 63110483b (1965). Chemical Abstracts, 65:282lg (1966). Chemical Abstracts, 73:48541m (1970).

ical particles had been immunologically bonded to their antibodies are observable as voids in the structure.

18 Claims, 4 Drawing Figures METHOD AND APPARATUS FOR IMMUNOLOGICAL DETECTION OF BIOLOGICAL I PARTICLES This invention relates to immunological detection of 5 reacting antibody proteins. This invention further has particular utility in the immunologic detection of particles whose presence in physiologic fluids in very low concentrations is of diagnostic interest, as for example, viruses.

This application is related to the copending application of Giaever, Ser. No. 266,278, filed June 26, 1972, and the copending application of Giaever, Ser. No. 384,1 13, filed July 30, 1973. These copending applications are commonly assigned with this application.

The related copending applications cited above disclose that an arbitrary protein will adhere to a substrate in a monomolecular layer only and that no other arbitrary protein will adhere to the protein layer. On the other hand, the specifically reacting protein to the first protein adsorbed onto the substrate will immunologically bond thereto. This discovery is exploited for diagnostic purposes in accordance with the teachings of the aforementioned related copending applications by means disclosed therein for efficiently and economically distinguishing between a monomolecular protein layer and a bimolecular protein layeron a substrate.

The diagnostic methods disclosed in the aforementioned copending applications require the formation of a substantially complete biomolecular protein layer for detection. While in theory, any immunologically reactive particle will form a detectably complete bimolecular layer when exposed to a slide coated with a monomolecular layer of its specifically reacting protein, in cases in which the particle to be detected is present in very dilute concentration in a fluid, detection in accordance with the teachings of the referenced copending applications may be unacceptably time-consuming.

A number of biologic particles are of significant diagnostic interest in substantially dilute concentrations. A prime example of these are the viruses. It is important to be able to detect the presence of viruses in body fluids in concentrations on the order of several thousand virus particles per cubic centimeter. Viruses are uniformly immunologically reactive. Viruses are not detectable by means of optical microscopic examination.

It is accordingly an object of this invention to provide method and apparatus for the immunologic detection of large biological particles. 55

A further object of this invention is to provide such 60 method and apparatus which provides for both the detection of such biological particles and for a determination of the concentration thereof in a fluid specimen.

Yet another object of the invention is to provide such 65 method and apparatus which is sufficiently sensitive to provide for the detection of a singlebiological particle.

Briefly, and in accordance with one embodiment of this invention, a wafer of substrate material has a film of etchable metal on one surface thereof. The metal film is coated with a monomolecular layer of antibodies to the biological particle to be detected. The substrate is then immersed in a fluid specimen to be tested for the presence of the particular species of biological particle of interest. Subsequently, a layer of etch-resistant metal is applied to the substrate over the layer of antibodies I and any biological particles bonded thereto. The substrate is then immersed in an etchant bath which dissolves the biological particles, those antibodies immediately underlying the biological particles, and a portion of the etchable metal film. After etching, the substrate is examined and points at which the biological particles to be detected had been bonded thereto are observed as voids in the non-etchable metal layer on the substrate.

The novel features of this invention sought to be patented are set forth with particularity in the appended claims. The invention, together with further objects and advantages thereof, may be understood from a reading of the following specification and appended claims in view of the accompanying drawing in which:

FIG. 1 is a sectional elevation view of apparatus in accordance with this invention showing a substrate having an etchable film thereon, a monomolecular antibody layer over the metal film, and a biological particle immunologically bonded to the antibody layer.

FIG. 2 is a sectional elevation view of the apparatus in FIG. 1 having additionally a non-etchable metal layer thereover and further illustrating means for applying the non-etchable layer in accordance with one embodiment of this invention.

FIG. 3 illustrates the etching of the apparatus of FIG. 2.

FIG. 4 is an isometric view, partially cut away, of apparatus in accordance with one embodiment of this invention for examining the etched apparatus of FIG. 3.

FIG. 1 is a sectional elevation view of a diagnostic slide in accordance-with this invention indicated generally at 30 and comprising a light-transmissive substrate member 10 which may be glass, mica, plastic, fused silica, quartz, or similar material, with glass being preferred, having a layer 11 of an etchable material which is preferably a film of an opaque etchable metal such as, for example, indium, and hereinafter, for convenience, referred to as metal, attached to one surface thereof. Metal layer 11 may be conveniently appled to substrate member 10 by evaporation as one example. The metal coated slide is then immersed in a solution of antibodies to the biological particle to be detected to adsorb a monomolecular layer of antibodies 12 on the surface of etchable metal film 11; this completes the fabrication of a diagnostic slide. The diagnostic procedure utilizing this slide comprises immersing the slide into a physiologic fluid specimen to be tested for the presence of biological particles of the species of interest. If the specimen contains particles of the species of interest, some of the particles, as 13 in FIG. 1, become immunologically bonded to antibody molecules 12 on the slide. Only particles specifically reactive with the antibodies will immunologically complex therewith.

