GB2123146A - Dual parameter flow immunoassays - Google Patents

Abstract

The invention encompasses an immunoassay technique based on the agglutination of two different particles having measurably different properties and coated with a substance specifically bindable to a substance to be determined. The agglutination is measured by using an optical or electrical counter, such as those designed to count blood cells, to measure the aggregate formed by measuring a distinct property associated with each particle, generally, size and fluorescense.

Description

SPECIFICATION Dual parameter flow immunoassays Particle counting immunoassay is well known nonradioimmunoassaytechnique,J. IMMUNOL.

METHODS, 18,33-44, (1977). This technique is based on the agglutination of latex or similar particles coated with a substance specifically bindable to a substance to be determined. The agglutination is measured by using an optical or electrical counter, such as those designed to count blood cells, to determine the reduction in the number of nonagglutinated particles.

The technique has been used to determine antigens, antibodies and immune complexes. J. AUTOM.

CHEM., 2,149-152, (1980),; J. IMMUNOL. METHODS, 23,29-50, (1978); and J. IMMUNOL. METHODS 28, 13-23, (1979). Avariation ofthe technique has been used to determine small molecules such as digoxin, CLIN. CHEM. 27/1,1205-1209, (1981). Counting of the number agglutinated particules is also well known.

Variations of particle counting techniques have been developed to overcome problems which became apparent. For example, the use oftwo incubation steps and particles of different sizes has been describes to overcome the so-called prozoning problem (samples containing very high levels of antigen appearto be negative since there is minimal agglutination). German patentapplication P3006899.6, September 1980, and P 29 18 342.4, November 20, 1980.

The present invention is particularly distinguished from the prior art in thatthe invention employs particles which have detectably different properties, such as size and fluorescence. Ratherthan measuring the disappearance of nonaggregated particles, the formation ofthe aggregate ofthetwo different particles and the substance to be assayed is measured by determining within the aggregate different particle properties associated with for example a large particle (i.e., size) and a small fluorescence particle (i.e., fluorescence). This measurement is accomplished in a dual channel optical-electrical cell counter of the type described in U.S. Patent 4,198,160 or in a fluorescence microscope.

This invention encompasses a methodfordetecting a multivalent specific binding substance in a biological fluid. First and second microscopic particles having different detectable properties and coated with a substance bindableto the multivalent specific binding substance wherein aggregates containing said first and second microscopic particles reformed in the presence of the multivalent binding substances are detected by the measurement of different properties associated with the first and second microscopic particles within the aggregate.Typically, the invention involves the detection of multivalent antigens in the solution using antibody-coated particles oftwo different sizes- nonfluorescent particles in the size range of 4-501l (micron) and fluorescent particles in the size range of 0.8p 1 or. In a sample without antigen, the fluorescent and non-fluorescent particle are not attached while in an antigen-containing sample the large, nonfluorescent particles have attached fluorescent particles to form aggregates.

The invention includes the novel aggregates of 4-501l nonfluorescent particles and 0.08-lOufluores- cent particles each coated with a substance bindable to a multivalent specific binding substance and bound together by such multivalent specific binding substance.

The invention also includes methods for detecting monovalent specific binding substances.

The term multivalent specific binding substance in the context of this invention refersto antigen, antibodies, and other biological molecules having more than one specific binding site. Viral antigens, such as those associated with hepatitis, (hepatitis B surface antigen, hepatitis A, hepatitis core antigen) herpes and other viral associated diseases, as well as their antibodies are examples of multivalent specific binding substances. Bacteria and bacterial antigens such as Neisseria gonorrheae and Streptococcus are other multivalent specific binding substances. Antigens associated with cancer such as carcinoem bryonicantigen (CEA) are likewise multivalent specific binding substanceswhich can bedeterminedbythe methods of this invention.Those skilled in the immunoassay arts will recognize wide variety of multivalent specific binding substances.

Monovalent specific binding substances can also be determined by methods and reagents of thins invention using polymers having a multiplicity of bound monovalent specific binding substances and particles coated with a substance bindable to the monovalent specific binding substance. The monovalent specific binding substances in the unknown will compete for binding sites on the particles and inhibit agglutination between the particle and the polymer having monovalent specific binding substance bound to it.Thus drugs and hormones are conjugated to a polymer, for example, proteins such as bovine serum albumin or polysaccharides such as dextran to form synthetic multivalentspecificbinding substanceswhichare capable of binding the microscopic particles of the type earlier described, preferably a nonfluorescent microscopic particle coated with a substance bindable to the synthetic multivalent specific binding substances and a small microscopicfluorescent particle coated in a similar manner. The resulting aggregate is detected as earlier described.

The invention utilizes at leasttwo types of microscopic particles each having detectably distinct prop erties. The particles are generally synthetic polymers such as polystyrene, polypropylene, polyacrylate, and the like. Red blood cells ofthetype described in U.S.

Patent 3,715,427 are also suitable particles for use in this invention. A preferred particle material is polystyrene, also referred to as polystyrene latex particles.

