Memoranda are state- Les Memorandums ments concerning the exposent les conclu- /e , conclusions or recom- sions et recomman- M e mmooranrantedaa mendations of certain dations de certaines / a t w / /WHO scientific meet- /reunions scientifiques ings; they are signed de /'OMS; ils sont Memoranaurns by the participants in signes par les partici- I the meeting. pants a ces reunions.

Bulletin ofthe World Health Organization, 62 (2): 217-227 (1984) © World Health Organization 1984 Immunodiagnosis simplified: Memorandum from a WHO Meeting*

Technologies suitable for the development ofsimplified immunodiagnostic tests were reviewed by a Working Group of the WHO Advisory Committee on Medical Research in Geneva in June 1983. They included tests and use ofartificialparticles coated with immunoglobulins, direct visual detection of antigen-antibody reactions, enzyme- immunoassays, and and fluoroimmunoassays. The use of mono- clonal antibodies,in immunodiagnosis and of DNA /RNA probes to identify viruses was also discussed in detail. The needfor applicability of these tests at three levels, i.e., field conditions (or primary health care level), local laboratories, and central laboratories, was discussed and their use at thefield level was emphasized.

Classical serological techniques have been used for All these tests can be carried out in laboratories that a long time for diagnostic purposes, e.g., for con- are equipped with basic instruments as well as special- firmation of clinical diagnoses, epidemiological ized apparatus (e.g., gamma counters for RIA, ultra- studies, testing of blood donors, etc. Some of these violet microscopes for IMF, etc.), which are usually techniques have been standardized to a high degree of available only in the larger, central laboratories. reproducibility, e.g., the The idea of developing simplified immunodiag- (CFT) and the indirect haemagglutination test, and nostic tests for use at the primary health care level or have been used in spite of difficulties reported by for field studies was considered a long time ago. The many laboratories (e.g., the anticomplementary introduction of artificial particles (latex, bentonite, properties of sera for the CFT). beads) and stabilized red blood cells coated with anti- Techniques using antibodies or antigens labelled gens has initiated a wide range of simple tests (drop with different markers have been introduced into slide tests) for detection of rheumatoid factors and immunodiagnosis during the past few decades and antinuclear antibodies, for the diagnosis of Chagas' three of them have been quite widely used in routine disease, echinococcus, etc. Some of these tests are testing, i.e., radio- (RIA), immuno- still in use as screening and/or qualitative tests. fluorescence (IMF), and enzyme-immunoassays The visualization of antigen-antibody reactions has (EIA), e.g., ELISA. The high degree of immuno- recently been achieved by other techniques, described chemical sensitivity of these tests makes them useful in this report, which could lead to the development of for the estimation of not only antibodies but also new simple tests. antigens or other substances that are present in The specificity of most immunodiagnostic tests minute quantities in the blood or in the urine, stool, depends on the purity of the reactants used for the etc. estimation. Some tests have shown very high degrees of specificity (e.g., the estimation of hormones by * This Memorandum is based on the report of the ACMR (WHO RIA), while others have been less effective because of Advisory Committee on Medical Research) Working Group on Diag- to nostic Tests for Use at the Primary Health Care Level, which met cross-reactivity (e.g., the estimation of antibodies in Geneva on 14-16 June 1983. The participants at this meeting are parasites using partially purified antigens). The intro- listed on page 227. Requests for reprints should be addressed to duction of monoclonal antibodies will undoubtedly , Division of Communicable Diseases, World Health Organization, 121 1 Geneva 27, Switzerland. A French translation of improve the immunochemical specificity of some this Memorandum will appear in a later issue of the Bulletin. immunodiagnostic tests, as already demonstrated. 4395 -217 218 MEMORANDUM

The ideal diagnostic test should be sensitive, is mainly to use mobile teams for screening the popu- specific, reproducible (precision of ), inexpen- lation on the basis of lymph node enlargement and sive, rapid and simple to perform. In addition, in presence of trypanosomes in the blood and lymph some situations, e.g., infectious diseases, it should be node juice. These methods are time-consuming and able to discriminate between active infections and require an important investment in terms of equip- past infections and indicate the effect of therapy and ment, skilled personnel, and running costs. In add- management of patients. The detection of antigens ition, the proportion of false-negative results from may be one of the ways to fulfil the latter require- this type of screening can be considerable - accord- ments. ing to a recent experiment in West Africa, as high as 80%. With the introduction of the fluorescent anti- body test, it was demonstrated that serodiagnosis would increase the efficacy of screening remarkably TECHNOLOGIES (AVAILABLE OR PROPOSED) (threefold increases of confirmed positive cases were reported in Zaire and Congo). However, these tests Agglutination using microorganisms are usually carried out in a laboratory far away from Simple agglutination reactions have been used for the endemic areas and so there is a delay between the many years to identify various species of bacteria. time of sampling and obtaining the results. It is thus Typhus fever (a rickettsiosis) is identified by the Felix- difficult to retrace later the patients with positive Weil tube-agglutination test and depends on agglutin- diagnoses and refer them to hospitals for treatment. It ation of a strain of Proteus bacillus containing an also adds a further burden on the limited personnel antigen that cross-reacts with the rickettsial antigens. and transport resources. Similarly, typhoid fever is detected by an agglutin- What is needed for the diagnosis of trypanosomi- ation assay, the Gruber-Widal test, which depends on asis, therefore, is a simple technique that will give flagellar and somatic antigens on the Salmonella results rapidly and will permit the recognition of organisms. Brucellosis in infected cattle is diagnosed trypanosomiasis suspects on the spot. by a tube-agglutination ring test; milk from an The direct agglutination test for Trypanosoma infected animal is mixed with stained Brucella brucei gambiense (or