ELSEVIER Biochemical Education 26 (1998) 157-160

Biochemical Education

Experimental Section

A simple immunoassay-based system capable of detecting antibody raised against human IgG

Gary Walsh”, Brian O’Shaughnessy”, Nancy Shanleyb, John J. Tobin” “Industrial Biochemistry Programme, Universiry of Limerick, Castletroy, Limerick, Ireland ‘Department of Science, Limerick Regional Technical College, Moylish, Limerick, Ireland

‘Olympus Diagnostica GmbH, O'Callaghan ‘s Mills, Co Glare, Ireland

Abstract

This experiment serves to underline the following concepts: (a) the inherent specificity of antibody-antigen binding, (b) the bivalent nature of antibody binding, and (c) the value of antibodies as diagnostic reagents. The experiment is simple and relatively inexpensive to perform, and serves to underpin the theory of antibody structure and function as taught in many basic biochemistry and immunology courses. 0 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved

1. Introduction

Antibodies form part of the body’s defence mechanism against infection and disease. They function through their ability to bind to ‘foreign’ proteins and other macromolecules (antigens). Antibody-antigen binding is an extremely biospecific interaction, with antibodies generally only being capable of binding the specific antigen against which they were raised. Central to their effective functioning in viva is their bivalent nature. This allows them to form antibody-antigen complexes, which are in turn more easily recognised and digested by phagocytic cells. In vitro this may often be visualised as the ‘precipitin’ reaction, which occurs in free solution when antibody and antigen are mixed together at appropriate concentrations.

The specificity of the antibody-antigen interaction has been exploited to develop a wide range of diagnostic tests (immunoassays), which detect either antigen or antibody. As antibodies lack an inherent characteristic which facilitates their easy detection in an immunoassay, an easily detectable marker (label) must normally be incorporated into the assay system. Markers commonly used include radioisotopes (radioimmunoassay) or enzymes (enzyme immunoassay).

In an alternative immunoassay format, latex particles have been used as the indicator system to provide a range

of simple, easy-to-use diagnostic reagents [l]. Latex beads are small uniform spherical particles normally manufactured by polymerisation of styrene in an aqueous environment [2]. Particle diameter can be controlled during synthesis, and particles in the range 0.1-1.0 pm have proved most suitable for diagnostic applications. Individual particles of such diameter are not visible to the naked eye, but appear as a milky solution when in suspension. Incubation of latex particles with a protein solution leads to adsorption of the protein onto the latex surface. (Alternatively, modified latexes containing reactive chemical groups can be manufac- tured, which facilitates covalent attachment of the protein molecules to the particle surface). Latex coated with antibody can then be used to detect antigen in any sample provided [Fig l(a)]. Alternatively, latex coated with antigen can be used for detection of antibodies [Fig l(b)]. The bivalent nature of antibodies means that as antigen-antibody binding occurs, large aggregates or clumps of latex particles are formed. This process can be visualised by the appearance of white granules in what was a smooth milky latex suspension.

In their simplest format, latex-based immunoassays are performed on glass or plastic test cards. The cards contain a number of test cells, or wells, in which the reaction can take place. The coated latex particles and sample to be assayed are mixed together in one such well

0307-4412/98/$19.00 + 0.00 0 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PII: SO307-4412(98)00049-l

158 G. Walsh et aL/Biochemical Education 26 (1998) 157-160

and then incubated with gentle rocking. The presence of analyte will promote agglutination within 3-5 min [Fig.

l(c)]. Numerous latex-based assays have found commercial

application [l-3]. Advantages include assay speed, ease of use, lack of a requirement for sophisticated laboratory equipment and relatively low cost of manufacture. Hence, the tests find widespread application, particularly in poorer regions of the world. Amongst the most popular latex-based tests are those capable of detecting pregnancy. Pregnancy detection is based upon incubating a urine sample from the prospective mother

with latex particles coated with antibody raised against hCG (human chorionic gonadotrophin - the hormone produced only by pregnant women, and found in their blood and urine). Latex particles coated with antigens or antibodies associated with specific infectious agents are also often used to detect the presence of these infectious agents in biological samples such as blood (e.g. latex coated with antibodies against Hepatitis B surface antigen to screen for Hepatitis B virus, or latex coated with HIV antigen to screen for antibodies to HIV - the AIDS virus). The practical outlined below, which can be completed in under two and a half hours, entails coating

Latex particles coated with antibody +

-_)

Antigen present in biological sample

Agglutination of latex parlicles

Latex particles coated with antigen +

Antibody present in Agglutination of biological sample latex patlicles

Fig. 1. Latex agglutination assay system. In (a) latex particles have been coated with antibody. The presence of appropriate antigen in the sample tested results in agglutination of the latex particles. The antigen present effectively acts as a bridge between adjacent latex particles. In (b) the latex particles have been coated with antigen. In this case the presence of appropriate antibody in the assay sample results in agglutination of the latex. Agglutination can easily be detected visually as illustrated in (c). In this case the well on the left hand side of the plastic test card contains unagglutinated latex particles, whereas the well on the right contains agglutinated latex. Reproduced with permission from Walsh, G., Headon, D., Protein Biotechnology, J Wiley and Sons, Chichester, 1994.