The next step in accordance with this invention is illustrated in FIG. 2 and comprises the deposition of a non-etchable layer over the structure of FIG. 1. The non-etchable layer is preferably a light-transmissive, non-etchable material and may be glass or other suitable'oxide or a metal such as gold. In accordance with one embodiment of this invention, diagnostic slide 30 is immersed in an electrolyte bath (not shown) and an electrical potential is applied by a source 17 thereof between metal film 11 and a quantity 16 of non-etchable metal which is also immersed in the electrolyte solution to plate a quantity 14 of non-etchable metal onto the structure shown in FIG. 1. For reasons more fully set forth hereinafter, metal 16 is preferably gold. It is important in practicing this invention to carefully control the deposition of non-etchable metal coating 14 to insure that the deposited metal is discontinuous about the periphery of particle 13 as shown in FIG. 2 by gap 15. In analternative embodiment of this invention, nonetchable metal may be applied by evaporation, again preferably gold. In this case, also the deposition process is monitored to insure discontinuties of the nonetchable metal coating about the peripheries of each biological particle bonded to the slide.

FIG. 3 illustrates the next step in the practice of this invention and comprises the immersion of diagnostic slide 30 after the deposition of the non-etchable metal layer thereon into a container 19 of a quantity 20 of an etchant solution, such as an acid. Etchant 20 dissolves the biological particles bonded to the slide, the immediately underlying antibody which had been complexed therewith and the underlying portion of etchable metal film leaving a void 18 in the slide structure and exposing a portion of light-transmissive substrate 10. Etchant 20 further undercuts a portion of the structure surrounding void 18 by dissolving additional quantities of etchable metal 11 to produce an expansion of void 18 into area 21. This undercutting serves to provide amplification of the detectability of void 18 when the nonetchable metal is gold. This is the reason for preferring gold as the non-etchable metal and results from the fact that a thin gold layer such as is applied in accordance with this invention is quite light transmissive in the absence of an underlying layer of another metal but is substantially opaque when the underlying layer is present. Accordingly, after removal from the etchant bath, slide structure exhibits light-transmissive regions corresponding to the points at which biological particles have been bonded thereto, which lighttransmissive regions have dimensions determined by the diameter of regions 21 which are substantially greater than the dimensions of voids 18. At other regions diagnostic slide 30 remains opaque.

Following the etch procedure, slide 30 is examined to determine whether or not biological particles had been immunologically bonded thereto and consequently whether or not the test specimen contained the specific biological particles of interest. A virus particle, for example, is typically 1,000 A in diameter. By the use of gold metal backed by etchable metal which is undercut in the etch process as discussed above, light- .transmissive regions in excess of 10,000 A in diameter are produced at sites at which biological particles had been immunologically bonded to their specific antibodies on the slide. This provides for reading of the slide to detect particles in the specimen by optical microscopy. Furthermore, if desired, a counting grid may be superimposed over the microscopic image to thereby provide for determination of concentration of the particles of interest in the specimen.

FIG. 4 illustrates alternative examination procedures to optical microscopy in accordance with this invention. In FIG. 4 an enclosure 31 has a bracket 32 on one side thereof for supporting slide 30 adjacent an opening (not shown) in enclosure 31. An illumination source such as incandescent lamp 33 is positioned within enclosure 31 to direct light against slide 30. If desired, a reflector 34 may be employed to increase the light intensity impinging on slide 30. By use of such a light box, a diagnostic slide 30 processed as hereinbefore described may be examined by unaided visual observation in a slightly darkened room. The sites at which biological particles have been immunologically bonded to the slide appear as clearly distinct speckles of illumination when so viewed. Obviously, quantitative information regarding the concentration of particles in the specimen may be obtained by counting the speckles ofillumination and this may be aided by placing a grid structure over the outer surfaces of slide 30 in retaining means 32. Alternatively, an integrating, indicating photoresponsive device 35 may be employed to provide quantitative information on the concentration of biological particles in the specimen. Device 35 may, for example, conveniently comprise a lens system for focusing light transmitted through slide 30 onto the photocathode of a photomultiplier tube operating in the current mode having a millimeter connected in series with the photomultiplier tube.