Card Agglutination Test for organisms and if antibodies are present in the milk, Trypanosomiasis (CATT)) is a remarkable improve- the organisms agglutinate and form a ring of clumps ment and has been adopted by WHO as the new floating on the fat layer at the surface of the milk. All screening system. The antigen used is a carefully these tests are used in laboratories but not in the field selected trypanosome clone with a variant antigen despite their simplicity. type which is seen frequently in many strains of Slide-agglutination tests are used for the identifi- T.b.gambiense in West and Central Africa. It may cation of many microorganisms, such as Neisseria also cross-react With other trypanosomes. The organ- meningitis serogroups A, B, C and D, and are of great isms are highly concentrated, stained blue, and lyo- use in epidemiological studies. Typing of the Sal- philized. The procedure is performed simply by mix- monella serotypes is also routinely performed using ing a drop of whole blood (fingerprick) with the re- slide agglutination with a battery of specific anti- suspended trypanosomes, shaking it for 3 minutes sera. (preferably in a standard fashion by using a mech- The bacterial agglutination tests are extremely anical device), and then reading it. For users who only simple, inexpensive to perform, and useful for the rarely apply the test (dispensaries, for instance), the identification of many microorganisms. For these use of plasma or serum is recommended since it makes reasons they continue to be used today and serve as reading easier. From preliminary results of a series of model systems for the development of other tests several hundred tests in various foci of T. b. gam- where simple assays are absolutely required. Indeed, biense, it seems that the number of false-negative and if other diagnostic tests could be as simple as these ag- false-positive reactions is below 20o. The cost of the glutination reactions, a great need would be satisfied. test will be approximately US$ 0.11 per test when A specific example of the need for simplifying diag- large-scale application permits regular production. nostic tests is the surveillance for African trypano- somiasis. In the endemic areas, such tests have to be The use ofartificial particles applied regularly on as large a proportion of the popu- Artificial particles coated with immunoglobulins lation at risk as possible. This implies that most of the have been used for many years in agglutination reac- tests will be used by mobile teams or by medical per- tions to detect and measure antiglobulins. In one sonnel of the small rural health centres. In the latter, sense, the sheep red blood , coated with rabbit there is usually no laboratory know-how nor strong antibody, can be considered a readily available "arti- motivation for laboratory work. The current practice ficial" particle since its underlying biological prop- IMMUNODIAGNOSIS SIMPLIFIED 219 erties are not central to the intended use in this system, macroscopic (approximately 8 mm diameter) poly- which is the detection of rheumatoid factors. The sub- styrene beads that are internally impervious to reac- stitution of human Cohn-fraction II for the rabbit tants. immunoglobulin, and polystyrene latex particles of The DASS system is only mentioned in this dis- microscopic dimensions for the red cell somewhat cussion of "simple" assays for historical reasons improved "standardization" of such tests but hardly because it necessitates the non-automated use of a changed the fundamental nature of these immuno- prohibitively expensive (approximately $40 000) chemically sensitive tests for rheumatoid factor. microfluorometer. This is used to make time-con- Many obvious extensions of such agglutination suming measurements on many individually viewed tests have been described wherein artificial particles microscopic beads of variable brightness that are slow have been coated with microbial antigens of interest to equilibrate and wash because the immunochem- in order to assay specific antibodies, or with specific istry occurs within the pores rather than at the surface antibodies to assay for microbial antigens. Although of relatively large beads. such assays certainly can be described as simple to use With the solid (hard) macroscopic bead (approx- since little other than the reagents are required to per- imately 8 mm), a single bead per test specimen is form rapid tests, at the molecular level quite compli- usually used; it can be separated easily from the incub- cated reactions may be represented, with assay results ation mixture by decantation and washed by pipetting importantly influenced by the presence of rheumatoid the buffer over its surface. However, a very much factors, immune complexes, C I q, staphylococcal larger total surface is available per unit test volume protein A, the valency of antibodies, and physico- and the average diffusion distance for soluble antigen chemical properties of the buffers and specimens. molecules to a bead surface is greatly diminished if Also, an accurate determination of end-point titres many microscopic beads (instead of one macroscopic may be difficult in some instances, leading to reduced bead) are used per test. Typically, immunofluoro- clinical sensitivity and specificity and consequently a metric assays may employ 1.5 x 108 polystyrene beads poor predictive value of positive test results. The (1.1 tm diameter) per 0.1 ml test specimen. Separ- diagnostic consequences of false-positive and false- ation of bound from free antigen is by low-speed negative assay results must always be considered centrifugation, in a centrifuge that can be used for when using such "simple" tests. many routine purposes. Magnetic separation systems Apart from their use in agglutination assays, arti- also are available that use magnetic artificial particles ficial particles have been extensively used to provide to separate bound from free reagents. one form of a solid phase reactant to facilitate the separation of immunochemical bound antibody (or Direct visual detection of antigen-antibody reactions antigen) from "free" antigen (or antibody) in ligand In the methods mentioned below involving identifi- binding assays of soluble analytes (either antigens or cation and quantitation of antigens or antibodies, the antibodies). test materials used are serum, plasma, whole blood, The surface of the artificial particle is analogous to and other biological fluids sampled by, for example, the surface of the plastic microtitre plate in ELISA venepuncture or capillary tubes with anticoagulant or assays. As with microtitre plates, the binding of a finger-tip blood collected and dried on filter-paper reagent