Suitable particles are generally known in the microparticle arts. It is also preferred to use particles of two different sizes, larger particles being in the p micron range and a smaller particle being in the .08-1 Op micron range. It is also preferred that the small particle befluorescently labeled. The later is accomplished by incorporating fluorescent material into the particle or affixing fluorescent molecules to the surfaceofthe particle.

Each particle is coated with a substance specifically bindabletothe multivalent binding substance. Typi cally, if a multivalent antigen is being detected the particles are coated with antibody to the antigen. More specifically, if hepatitis B surface antigen is to be detected the particles will be coated with antibody or F)ab')2fragment of the antibodyto hepatitis B surface antigen.Common systems are as follows: Large Multivalent Small Particle Coating Binding Substance Particle Coating 6 - guinea pig Hepatitis B 0.4911 goat antianti-HBsAg IgG surface antigen HBsAg F(ab')2* 611 - monoclonal CEA 0.49p-monoclonal antibody I anti- antibody II anti-CEA CEA 611-Hepatitis B Anti-HBsAg 0.49p Hepatitis B surface antigen surface antigen monoclonal anti- Chlamydia 0.911 monoclonal body I anti- antibody anti Chlamydia Chlamydia Fixed red blood HBsAg 0.624 goat anticell-guinea pig HBsAg F(ab')2* anti-HBsAg IgG 6,u - Antibody to bovine serum 0.491l antibody to digoxin albumin having digoxin a plurality of digoxin molecules bound thereto 6p - antibody to dextran having O.49p-antibody to digoxin a plurality of digoxin digoxin molecules bound thereto *F(ab')2fragment of anti-HBsAg IgG having activity against HBsAg The particles are coated by adsorption or by covalent linkage using anyone of a number of commonly known coating methods.

In practice, the presence of the multivalent specific binding substance in a test sample causes the particles to agglutinate (aggregate). Thus, particles having detectably different properties are present in the aggregrate and the aggregrate is detected by measuring those properties. Most typically, a num ber of small fluorescent particles are bound to a large nonfluorescent particle and the aggregate is detected by measuring its unique combination of size and fluorescence.The aggregate can also be detected by labeling the large and the small particle with differentfluorescent dyes and detecting the dual fluorescenceoftheaggregate.Thetwo parameter system (volume + fluorescence ortwo fluorescent particles) is particularly advantageous in that it avoids false-positive results due to nonspecific aggregation of each type of particle.

False-positive results are also reduced by pretreat ing the serum with pepsin to digest nonspecific serum interferences. Thus, incubating equal volumes of serum and 1 % pepsin in 0.15 molar hydrochloric acid for 15 minutes at 37 C then neutralizing the solution with 0.5 molar sodium hydroxide is an effective pretreatment. Pepsin treatment is particular ly useful in hepatitis measurement in that the hepatitis antigen is resistant to pepsin digestion.

It has also been found that nonspecific agglutina tion ofthetwo particles during storage as well as during the assay is reduced by suspending the particles in an optimized buffer. For example, a 0.1 molar glycine,0.15 molar sodium chloride buffer at pH 9.2 and containing 1% bovin serum albumin, 1 molar NaCI and 10% dimethylformamide reduces nonspecific agglutination and improves the assay.

The invention is illustrated bythefollowing exam ples.

EXAMPLE I Coating of Microparticles with Anti-HBsAgAnti- bodies Polystyrene particles (6,u,10% suspension) are washed free of surfactants by repeated washes in water followed bythreewashes in methanol. Guinea pig anti-HBsAg IgG (6 mg) is added to a suspension of 6x 108 particles in phosphate buffered saline (PBS), pH 7.4. (total volume 14 m Ls). The mixture is stirred overnight (20 hours) at roomtemperatrure using a magnetic stir bar. The particles are washed twicewith PBS and resuspended to 6 x 106 particles/mL in PBS containing 1% bovine serum albumin and 0.1% sodium azide.

In a similar manner,0.4911fluorescent particles (Polysciences, Inc. x 1010 total) are coated with 300 g affinity purified goat antilHBsAg F (ab')2 (one mL total coating volume). After coating,the particles are washed twice with PBS and resuspended to a density of 6 x 108 particles per mL in PBS containing 1% bovine serum albumin and 0.1% sodium azide.

EXAMPLE II HBsAg ASSAY PROCEDURE Pretreatment: To 100 L 10011Lserum orplasma is added lOOp L1% pepsin solution in 0.15 HCI. The mixture is allowed to digest for 15 minutes in a 37 C water bath before being neutralized with 0.5M NaCH.

Assay: 50 L of 6 particles coated with guinea pig anti-HBsAg lgG (3 x 105 particlestotal) is added to the pretreated sample. The mixture is shaken (220 rpm) for one hour at room temperature on a Tektator rotary shaker. Small fluorescent microparticles (0.49 p, 3 x 107total) coated with goat anti-HBsAg F (ab')2 fragments are then added. The mixture is centrifuged at room temperature at approximately 1500 x g for five minutes to mix the large and small particles.

In samples containing HBsAg, aggregates of 6p particles and small fluorescent particlesareformed.