While this invention has been described with reference to particular embodiments and examples, other modifications and variations will occur to those skilled in the art in view of the above teachings. Accordingly, it should be understood that within the scope of the appended claims, the invention may be practiced otherwise than is specifically described.

The invention claimed is:

1. Apparatus for detecting biological particles of a particular species in a fluid comprising:

a substrate;

a metal film on a surface of said substrate;

a layer of antibodies to said species of biological particle, said antibodies being adsorbed on said metal film;

means for applying a second metal layer over said layer of antibodies and any of said biological particles bonded thereto; and I means for selectively etching said biological particles, said metal film, and said antibodies.

. 2. The apparatus of claim 1 wherein:

said means for selectively etching comprises an acid;

said metal film comprises a metal which is etchable by said acid; and

said second metal layer comprises a metal which is resistant to etching by said acid.

3. The apparatus of claim 2 wherein said second metal layer has voids therein, said voids being disposed about the peripheries of said biological particles.

4. The apparatus of claim 3 wherein said substrate comprises a light-transmissive material which is resistant to etching by said acid.

5. The apparatus of claim 4 including additionally means for directing light onto a surface of said substrate.

6. The apparatus of claim 5 wherein said means for directing light onto a surface of said substrate comprises:

an enclosure having first and second end members,

said second end member having an aperture therein; I

a lamp within said enclosure adjacent said first end member; and

means attached to a surface of said second end member external to said enclosure for receiving said substrate and for positioning said substrate adjacent said aperture.

7. The apparatus of claim 6 further including means external to said enclosure for receiving light transmitted through said substrate and for indicating the quantity of light energy received.

8. A method for detecting biological particles of a particular species in a fluid comprising the steps of:

immersing a substrate having a metal surface into a solution of antibodies to said species of biological particle to adhere a layer of said antibodies thereon;

immersing said substrate having said antibodies thereon into said fluid whereby a plurality of said biological particles, if present in said fluid, immunologically bond'to said antibodies;

applying a second metal layer over said layer of antibodies and any of said biological particles;

immersing said substrate in an etchant solution; and

examining said substrate to determine whether any of said biological particles had bonded to said substrate.

9. The method of claim 8 further including the step bonding a metal film to a wafer of light-transmissive material to form said substrate.

10. The method of claim 8 wherein said applying step more particularly comprises the steps of:

immersing said substrate in a solution of a salt of said second metal;

immersing a block of said second metal in said solution of salt; and

applying an electrical potential difference between said block and said metal surface to electroplate said second metal onto said substrate.

11. The method of claim 8 wherein said applying step comprises evaporating said second metal onto said substrate. g

12. The method of claim 8 wherein said applying step further includes the step of monitoring the deposition of said second metal to insure that-said second metal layer is discontinuous about the peripheries of said biological particles.

13. The method of claim 8 wherein said examining step comprises microscopic examination of said substrate.

14. The method of claim 8 wherein said examining step more particularly comprises:

backlighting said substrate; and

visually observing as points of transmitted light locations on said substrate at which said biological particles had been bonded.

15. The method of claim 8 wherein said examining step more particularly comprises:

backlighting said substrate;

receiving and integrating light transmitted through said substrate; and

indicating the integrated quantity of said light to provide a measure of the concentration of said biological particles in said fluid.

16. Apparatus for detecting biological particles of a particular species in a fluid comprising:

a substrate:

an etchable film on a surface of said substrate;

a layer of antibodies to said species of biological particle, said antibodies being adsorbed on said etchable film;

means for applying a layer of non-etchable material over said layer of antibodies and any of said biological particles bonded thereto; and

means for selectively etching said biological particles, said etchable film, and said antibodies.

17. The apparatus of claim 16 wherein:

said means for selectively etching comprises an acid;

said etchable film comprises a metal which is etchable by said acid; and

said layer of non-etchable material comprises a metal which is resistant to etching by said acid.