to the "clean" surface of an artificial particle discs. may be passively obtained through unknown "ab- Several methodological variants have been devel- sorptive forces", or more actively pursued through oped. They have, however, the following principal knowledge of some specific chemistry; in either case, steps in common, (a) primary coating of solid flat a desired or undesired selective macromolecular surface, (b) application of sample, (c) visualization orientation may result. Obviously, different indi- of antigen-antibody reaction. cators (radioisotopes, fluorochromes, enzyme-sub- strates) can be used to follow the bound virus-free (a) Various materials giving a solid flat surface reactants in artificial particle systems. have been tried for their suitability. At an early stage, Various kinds of artificial particles have been used untreated glass was found unsatisfactory unless pre- for ligand assays of soluble antigens, e.g., bentonite coated with a thin layer of metal, e.g., indium, gold- surfaces, the surface of microscopic fibres of re- indium, or tantalum. Since then, the general trend generated cellulose, the relatively large internal pore has been to turn to a surface of polymer material, surface of microscopic (approximately 30 Am di- e.g., polystyrene and polyvinyl or to employ metal- ameter) agar-gel beads (or Defined Antigen Substrate based silicone oxide or dioxide. These have not so Spheres (DASS)), the outer surface of somewhat far been applied extensively in clinical situations. smaller (approximately 10 Am diameter), small pore The primary coating of the surface by the relevant polyacrylamide-gel beads, and the outer surface of serological reagent (antigen or antibody) is achieved microscopic (approximately lym diameter) and by either spontaneous adsorption (hydrophobicity) or 220 MEMORANDUM

by physicochemical binding (covalent). A method- colour observation, e.g., for indium plates, grades of ological requirement is the establishment of a thin darkness of the colour brown, which result from ''mono' molecular layer of the reagent adequately effects obtained with thin films. In the metal-based attached to the surface, with low serological reactivity silicone dioxide system, detection is dependent on a and with a sufficient density of reacting sites. The series of colour changes. However, these are some- material used for coating is either a solution of anti- what difficult to differentiate. gen (preferably purified) or a solution of antibody, There are quite a few reports published about the preferably an affinity-purified Ig-preparation from use of these methods for assaying specific antibodies hyperimmune serum. For special purposes specific in serum or blood samples in relation to various infec- ligands may be used for the primary coating. tious and non-infectious diseases. Less experience has (b) There are in principle two ways of placing a test been gained, so far, about the suitability of these sample on the primary coated surface. One is to do it methods for detection of antigens. As the assaying dropwise; a single drop for a qualitative assay or a techniques mentioned are quite simple, versatile, and series of drops representing a serial dilution for inexpensive, and as no particular instrumentation is quantitation (end-point titration). The other way of needed, they should be suitable for serodiagnostic use placing the sample is to use an agar layer established in, for example, field investigations in developing on top of the coated surface. The sample is placed into countries at the level of local community health punched holes in the agar and dilution of the reagent centres. Further trials of their application in this way occurs by the concentration-gradient principle. should be promoted if a constant supply of the suit- (c) Visualization of an area where binding of sero- able slides (e.g., indium) could be arranged. logically corresponding reagents (antigen and anti- body) has taken place can be achieved by various Enzyme-immunoassays techniques: Enzyme-immunoassays (EIA), e.g., enzyme-linked - In the water vapour condensation technique, the immunosorbent assays (ELISA), are techniques rou- cool coated surface is exposed to an increased humid- tinely used for the detection of minute amounts of ity, by vapour from heated water or, as a simpler constituents that are present in biological fluids or alternative, the investigator's breath. In an area of cells. They are based on antigen-antibody reactions in antigen-antibody binding an increased wettability is which one of the constituents has been linked to an visualized as drops of greater size than those of the enzyme. The enzymes are not directly detectable but background layer area with unaltered primary coat- can be detected indirectly after reaction with appro- ing. Thus, this type of differentiation is based on a priate substrates. This step offers the advantage of simple physical phenomenon which is easy to create making it possible to visualize the enzyme activity and to record. through a coloured product. - Another and somewhat more sensitive mode of Enzyme-immunoassays have been devised either visualization can be obtained by applying to the for quantitative tests, using procedures similar to surface a suspension of small polymer beads, either those described for radio-immunoassays, and for the uncoated (nonspecific adsorption) or coated with a detection and localization of material in tissues or suitable serological reagent or combination of re- cells, as with fluorescent reagents. agents, e.g., according to the mixed haemadsorption Two types of assays have been devised. (1) The principle. homogeneous assays, which make use of antigen - For the identification of bound antibodies and coupled to enzyme, are based on antibody-mediated their Ig isotype differentiation, a reinforcement changes of enzyme activity and do not require the visualization technique can be applied. Prior to the separation of the antigen-antibody complexes. How- visualization an additional step is introduced where ever, the present state of development of these assays the surface is exposed to anti-Ig or class-specific anti- limits their use to substances of low relative molecular Ig reagents. mass, like drugs and hormones. (2) In the heter- - Still another mode of visualization of areas ogeneous assays, the antigen-antibody complexes where an antigen-antibody reaction has taken place have to be separated from free antigen and/or anti- on the solid surface is the use of a sero-enzymatic in- body, and thus solid-phase bound antigen or anti- dicator system, e.g., horse-radish peroxidase linked body is necessary. These assays allow the quantitation anti-Ig or isotype-specific anti-Ig. An area of antigen- of both antigen and antibody. antibody binding induced by a test sample is regis- For the quantitation of antigens, competitive or tered as an easily recognized change of colour. non-competitive procedures are available. Satisfac- - When metal or metal-based silicone surfaces are tory results are obtained by the non-competitive employed in thin layer analyses, the identification of "sandwich" procedure, using an immobilized anti- areas with bound antigen-antibody is also based on body to trap the antigen from the biological fluid and IMMUNODIAGNOSIS SIMPLIFIED 221

