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Enzyme Immunoassays in Diagnostic Medicine Theory and Practice *

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Enzyme Immunoassays in Diagnostic Medicine Theory and Practice * Enzyme immunoassays in diagnostic medicine Theory and practice * A. VOLLER,' D. E. BIDWELL, & ANN BARTLETT Serological methods are playing an increasingly important role in the diagnosis and epidemiological assessment ofdiseases. Simple, inexpensive methodsfor large-scale applica- tion are urgently needed. The enzyme immunoassay methods developed recently and reviewed here hold great promise for application in a wide variety of conditions. Under laboratory conditions they can be as sensitive as radio-immunoassay, but they can also be adapted as simple field screening procedures. These methods are based on the use of antibodies or antigens that are linked to an insoluble carrier surface. This is then used to " capture " the relevant antigen or antibody in the test solution and the complex is detected by means of an enzyme-labelled antibody or antigen. The degradation of the enzyme substrate, measured photometrically, is proportional to the concentration of the unknown " antibody " or " antigen " in the test solution. The application of these techniques to en- docrinology, immunopathology, haematology, microbiology, and parasitology is reviewed. At present there are two widely accepted assays Weemen & Schurs (31, 32), offered an attractive that employ labelled antibodies and antigens. They alternative to the labelled antibody/antigen methods are immunofluorescence, in which a fluorescent dye mentioned previously. Enzyme immunoassays is conjugated to the antibody, and radio-immuno- employ antibodies or antigens conjugated to assay, in which isotopes are attached to antibodies enzymes in such a way that the immunological and or antigens. In practice immunofluorescence is not enzymatic activity of each moiety is maintained. easy to quantify for antibody assays, since it depends These assays give objective results and are extremely on subjective visual assessments of fluorescence and sensitive, although, at present, they are not usually the results are usually expressed as the serial dilution quite as precise as radio-immunoassay. The reagents of serum that gives the least fluorescence. Radio- present no health hazards, they are stable, and have immunoassay, on the other hand, is highly sensitive long shelf-lives. Moreover, the estimation of results and permits precise quantification. However, the can either be visual or be made with a rather simple isotope labels may decay rapidly, so that the con- spectrophotometer of the type found in most labora- jugates have a short shelf-life; complex equipment is tories. The range of application of enzyme immuno- necessary for their assessment; and, because of the assays is potentially as wide as that of radio-im- medical hazards, they must be handled only by munoassay and they may also reinforce or replace highly trained personnel. other serological tests, such as complement-fixation, The introduction of enzyme immunoassays, pion- haemagglutination, and immunofluorescence. eered by Engvall & Perlmann (11, 12) a and Van * From the WHO Collaborating Laboratory, Nuffield PRINCIPLES Institute of Comparative Medicine, The Zoological Society of London, Regent's Park, London, N.W.1., England. ' Also at the Department of Clinical Tropical Medicine, Detection of antibody using enzyme-labelled anti- London School of Hygiene and Tropical Medicine, Keppel globulins (indirect method-Fig. 1) Street, London, W.C.1, England. Requests for reprints should be addressed to this author. The antigen is coupled to a solid-phase support a These assays are generally known as ELISA (enzyme- and the sera thought to contain antibody are incu- linked immunosorbent assays), but for the purposes of this paper the generic term enzyme immunoassays will be bated in this sensitized carrier. Excess serum compo- employed. nents are washed away and then the enzyme-labelled 3418 -55- BULL. WORLD HEALTH ORGAN., Vol. 53, 1976 56 A. VOLLER ET AL. ANTIGEN ADSORBED TO PLATE I ANTIBODY ADSORBED TO PLATE WASH WASH 2 ADD SERUML ANY SPECIFIC 2 TEST SOLUTION CONTAINING ANTIBODY ATTACHES 10 ANTIGEN ANTIGEN ADDED WASH WASH a ADD ENZYME-LABELLED ANT3LOBULIN 3. ADD ENZYME-LABELLED WHICH ATTACHES TO ANTIBODY SPECI FIC ANTIBODY WASH WASH 4. ADD SUBSTRATE 4. ADD ENZYME SUBSTRATE AMOUNT HYDROLYSED= AMOUNT AMOUNT HYDISISMAMOUNT ANTE ANTIBODY PRESE PRESENT Fig. 1. The principle of the enzyme-labelled antiglobulin Fig. 2. The principle of the double-antibody method method for measuring antibody (indirect method). for measuring antigen. antiglobulin (conjugate) is added. The conjugate will enzyme-labelled specific antibody is then added and become attached to the antigen-antibody complexes this becomes attached to the antigen already " cap- on the carrier surface, and the amount of conjugate tured " by the sensitized surface. After incubation, attached is measured by the amount of substrate that the excess conjugate is washed away and the amount it degrades. attached is measured by the rate at which it degrades This is a very useful method, since a single