G. Walsh et al.lBiochemical Education 26 (1998) 157-160 159

human IgG (in this case representing antigen) onto latex beads. This is subsequently used to screen samples for the presence of mouse antibody raised against human IgG.

2. Materials

2.1. Equipment required includes

Pipettes (0.1 and 1.0 ml), test tubes or microfuge tubes, water bath (45X), bench-top centrifuge or micro- fuge, vortex, plastic test cards and mixing sticks (e.g. toothpicks).

2.2. Reagents required include

Latex, Glycine Buffered Saline (GBS; 0.9% NaCl in 0.1 M glycine buffer, pH 8.2) antigen solution (1.0 mg ml-’ human IgG, also containing 4.0 mg mll’ BSA in GBS), and blocking solution (5% BSA in GBS); Negative control solution (GBS). Positive control solution (1 mg ml-’ mouse antibody against human IgG in GBS). Four ‘unknown’ solutions (e.g. casein, hemoglobin, human albumin, and mouse antibody raised against human IgG, made up separately; 1.0 mg ml-’ in GBS). Latex (unmodified) suppliers include Polymer Laboratories, Shropshire, UK, Tel. +44(O) 1694 723581, or Sigma Chemical Co. Agglutination cards are obtain- able from many medical reagent suppliers.

3. Method

Pipette 0.5 ml of the 1% latex into a test tube and add 0.5 ml of the human IgG. Incubate at 45°C for 30 min. Add 0.1 ml of the blocking solution and incubate at 45°C for a further 30 min. Centrifuge the mixture in the bench-top centrifuge at medium speed for 2-4 min. Pour off the supernatant and resuspend the coated latex in 1 .O ml of GBS using the vortex mixer (the latex may also be resuspended by repeated aspiration with a Pasteur pipette).

3.1. Assay of samples

Pipette 40 ~1 of the controls or unknown samples into separate wells on the test card. Pipette 40 ~1 of coated latex into each of the wells. Mix the latex with the samples using mixing sticks. Gently tilt the card from side-to-side for 2-3 min. Record and interpret results.

4. Notes

(a) Latex beads of varying diameter may be used. In our experience, 0.8 pm particles are most suitable as they

(b)

(c)

(4

W

m

k)

c-4

are easy to sediment and yield clearly identifiable agglutination patterns. Latex obtained commercially is usually sold at lo-30% (w/v). The latex may be diluted to 1% with water prior to the practical class. In our experience, the coated latex may be resus- pended adequately by vortexing (Methods). However, if difficulty in resuspension is experienced, or if the latex appears grainy when placed alone on a slide, repeated aspiration with a Pasteur pipette may be employed for more effective resuspension of the latex. Alternatively a sonicator may be used, if available. Home-made assay cards may be prepared by lightly painting circles (approx. 15-20 mm in diameter) on a plastic card surface, using typing correction fluid (eg Tipp-Ex). Plastic cards should be dark/black in color to aid visualization of agglutination. If automatic pipettes are not available for class use, Pasteur pipettes (1 drop) may be used for applying the samples and latex to the test cards. Volumes of sample/latex of less than 40 ~1 however should not be applied, as the use of such smaller volumes renders visualization of the agglutination process more difficult. The manufacturing parameters listed in the methods section have been optimized. However, the experi- ment can be modified to investigate the effect of changing parameters such as bead diameter, coating times, etc on assay results. Polyclonal antibody preparations are most often used in latex-based immunoassay systems. Monoclonal antibodies may be used provided the antigen displays two or more identical epitopes against which the monoclonal has been raised. Alternative latex-based immunoassay systems may be constructed by applying appropriate antigen- antibody pairs to the protocol outlined. (We have used anti-hCG polyclonal antibodies to construct a pregnancy detection kit). The most satisfactory results are obtained in cases where the antigen is a macromolecule.

5. Question

Latex immunoassays are basically qualitative, giving a ‘yes’ or ‘no’ answer. Once the antigen is present above a certain minimal concentration in the assay, agglutination will be observed. Although agglutination may occur slightly more rapidly at higher antigen concentrations, in practice this cannot be used as the basis of a quantitative/ semi-quantitative test. If you were given a positive standard of known antigen concentration and a sample of antigen of unknown concentration, could you use the

160 G. Walsh et al./Biochemical Education 26 (1998) 157-160

assay system to quantify the antigen concentration present in the unknown sample?

(Answer: The minimum concentration of antigen required to promote agglutination can be determined by assaying a series of dilutions of the antigen standard. Various dilutions of the sample of unknown antigen concentration can then be assayed in order to determine the maximum dilution still capable of promoting aggluti- nation. The antigen concentration in the unknown can be determined, semi-quantitatively at least, by multiplying this dilution factor by the minimum concentration of

antigen that was determined to be capable of inducing agglutination).

References [l] L.B. Bangs, Latex immunoassays, Journal of Clinical Immuno-

assays 13 (1990) 3127-3131. [2] L.B. Bangs, Uniform Latex Particles. Seragen Diagnostic Inc.,

U.S.A., 1984. [3] J.M. Singer, CM. Plotz, The latex fixation test. I. Application to the

serologic diagnosis of rheumatoid arthritis, American Journal of Medicine 21 (1965) 883-892.