the same or another enzyme-labelled antibody for ogenic substrates exist which require the use of special measurement. ultraviolet light-emitting equipment. The substrates The quantitation of antibodies is performed using used for the detection of peroxidase activity are the immobilized antigen. The amount of antibody bound most rapid, but they can be easily oxidized by any to the antigen is then evaluated with an enzyme- traces of oxidizing agents (other than the enzyme and labelled anti-immunoglobulin. The values obtained hydrogen peroxidase) in the vessels and they are light- are referred to a standard curve for quantifying. sensitive. Beta-galactosidase and alkaline phos- Thus the sensitivity and reproducibility of the vari- phatase substrates are relatively stable. ous enzyme-immunoassays depend mainly on four The solid-phase is the major problem. The im- "more or less controllable" parameters: enzymes, mobilization of the antigen or antibody can be made substrates, conjugates, and the solid-phase used for either by covalent binding or by adsorption through immobilization. non-covalent interactions. Supports can be agarose, Enzymes and conjugation procedures have been cellulose polyacrylamide, or polyacrylamide-agarose well investigated. Mainly, four enzymes of high turn- particles. They are separated from the liquid phase over activity are currently used: horse-radish per- by centrifugation. More convenient are particles oxidase, alkaline phosphatase from Escherichia coli rendered magnetic, which can be easily and quickly or calf intestine, and beta-galactosidase from E. coli. isolated with a magnet. The most widely used sup- Glucose-oxidase from Aspergillus niger is less ports are polystyrene plates with 96 wells for which commonly used. automatic readers are commercially available. Poly- Conjugates prepared with antibodies directed styrene balls have also been used. All these supports against the immunoglobulins of many species (and have their advantages and disadvantages: particles their isotypes) are commercially available. The give good reproducibility but necessitate multiple quality of antibody conjugates depends mainly on the handlings, magnetic polyacrylamide beads are useful avidity or affinity of the antibody. They have to be and can be re-used after regeneration, but the lack of purified by affinity chromatography or immuno- homogeneity in the bead size may produce irregular adsorbents in order to obtain conjugates which give results. the best results in terms of positive signal over In conclusion, evaluation of antigen or antibody background. However, immunoglobulins prepared in biological fluids can be performed by various by salt fractionation can in some cases be useful, immunoassays. The choice of the test must be dic- especially when monoclonal antibodies obtained tated by individual considerations. from ascites are used. Antigen-enzyme conjugates In the current state of development of these tech- have been less commercialized, but some already exist niques, the following simplifications are already prac- in kits. tised in some laboratories: Conjugates prepared under the same conditions with all these different enzymes give the same range of (i) Simplification in terms of "reading the test". In detection, but the time for the enzyme activity reac- many instances there is no need for a sophisticated tion varies. instrument for detecting the reactions. A naked-eye appreciation can be roughly estimated by reference to There exist some alternatives to the use of enzyme- coloured standards. These standards have, however, antibody conjugates which in some cases increase the to be prepared for the test since the reproducibility of sensitivity of the test. These include: the enzyme assays depends on the time and tempera- ture at each step. - use of peroxidase-antiperoxidase (PAP) com- plexes; (ii) Simplification in terms ofrapidity. The differ- - use of synthetic hybrid antibodies with double ent times of incubation (immune-complex formation specificity directed against immunoglobulins and and enzyme activity) can be shortened without dram- either enzyme or another marker such as red blood atically decreasing sensitivity by slightly increasing cells; the concentration of the enzyme-conjugate. - use of enzyme protein A conjugate; (iii) Simplification concerning the solid-phase. The - use of biotinylated antibody or antigen and most convenient are the 96-well polystyrene micro- enzyme-labelled avidin. plates. They offer the following advantages: Various substrates for each enzyme are available: - they can be previously coated with almost all ,chromogenic substrates, the product of which can antigens or antibodies at low concentrations; be either soluble (useful for spectrophotometric - they do not require large amounts of material measurement) or insoluble (giving a coloured pre- (50-200 Id); cipitate allowing the precise localization of the site of - they are easily washable (by immersion for the enzyme reaction). For a more sensitive test, fluor- example); 222 MEMORANDUM

- they are easily used with commercially-available the tests, even if carried out in central laboratories, multiple pipettes adapted to this kind of plate. may have a great impact on primary health care. But there are some disadvantages: Immunofluorescence. Indirect immunofluores- cence is one of the best methods available today for - reproducibility varies between different lots; the rapid diagnosis of many organisms. The reagents - they are expensive; are often made commercially and are quality con- - small amounts of antigen must be coated to avoid release during further incubations (this could be trolled. Good fluorescence microscopes are absol- utely essential for reading the tests and their main- a limitation when the antigen is only part of the mix- tenance is a necessary condition for their use. The ture); training of experts is also needed. A training course of - they require a large volume of washing medium (PBS-Tween), which may be considered quite compli- 2-3 weeks may not be sufficient for beginners, but further training