added substrate. enzyme-labelled antihuman globulin can be used to detect any human antibodies regardless of the dis- Detection of antigen by the labelled antigen competi- ease state that is being investigated. For more so- tion method (Fig. 3) phisticated investigations it is of course possible to Again, immunoglobulin containing specific anti- use separate enzyme-conjugated antisera to human body is attached to the solid carrier surface. This IgG, IgM, and IgA, and so class-specific antibodies time a mixture of solution thought to contain anti- can be detected and measured. gen and enzyme-labelled antigen are incubated in various proportions on the carrier. The amount of Detection of antigen by the double-antibody method enzyme-labelled antigen attached is again measured (Fig. 2) by the rate of hydrolysis of its substrate. The more In this variation immunoglobulin containing spe- antigen there is in the unknown solution, the less cific antibody is used to sensitize the carrier surface. labelled antigen will be attached. The solution containing the antigen is then incu- In all these methods the end result is a change in bated with the sensitized surface and the excess colour of the enzyme substrate. This can be mea- solution is washed away. A conjugate consisting of sured accurately in a spectrophotometer. ENZYME IMMUNOASSAYS IN DIAGNOSTIC MEDICINE 57 1. ADSORB ANTIB6DY TO SURFACE antigens are available, absolute measurements of the optimum coating quantities can be determined and used for later batches of antigen. Similarly, for antibody coating of plates, if specffic antibody is used, an absolute, measured quantity can be used LrI each time. However, for convenience it will often be 4 found that the whole immunoglobulin fraction of an 2a.ADD ENZYME-LABELLED 2b. ADD ENZYME-LABELLED ANTIGEN +tJNKNOWN ANTIEN ANTIGEN antiserum can best be employed. In the latter case, a chequer-board titration must be carried out on each new antiserum. Test parameters It is easiest to determine the optimum incubation times and temperatures for each new system by trial and error. For convenience, in our laboratory, plates I I are usually sensitized overnight at 4°C and serum 3a. 3b. and conjugate incubation times of 2 h each are used. 13 ~~~ADD However, in many systems, adequate sensitization is ir j f ENZYME SUBSTRATE achieved after 1 h at 37°C and an incubation time of 1 h at 37°C is often satisfactory. Shorter times tend to yield less accurate results. SUBSTRATE HYDROLYSISa LABELLED ANTIGEN] The washing procedures are critical and it is important to ensure that all wells or tubes are DIFFERENCE BETWEEN 3a &3b 2JNKNWOWWNWKT1GE? treated in exactly the same way. Fig. 3. The principle of the enzyme-labelled antigen Conjugates competitive assay for measuring antigen. Engvall & Perlmann (11, 12) favoured alkaline phosphatase as an enzyme marker. It has high activity, and the chosen substrate is cheap and GENERAL CONSIDERATIONS nontoxic, with a bright yellow reaction colour that The carrier surface can be assessed visually or in an inexpensive spectro- The carrier may be beads, tubes, or plates. Some photometer. Conjugates of alkaline phosphatase can workers have used Sepharose beads, since these be stored at 4°C with sodium azide preservative. permit covalent linking of antigen or antibody to the Peroxidase was shown long ago by Avrameus & surface (7, 8); however, they are less suitable for large- Uriel (1), Nakane & Pierce (24), and Nakane (23) to scale use than are the polystyrene tubes used by be a good choice of enzyme for conjugation. Again, Engvall & Perlmann (11, 12) or polystyrene micro- it has high activity, is cheaper than alkaline phos- haemagglutination plates (35). The latter are parti- phatase,and yields a visible (brown) reaction product. cularly suitable for large-scale use because they are The choice of enzyme is largely a matter of cheap and small quantities of reagents can be used. personal preference. Conjugates of either that are Both polystyrene tubes and plates are satisfactorily available at present are not entirely satisfactory, coated with antigen or antibody by passive adsorp- since they contain varying amounts of polymers and tion. Solutions containing 1-10 mg/litre of the speci- unconjugated proteins. fic protein usually give adequate coating. In tests employing labelled antibodies, it is often possible to label the whole immunoglobulin fraction Coating the carrier with antigen and antibody with the enzyme. However, to ensure specificity, For most tests, only crude mixtures of antigens pure antibodies, prepared by affinity chromato- are available. For these, the only practicable way of graphy, may be preferable in some instances. determining the correct amount for coating is to It is always best to use the most highly titred carry out chequer-board titrations against positive antisera that are available, because this allows the and negative sera. The antigen dilution that gives the conjugates to be used in a more dilute form and so best discrimination between the positive and nega- requires less enzyme, which is the most expensive tive sera is used in subsequent tests. Where pure reagent in these tests. 58 A.
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