18. The apparatus of claim 16 wherein said etchable film is opaque and said non-etchable material is light transmissive.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3250596 *8 Apr 196310 May 1966Boehringer & Soehne GmbhMethod for determining the virus adsorptive capacity of aluminum oxide
US3492396 *13 Mar 196727 Jan 1970Becton Dickinson CoAgglutinate separation method and apparatus
US3646346 *26 Dec 196829 Feb 1972Pharmacia AbAntibody-coated tube system for radioimmunoassay
US3770380 *19 Apr 19716 Nov 1973Us ArmyArticle and method for multiple immune adherence assay
Non-Patent Citations
Reference
1 *Chemical Abstracts, 63:10483b (1965).
2 *Chemical Abstracts, 63:3532d (1965).
3 *Chemical Abstracts, 65:2821g (1966).
4 *Chemical Abstracts, 73:48541m (1970).
5 *G. M. Edelman et al., Proc. Nat. Acad. Sci., 68, (9), 2153 2157 (Sept. 1971).
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3960488 *1 Apr 19741 Jun 1976General Electric CompanyMethod and apparatus for quantitative surface inhibition test
US3960489 *1 Apr 19741 Jun 1976General Electric CompanyMethod and apparatus for determination of concentration of immunologically reactive biological particles
US3960490 *1 Apr 19741 Jun 1976General Electric CompanyMethod and apparatus for detecting immunologic reactions by diffusion in gel
US3960491 *1 Apr 19741 Jun 1976General Electric CompanyMethod and apparatus for detecting immunologically reactive biological particles
US3975238 *26 Jun 197517 Aug 1976General Electric CompanyMethod and apparatus for detecting molecules in solutions
US3979184 *27 May 19757 Sep 1976General Electric CompanyProteins
US3979509 *3 Sep 19747 Sep 1976General Electric CompanyOpaque layer method for detecting biological particles
US4090849 *20 Dec 197623 May 1978General Electric CompanyDiagnostic device and manufacture thereof
US4092116 *27 Aug 197530 May 1978General Electric CompanyMultilayer, biochemical immobilization
US4219335 *18 Sep 197826 Aug 1980E. I. Du Pont De Nemours And CompanyImmunochemical testing using tagged reagents
US4238757 *19 Mar 19769 Dec 1980General Electric CompanyField effect transistor for detection of biological reactions
US4280815 *18 Jun 197928 Jul 1981Technicon Instruments CorporationElectrochemiluminescent immunoassay and apparatus therefor
US4328183 *18 Jul 19804 May 1982Mt. Sinai School Of Medicine Of The City University Of New YorkBlood cell typing and compatibility test procedure
US4592894 *22 Nov 19833 Jun 1986The United States Of America As Represented By The United States Department Of EnergyField emission chemical sensor for receptor/binder, such as antigen/antibody
US4612281 *5 Nov 198216 Sep 1986Palo Alto Medical Foundation Research InstituteImmunoassay for detecting immunoglobulins and test kit
US4794089 *25 Mar 198627 Dec 1988Midwest Research Microscopy, Inc.Method for electronic detection of a binding reaction
US4877747 *4 Apr 198631 Oct 1989Plessey Overseas LimitedOptical assay: method and apparatus
US4911782 *28 Mar 198827 Mar 1990Cyto-Fluidics, Inc.Method for forming a miniaturized biological assembly
US4931384 *20 Oct 19845 Jun 1990Ares-Serono N.V.For chemical,biochemical samples; coating substrate with material to bind species, reflect polarized light, measuring reflective dip
US4995402 *12 Oct 198826 Feb 1991Thorne, Smith, Astill Technologies, Inc.Analyte sensor
US5089387 *7 Jul 198818 Feb 1992Adeza Biomedical CorporationBiological diffraction grating design of lines of active hybridizing reagent
US5108889 *12 Oct 198828 Apr 1992Thorne, Smith, Astill Technologies, Inc.Assay for determining analyte using mercury release followed by detection via interaction with aluminum
US5118608 *22 Sep 19892 Jun 1992Ares-Serono N.V.Optical assay technique
US5169599 *30 Aug 19908 Dec 1992Teknekron Sensor Development CorporationMethod and apparatus for optically detecting presence of immunological components
US5397568 *25 Feb 199314 Mar 1995Whitfill; Craig E.Method of treating infectious bursal disease virus infections
US5397569 *25 Jan 199314 Mar 1995Whitfill; Craig E.Method of treating Infectious Bursal Disease Virus infections
US5413939 *29 Jun 19939 May 1995First Medical, Inc.Solid-phase binding assay system for interferometrically measuring analytes bound to an active receptor
US5679579 *29 Jan 199621 Oct 1997First Medical, Inc.Immunofluorescence measurement of analytes bound to a substrate and apparatus therefor