may be organized by making avail- cated for field use. able positive and negative control specimens (fixed on The simplifications already described should be slides) for staining and reading in local laboratories. adapted because they have so far not been well studied High quality reagents are also absolutely mandatory in routine test procedures. for reliable immunofluorescence tests. (iv) Use ofpaper sheets. Nitrocellulose sheets have It is thought that, in the future, many immuno- been found suitable for spotting antigens (various fluorescence assays may be replaced by enzyme- proteins, liposaccharides, or DNA), which are after- immunoassays. wards detected by antibody-enzyme conjugates. Fluoroimmunoassays. It is possible that in a These could be used either as antigen carriers for anti- few years fluoroimmunoassays will replace radio- body diagnosis or as material carrier (for the collec- immunoassays and enzyme-immunoassays in well- tion of blood or gland extract, etc). equipped laboratories, though this will mean new The advantages are: investment in equipment. The following types of fluoroimmunoassays (FIA) are available: - they can be stored dry or wet in small, capped tubes; (1) Solid-phase FIA. This is similar to the two-site, - they can be washed easily with a small volume of "sandwich", (IRMA) in washing medium; that the antibody carries the label, in this case a - they can be the carrier of many different anti- fluorochrome. For viral antigen detection, "capture" gens and can be incubated in one step with the antibodies are bound on solid phase, usually a plastic patient's serum; material, which is efficient for binding the antibody. - they can be used with precipitating substrates High backgrounds, including that from the plastic and stored. may result in unacceptably low sensitivities for anti- gen detection. However, the sensitivity may be high The disadvantages are: enough for the assay of viral antibodies. - they are very expensive at present; (2) Fluorescence-polarization method. In this - they are difficult to handle as a sheet and should assay, the labelled antigen is excited with polarized be adapted to something solid (a stick, for in- light and the degree of polarization of the fluorescent stance); emission is measured. If antibodies bind to the anti- - no systematic work has been done concerning gen, brownian rotation diminishes and the degree of the stability of the spotted material. polarization increases. The assav is simple and rapid, New developments of tests based on competition to requiring no separation steps, but unfortunately it has eliminate one step of incubation and to shorten the some major disadvantages. Sensitivity is limited by assay should be encouraged. In addition, the develop- background problems, the polarization response is ment of heat-stable antibodies and enzymes would be non-linear, and the relative molecular mass of the an advantage. detectable antigen cannot be larger than about 20 000 (e.g., whole viruses cannot be detected by this Immunofluorescence andfluoroimmunoassays method). None of the tests are useful in the field and most (3) Fluorescence-quenching methods. In direct likely will not be in the near future. Immunofluor- fluorescence quenching, a fixed amount of antigen escence can be used in the local diagnostic labora- that is labelled with a fluorescent probe (e.g., fluor- tories. Fluoroimmunoassays require the facilities of a escein isothiocyanate) is mixed with an antibody that central laboratory, including maintenance. However, is labelled with a fluorescent quencher (e.g., rhod- IMMUNODIAGNOSIS SIMPLIFIED 223 amine) which adsorbs the fluorescent emission from delay of 400 As, the single-photon emission is counted a proximal probe. If unlabelled antigen (from the for 500 As at 613 nm. After another delay of 100 As, specimen) is present, it binds the quencher-labelled the cycle is repeated for about 1000 times during the antibody to prevent quenching. A modification is that total counting time of 1 second. The results are ex- half of the antibody molecules are labelled with a pressed as counts per second (cps) values. In each fluorescent probe and the other half with a quencher. assay, the background fluorescence of the counting When unlabelled antigen is present, the two types of solution is measured in triplicate and the mean value antibodies are brought together causing quenching of may be deducted from the cps values in the assay. In a fluorescence. Another type of quenching makes use further developed, direct, one-incubation TR-FIA, of fixed amounts of: (i) antigen labelled with a fluor- the specimen is added simultaneously with the escent probe, (ii) antibody to the fluorescent probe labelled viral antibody to polystyrene tubes or micro- which quenches its fluorescence, and (iii) antibody to titre strips coated with the same antibody as in the epitopes on the antigen that are close enough to the label. After a one-hour incubation and washing, the fluorescent probe to sterically block the action of the counting solution is added and the fluorescence is antibody to the fluorescent probe. Excess antigen measured. The sensitivity of the direct TR-FIA with added to the system removes the blocking antibodies one incubation is the same as the sensitivity of the in- resulting in quenching of fluorescence. direct TR-FIA or the indirect radio-immunoassay (4) Fluorescence-enhancement methods. In excep- with three incubations and washings. tional cases, an increase in the fluorescence of labelled The TR-FIA is a good candidate for replacing antigens has been found after binding to antibody but radio- and enzyme-immunoassays. The disadvantage there are no reports indicating that this method is of the technique, especially in developing countries, is applicable to microbial antigens. that it requires a single-photon counting photometer and even simple non-automated equipment without (5) Time-resolved fluoroimmunoassays. A new printer may be more expensive than spectrophoto- type of FIA, called time-resolved fluoroimmunoassay meters used for EIA reading. (TR-FIA), has recently been developed and applied to In all the technologies discussed so far, it is clear viral antigen and antibody assays. The TR-FIA is that the sensitivities and specificities of immuno- based on a fluorescence probe with a long life-time. assays, whether applied for antigen or antibody It is excited by a short light pulse and the specific assays, are not dictated by the probes used (isotope, fluorescence is measured after a selected time delay. enzyme, fluorochrome) but by the quality of the During the delay the background fluorescence, which immunoreagents used. The main difference in these