In HBsAg negative samples, only a low level of 6p 0.49,uaggregates areformed. The presence or absence of such aggregates can be enumerated by use of a fluorescent microscope or by instru mentation designed to measure coincident volume and fluorescence signals.

EXAMPLE Ill Coating ofMicroparticles with Anti-CEA Monoclonal Antibodies Microparticles (6 ,2 x 108 total, Microspheres Research) were washed five times in PBS, pH 8.3. To the washed beads were added 200 g monoclonal antibody I anti-CEA and 200 l PBS pH 8.3. The particles were stirred overnight at room temperature, washedthreetimes in 0.1M glycine, 0.15M NaCl, pH 9.2, containing 1% bovine serum albumin (GBS-BSA) and resuspended in 1 ml GBS-BSA.

Similarly, fluorescently dyed 0.49 p particles (1.6x 1010 total, Polysciences Inc) were added a coating solution containing 120111 monoclonal antibody anti-CEA (9Opg) and 120 pI PBS, pH 8.3. The mixture was stirred overnight, washed twice with GBS-BSA and resuspended to 1 ml in GBS-BSA.

EXAMPLE IV CEA Assay Coated microparticles were diluted 1:30 in GBS BSA. To 300 l serum were added 50 p 6 p particles coated with monoclonal antibody (3.18x 105total) and 200,u11 m glycine, 1.5m NaCI, pH 9. The mixture was incubated one hour at 45 C, then washed using 1 ml GBS. Fluorescentlydyed 0.4911 particles (50 l, 2.6 x 1 06 total) coated with monoclonal antibody II were added, and the mixture centrifuged at room tempera ture at approximately 1500 x g for 5 minutes to mix the 6 p and 0.49 p particles.The suspension was diluted with 1 ml normal saline and the numberof6 p -0.49p aggregates determined. This assay method allows the amount of CEAto be determined in a quantitative manners illustrated in the table below: Standard Curve with CEA Mixed Monoclonal Flow Assay CEA Conc Number of 611- 0.49 aggregates out {ng/ml) of 10,000 Total 6p particles Average 0 616 1 709 2 850 5 942 10 1154 20 1541 EXAMPLE V This invention is used to determine monovalent binding substances. Nonfluorescent6.0 particles are coated with antibody to digoxin. The test sample is incubated with the coated particles and bovine serum albumin having a plurality of coupled digoxin molecules. Afterthe incubation a small fluorescent particle coated with anti-digoxin antibody is added. In asamplewithoutdigoxin,the digoxin-BSA binds the large and small particles together. In a sample containing digoxin,thedrug competeswiththe digoxin-BSAfor binding sites on the first solid phase and thereby reduces the number of volume4luoresc- ence aggregates formed.

In a similar manner monovalent (one specific binding site) substances such as drugs, hormones and the like can be bound to bovine serum albumin, dextran, or similar synthetic polymers for similar determinations.

Claims (7)

1. A method for detecting a multivalent specific binding substance in a biological fluid comprising intermixing with the biological fluid first and second microscopic particles having different detectable properties and coated with a substance bindableto the multivalent specific binding substances, wherein aggregates containing said first and second microscopic properties are formed in the presence of the multivalent binding substance and detecting said aggregates by detecting different properties associ ated with thefirst and second microscopic particles in the aggregate.

2. A method for detecting a multivalent specific binding substance in a biological fluid comprising intermixing with the biological fluid nonfluorescent microscopic particles and smallerfluorescent mic roscopic particles wherein the fluorescent and nonfluorescent particles are coated with a substance bindableto the multivalent specific binding subst ance wherein aggregates containing said nonfluorescent particles and one or more fluorescent particles are formed in the presence of the multiva lent specific binding substance and detecting the aggregate through its size andfluorescence characteristics.

3. The method according to Claim 2 wherein the biological fluid is pretreated by pepsin digestion.

4. The method according to Claim 3 wherein the method is conducted in a solution which is about 0.1 molar glycine, 0.15 molar sodium chloride buffered at pH 9.2, and containing 1 % bovine serum albumin, 1 molar NaCl, and 10% dimethylformamide.

5. Aaqueoussuspension ofaggregatesofa nonfluorescent microscopic particle having smaller fluorescent microscopic particles bound thereto, each particle coated with a specific binding substance and bound together with a multivalent specific binding substance bindable to the specific binding substance coated on the particles.

6. A method for detecting a monovalent specific binding substance in a biological fluid comprising intermixing with the biological fluid a polymer having bound thereto a multiplicity of the monovalent specific binding substance: nonfluorescent microscopic particles and smallerfluorescent microscopic particles wherein the fluorescent and nonfluorescent particles are coated with a substance bindable to the monovalent specific binding substance in the biological fluid and the polymer bound monovalent specific binding substance wherein the monovalent specific binding substances in the biological fluid inhibits the formation of aggregates between the polymer having bound thereto a multiplicity of the monovalent specific binding substance and the fluorescent and nonfluorescent microscopic particles; and measuring the extent of aggregate formation by detecting the size and fluorescent characteris tic ofthe aggregates.

7. A method for detecting a multivalent specific binding substance in a biological fluid substantially as described with reference to the Examples herein.