US5705399 *20 May 19946 Jan 1998The Cooper Union For Advancement Of Science And ArtCrystal detector oscillator, measuring signals with resonant frequency, electrodes for detectors
US5871748 *21 Aug 199616 Feb 1999Embrex, IncAdministering to the subject a vaccine complex comprising a live vaccine virus and antibodies bound to said live vaccine virus
US6136319 *27 Jan 199824 Oct 2000The University Of ArkansasMethod of treating viral diseases in animals
US629987411 Jul 20009 Oct 2001University Of ArkansasAdministering vaccine conjugate consisting essentially of a live virus and a neutralizing factor bound to live virus; neutralizing factor is selected from the group consisting of antibodies and antibody fragments
US63954831 Oct 199928 May 20023M Innovative Properties CompanyMicroarays for use in gene sequencing, monitoring gene expression, gene mapping, bacterial detection and drug screening
US64826388 Nov 200019 Nov 20023M Innovative Properties CompanyHeat-relaxable substrates and arrays
US64921331 May 200010 Dec 20023M Innovative Properties CompanyA microorganism culture device comprising: discs attached to a substrate and a reflector on one surface of each of the discs to reflect electromagnetic energy of selected wavelengths after the energy has passed through the disc. the
US659308920 Jul 200115 Jul 20033M Innovative Properties CompanyPolymeric substrate, mask layer having projected surface area and topographical surface area that is greater than the projected surface area, linking agents on mask layer
US659717623 Jul 200122 Jul 2003Quantum Design, Inc.Method and apparatus for making measurements of patterns of magnetic particles in lateral flow membranes and microfluidic systems
US666406020 Jul 200116 Dec 20033M Innovative Properties CompanyArrays with mask layers and methods of manufacturing same
US690002810 Oct 200231 May 20053M Innovative Properties CompanyReflective disc assay devices, systems and methods
US718984211 Jun 200213 Mar 20073M Innovative Properties CompanyA relaxed oriented plastic substrate with reactants and labels directly attached at binding sites where the structures of the reactants are identifiable by their discrete location; the label amount is proportional to the amount of reactant; increase detection signal strength; high surface area; kinetics
US768283310 Sep 200323 Mar 2010Abbott Point Of Care Inc.Immunoassay device with improved sample closure
US772309910 Sep 200325 May 2010Abbott Point Of Care Inc.Generates an electrochemical signal based on the formation of a sandwich between a first immobilized antibody to a target analyte, the target analyte, and a labeled antibody; capable of being used in the point-of-care clinical diagnostic field, including accident sites, emergency rooms, and surgery
US798138713 Nov 200919 Jul 2011Abbott Point Of Care Inc.Immunoassay device with improved sample closure
US816843918 Feb 20101 May 2012Abbott Point Of Care Inc.Method for measuring an analyte in blood
US821685313 Jul 201110 Jul 2012Abbott Point Of Care Inc.Immunoassay device with improved sample closure
US830936418 Feb 201013 Nov 2012Abbott Point Of Care Inc.Method of performing an immunoassay in blood
US83773928 Jun 201219 Feb 2013Abbott Point Of Care Inc.Immunoassay device with improved sample closure
US846092218 Feb 201011 Jun 2013Abbott Point Of Care Inc.Immunosensor system for blood with reduced interference
US874777417 Dec 201010 Jun 2014Abbott Point Of Care Inc.Integrated hinged cartridge housings for sample analysis
US876507515 Aug 20121 Jul 2014Abbott Point Of Care, Inc.Immunoassay reagent composition
DE3022426A1 *14 Jun 19808 Jan 1981Technicon InstrElektrochemilumineszenz-immunoassay
DE3042535A1 *2 Jul 198011 Feb 1982Wang Wei KungVorrichtung zur feststellung von induzierten signalen
EP0364208A1 *10 Oct 198918 Apr 1990Thorne, Smith, Astill Technologies, Inc.Assay and sensing means for determining analyte
EP2551671A29 Sep 200430 Jan 2013Abbott Point Of Care, Inc.Immunoassay device with immuno-reference electrode
WO1980002077A1 *17 Mar 19802 Oct 1980Gen ElectricMethod and apparatus for measuring antibody levels
Classifications
U.S. Classification435/5, 435/7.32, 216/108, 436/806, 436/805, 436/525, 435/7.21, 428/433, 435/287.9, 216/84, 435/287.2, 422/504
International ClassificationG01N33/553, C12Q1/70
Cooperative ClassificationG01N33/553, Y10S436/806, C12Q1/70, Y10S436/805
European ClassificationC12Q1/70, G01N33/553