has a short decay time, is eliminated and the specific assays is that radio-immunoassays and fluoro- fluorescence is measured with similar or higher sensi- immunoassays, where the labelled antigen or anti- tivity than obtained by radio-immunoassays or body is measured directly after the immunological enzyme-immunoassays. reactions have completed, can be standardized more Rare earth metals, lanthanides, e.g., europium accurately. (Eu), usually have a long decay time, 100 to 1000 As. An additional advantage of lanthanide chelates is that DNA /RNA probes there is a large difference between the excitation and The techniques on how to use the analysis of DNA emission wavelengths (Stokes' shift), which causes and/or RNA probes of viruses or microorganisms for further reduction in background and improves the diagnostic purposes have not yet been worked out to possibility of detecting specific fluorescence. Binding such an extent that they can be applied in field labora- of Eu to antibody or antigen can be done via EDTA tories. However, there exist relatively simple and which binds efficiently to lanthanides, and an amino- rapid methods to identify or characterize virus strains phenyl derivative of EDTA has been synthesized by analysing their genomes. In the following, various which can be linked to proteins by the standard techniques will be mentioned which are applicable to coupling reactions used in protein chemistry. different virus systems or to microorganisms and Solid-phase assays have been used in TR-FIA for which are summarized in Table 1. the measurement of viral antigens and antibodies. After the immunoreactions have been completed, the (1) RNA-viruses a solution solid phase is washed, counting (2-naph- (a) Migration rate analysis by polyacrylamide gel toyltrifluoroacetate in aqueous detergent solution) is electrophoresis (PAGE) added, and the fluorescence is measured in a single- photon counting fluorometer equipped with a xenon Viruses with a segmented genome exhibit a specific flash lamp. The excitation wavelength is 340 nm and pattern of their RNA segments after PAGE. The the length of the excitation pulse is lts. After a time RNA can be labelled either in vivo or in vitro prior to 224 MEMORANDUM

Table 1. Molecular techniques for characterization of virus genomes and their applications

Virus Main fields Special Technical genomes Method of application requirements complexity Usesa

RNA (i) migration rate influenza virus, radioisotopes relatively D, E, R analysis in poly- rotavirus not simple acrylamide gels essential (ii) T,-oligonucleotide influenza virus, isotopes moderately D, E, R mapping poliovirus, complex flavivirus (iii) molecular influenza virus isotopes moderately D, E, R hybridization complex DNA (i) endonuclease herpes virus, radioisotopes, relatively D, E, R restriction cytomegalovirus, specialized simple mapping, varicella virus, enzymes polyacrylamide papilloma virus, gel migration poxvirus, analysis plasmids (ii) molecular herpes virus, radioisotopes, complex E, R hybridization hepatitis B, specialized onc-genes of enzymes RNA tumour viruses

a D = diagnostic use; E = use in epidemiological studies; R = use in research work.

PAGE (influenza viruses), or can be detected directly The isolated DNA of these viruses are treated by by staining the gel with ethidium bromide (rota- specific bacterial restriction endonucleases, and the viruses). resulting fragments are separated by PAGE and (b) Oligonucleotide fingerprinting of viral RNA visualized either by staining with ethidium bromide or by autoradiography. DNA cleavage patterns have Any viral RNA can be characterized by this tech- allowed the identification of different papilloma nique. The RNA is digested by Tl-RNase, which viruses, the typing of herpes simplex viruses (HSV) cleaves the RNA after G, and the oligonucleotides are and the identification of genetic variants of HSV separated by two-dimensional PAGE. The larger types 1 and 2 and cytomegalovirus, and the character- oligonucleotides are unique for each RNA and are ization of plasmids. used for characterization and determination of gen- etic relatedness. (b) DNA hybridization procedure (c) Molecular hybridization DNA is fragmented by specific restriction endo- nucleases, and the fragments are separated by PAGE If virus RNA (vRNA) is hybridized to its hom- and transferred by "blotting" onto nitrocellulose ologous complementary RNA (cRNA), forming a membranes, to which they are firmly bound. The double-stranded RNA, it is resistant against treat- immobilized DNA is identified by hybridization with ment with RNase A. In heterologous hybrids (vRNA radio-labelled virus-specific DNA or RNA probes of one strain, cRNA of a genetically-related strain), and visualized by autoradiography. For "dot hybrid- the regions of mismatching (indicating genetic di- ization" the DNA is directly immobilized on nitro- versity) will be digested. Thus, the RNase-protection cellulose filters and is detected using radio-labelled is a relative measure of genetic relatedness. Various DNA or RNA probes, while in the sandwich hybridiz- combinations of this technique are available: ation method, one of the reagents is on the filter. (i) direct RNA-RNA hybridization; (ii) compet- These procedures have been used, for example, to itive hybridization (iii) RNA-DNA hybridization; detect virus-specific sequences integrated into cellular (iv) PAGE analysis of hybrids after treatment with SI genomes. DNA can be hybridized also in situ, which nuclease. allows the detection and localization of virus-specific sequences in tissue sections or cells. (2) DNA viruses Future trends, applying DNA and/or RNA probes (a) Restriction endonuclease mapping of viral in diagnosis, are to prepare probes for hybridization, DNAs which are not labelled by radioisotopes but by other IMMUNODIAGNOSIS SIMPLIFIED 225 markers, e.g., enzymes. Furthermore, the availability markedly in their tolerance to heat, desiccation, and of individual genes, specific for the material under stability in solution, unlike antisera which can often investigation, might help in the application of withstand harsh treatment. methods that have been worked out for viruses to more complex organisms such as parasites. Selection of test systems Diagnostic assays are necessarily of three kinds: MONOCLONAL ANTIBODIES: those used in the central laboratory, those used in THEIR USE IN IMMUNODIAGNOSIS local laboratories, and those used in the field. In this order, these are expected to have a decreasing com- plexity and decreasing dependence on expensive Monoclonal antibodies have already proved their equipment. Because a good diagnostic assay should value in biology as highly specific probes for antigens have high specificity and high sensitivity, these should in complex mixtures. Their application to immuno- be a minimum prerequisite; the degree of simplicity diagnosis is thus twofold: (i) as probes for identifi- and the cost are next in importance. Fortunately, cation, purification, and characterization of relevant many immunological tests using monoclonal anti- antigens, and (ii) as reagents in diagnostic assays bodies can be made relatively simple and inexpensive themselves. Monoclonal antibodies have an excep- and thus will have all the attributes of a desirable tional degree of specificity and promising sensitivity, assay. and they could make a great impact on immuno- Many monoclonal antibodies have already been logical diagnosis; ways to adapt them to the simplest shown to have immunodiagnostic potential. Some possible diagnostic tests must be considered as a have been extensively tested using actual clinical priority. samples (although not in the field), and some use in- One of the most positive features of monoclonal novative assays which can probably be modified for antibodies is also responsible for great difficulties in use in simple field tests for large numbers of samples. their use: that is, their individuality. Each mono- clonal antibody is unique, not only in antigen-binding (a) Direct binding assays.a Suitable monoclonal specificity but also in its biochemistry. It is therefore reagents can be used to detect antigens that are bound important that the selection of monoclonal antibodies to a solid phase (latex, microplate wells, nitrocellulose should be performed at several levels if ultimately paper). In most assays, the monoclonal antibody they are to be used as a diagnostic reagent: first, selec- must be labelled with 1251 or with appropriate tion has to be for the desired specificity; second, for enzymes for ELISA procedures. Selection of the their ability to function in a particular assay; and monoclonal reagents will thus depend on high affinity third, for their stability, ease of purification, and for the antigen, and for their ability to cause direct ag- ability to be manipulated in biochemical reactions glutination, for example, in assays using direct ag- such as those encountered during coupling to glutination of latex particles to which the antigen is enzymes. adsorbed. Direct binding of specific monoclonal antibodies Selection of monoclonal reagents will also be useful in "sandwich" assays in which the monoclonal reagents are first used to bind the The majority of monoclonal antibodies utilized antigens from a mixture and then to detect the bound so far have been selected primarily on the basis of antigen, as previously mentioned. In some situations, specificity, often after primary screening only on the antibodies specific for two different epitopes will be immunizing antigen. The search for diagnostically required. This type of assay is likely to prove of great useful monoclonal antibodies should be approached value when the antigen to be measured is present in from two directions: (i) by selecting from the battery small quantities in crude mixtures. of reagents produced by fusions, and (ii) by specifi- cally selecting reagents using the exact assay in which (b) Inhibition assays. Both antigen and antibody they will ultimately be employed, including testing on can be detected in assays based on the inhibition by samples in the situation of intended use. In addition, test samples of binding of labelled monoclonal in either approach, the use of selected detection reagents to antigen bound to solid phase. Here it is reagents can aid in obtaining antibodies that are likely important to select monoclonal antibodies of the to have the functional properties desired for the "correct" affinity for antigen because it is possible ultimate assay and that are easy to purify and handle biochemically. a Direct binding assays in this report refer only to those assays in Another level of selection which is important for which antigen is detected by an unlabelled antibody (in the case of thin-layer assays), or labelled antibody with no need for a second the development of assays is concerned with the antibody directed to the first, as is often the case with "indirect" stability of the reagents, which are known to vary assays. 226 MEMORANDUM that antibodies of high affinity will compete too bodies, which can interact only in the desired manner successfully with the inhibitor antibody in the test through the two sterically remote epitopes on the anti- sample. If antigen is being detected, this may not be a gen molecule. Following this one-step incubation problem. In inhibition assays, consideration must be period, which is much shorter than with sensitive given to the selection of monoclonal reagents which competitive binding assays (because here the reaction will allow the assay to be performed in as few steps as is driven by antibody excess rather than deficiency), possible, e.g., by including the inhibitor and detecting the immune complex bound to the solid-phase is read antibody and antigen in one reaction mixture. with an appropriate sensor. By making use of two different epitopes, immunochemical specificity can A special type of assay based on anti-idiotype be markedly enhanced, as has been demonstrated monoclonal antibodies can be developed which de- by an immunoradiometric assay (IRMA) employing pends on the inhibition by antigen of the binding of two monoclonal antibodies, specific for the alpha two monoclonal antibodies, one of which is specific and beta chains of human chorionic gonadotrophin for an epitope in or near the antigen binding site of the (HCG). Biologically functional HCG is accurately other. The advantages of this type of assay are that assayed in the presence or absence of other hormones the reagents (both monoclonals) can be obtained in having the same alpha chain, or free alpha and beta extremely large quantities, there is no need for chains. Although many IRMAs employing polyclonal coupling of antigen to a solid phase, and the assay is antisera have been reported since the late 1960s when suitable for detecting antigens present in small IRMA was introduced as an alternative to RIA, amounts in complex mixtures. generally they have had the same order of immuno- chemical sensitivity to RIA. In general, the same anti- Improvement of assays using monoclonal antibodies sera are used for the solid phase and the labelled anti- Recent theoretical considerations and some body in two separate incubations with intervening practical demonstrations suggest that the immuno- wash. Experimental evidence shows that if both anti- chemical sensitivity and specificity, the stability, the bodies have the same epitope specificity, labelled anti- precision, and the rapidity of routine immunoassays body can compete with solid-phase antibody during can simultaneously be improved, possibly by several the second incubation to release, and thereby lose, orders of magnitude, if monoclonal antibodies that antigen from the system. Additional problems with have non-radioisotopic labels displaying higher IRMAs that employ the same polyclonal antibody for equivalent specific activities are used rather than the both incubations are that wasteful amounts of very currently used radioisotopes. It is claimed that con- pure antigen (when available) are needed to make ventional competitive binding radio-immunoassays polyclonal antisera monospecific (by affinity chrom- (which employ a fixed amount of radioisotope- atography), and IRMA uses much more antibody and labelled antigen and saturate binding sites on a limited its specificity requirements are much greater than number of antibody molecules with excess antigen) needed for RIA. In principle, monoclonal antibodies have approached a theoretical limit of immuno- would solve all these problems. However, the partici- chemical sensitivity of approximately 107 antigen pants at the meeting were unaware of any practical molecules per test. In practice, this limiting sensitivity use as yet of these interesting theoretical consider- has not been improved by increasing the specific ac- ations that would simultaneously and markedly im- tivity of the labelled antigen, nor by replacing it with a prove the sensitivity, specificity, stability, precision, signal-amplifying non-radioactive labelled antigen. and rapidity of routine immunoassays of antigens. However, the theoretical limiting sensitivity for non- Until more experimental evidence is forthcoming, a competitive, two-site "sandwich" immunometric judicious balance between caution and enthusiasm is assays is one antigen molecule per test! For this type needed. Although these non-isotope immunometric of assay, ideally, an unlabelled monoclonal antibody assays cannot be considered as "simple", they could of high avidity for one antigen site (epitope) and greatly simplify immunoassays. present in large excess over antigen concentration is Monoclonal antibodies, when properly selected, bound to a solid-phase surface to "immuno-extract" thus have a tremendous potential for use in diagnostic essentially all antigen molecules from the assay assays and may allow development of extremely specimen, whereupon they are detected by a second simple tests suitable for field use. They are of course monoclonal antibody that is labelled and is of high required (rather than antisera) in those many situ- avidity for a second antigen site (epitope), sterically ations where antigens cannot be purified and used to remote from the first site; this labelled monoclonal make antisera. Monoclonal antibodies have a high antibody also is present in large excess over antigen. If degree of specificity and the ability to be standardized these ideal conditions are met, the assay specimen can and made in large quantities, so that effort should be be added to an appropriate mixture containing both spent in their selection and adaptation towards simpli- the solid phase and the labelled monoclonal anti- fication of the tests. IMMUNODIAGNOSIS SIMPLIFIED 227

CONCLUSIONS ment of extremely simple test kits for detection of antibodies as well as antigens suitable for field use.

The Working Group reviewed the technologies * * available for performing several simple immunodiag- nostic tests and discussed possible simplifications P. Halonen, Department of Virology, University of and/or improvements of other technologies. Turku, Turku, Finland Direct agglutination of microorganisms (bacteria, 0. Ouchterlony (Chairman), Department of Bac- parasites) and indirect agglutination of particles teriology, Goteborg University, Goteborg, Sweden T. W. Pearson (Rapporteur), Department of Bio- coated with antigens are the only simple tests widely used for routine diagnosis. Several recently developed chemistry and Microbiology, University of Vic- toria, Victoria, BC, Canada simple tests, based on direct visualization of the C. Reimer, Biological Reagents Section, Center for antigen-antibody reactions either on solid modified Disease Control, Atlanta, GA, USA surfaces or in gels, are still at the research stage of C. Institute for Giessen, development and have not been validated in con- Scholtissek, Virology, trolled trials with the exception of the indium slide Federal Republic of Germany test, which has shown promising results in the diag- T. Ternynck, unit, Depart- nosis of schistosomiasis. ment of Molecular Biology, Pasteur Institute, The technologies using different markers (isotopes, Paris, France fluorochromes, enzymes) represent the main diag- nostic methods at present; however, they can only be WHO Secretariat performed in well-equipped laboratories. Possible V. Houba (Secretary), Immunology, Division of simplification and/or improvements have been dis- Communicable Diseases cussed. The enzyme tests (e.g., ELISA) are the only F. A. Assaad, Division of Communicable Diseases candidates for simple tests to be developed for the B. Mansourian, Office of Research Promotion and field level (naked-eye reading of the results in com- Development parison with coloured standards). Many modifi- V. R. Oviatt, Special Programme on Safety Measures cations discussed could improve these tests in terms of in Microbiology rapidity, sensitivity, specificity and economy, but T. A. Bektimirov, Virus Diseases, Division of Com- they will need complicated equipment. municable Diseases The RNA/DNA probes have been successfully G. Causse, Bacterial and Venereal Infections, applied for diagnostic purposes in several infectious Division of Communicable Diseases diseases and could be developed into field-level tests R. H. Morrow, Parasitic Diseases Programme in the future, especially if the isotopes could be P. de Raadt, Trypanosomiasis and Leishmaniasis, replaced by other markers, e.g., enzymes. Parasitic Diseases Programme Monoclonal antibodies have already proved their L. Martinez, Malaria Action Programme value in diagnostic procedures by increasing the B. 0. L. Duke, Filarial Infections, Parasitic Diseases specificity of the tests. In addition, they can be Programme prepared in large quantities at any time and place S. K. Noordeen, Leprosy, Division of Communi- worldwide by distibuting the clones which is an cable Diseases important factor for standardization. When properly A. Moncayo, Trypanosomiasis and Leishmaniasis, selected, monoclonal antibodies may allow develop- Parasitic Disease Programme