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we have found that chaotropic agents such as 9 M urea, 3.5 M MgCI2, and 0.2 N NaOH were also effective in eluting immunocomplexes from Staph A and reducing nonspecific backgrounds although the final recovery after dilution in Triton X-100-containing buffers is lower than for SDS. However, these agents may be useful for particular proteins, especially those susceptible to unfolding and aggregation following SDS treatment. The virtual elimination of nonspecific background probably reflects a constant proportion of nonspecific adsorption of radioactive proteins during each of the Staph A steps. At the second Staph A immunoadsorption, the starting radioactivity was low enough that the final nonspecific background was effectively eliminated. In agreement with a previous study, 3 the background at each Staph A immunoadsorption ranged between 0.1 and 0.3% of input radioactivity. The second immunoadsorption decreases cellular backgrounds by approximately 100-fold from about 2250 parts per million (ppm) to less than 25 ppm. Recovery of specifically bound polypeptides following the second immunoprecipitation step decreases by approximately 50% without qualitatively altering the electrophoretic pattern of immunoadsorbed proteins. Thus, the trade-off in this procedure is a 50% loss in specific recoverable polypeptides for a significant decrease in overall nonspecific background; the net effect being a 30- to 50-fold increase in signal-to-noise ratio, resulting in a more sensitive analysis of the immunoadsorbed proteins. Acknowledgments The authors thank Dave B. Alexander, Caroline P. Edwards, Nancy G. Forger, Cherie L. Holcomb, Emily J. Platt, and Melanie K. Webster for their helpful comments and Christina Cheng for her preparation and typing of this manuscript.

[53] I m m u n o a s s a y s By CHARLES W.

PARKER

Immunoassays use the binding specificity of an antibody for its specific antigen to measure either the antigen or antibody. ~ To quantitate the reaction either the antigen or the antibody is labeled. In theory, any label permitting sensitive measurements may be used, but frequently the label is C. W. Parker, "Radioimmunoassay of Biologically Active Compounds." Prentice-Hall, Englewood Cliffs, New Jersey, 1976.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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a radioactive isotope, and the term radioimmunoassay then becomes applicable. Alternatively e n z y m e s with high turnover numbers such as horseradish peroxidase, alkaline phosphatase, or fl-galactosidase can be attached to an antibody or antigen and used with fluorogenic or chemiluminogenic substrates for sensitive i m m u n o e n z y m e measurements. Immunoassays have provided a sensitive, reproducible, convenient, and generally applicable approach to the measurement of molecules of biologic interest. In the most sensitive systems measurements of femtomole or even atamole quantities of antigen is possible. The potential value of radioimmunoassays for analytical purposes was first pointed out by Betson and Yalow in their studies with insulin. 2 The radioimmunoassay concept has since been extended to a larger number of other polypeptides and proteins. Immunoassays have been shown to be very useful for discriminating between closely related protein species as well as for determining their absolute concentrations. My colleagues and I extended the radioimmunoassay concept to low-molecular-weight drugs (digitalis, opiates) and metabolites (cyclic nucleotides, prostaglandins) which must be chemically coupled to proteins in order to produce antibodies and pointed out the remarkable degree of sensitivity and specificity that was possible even with small chemical determinants.l'3-1° In most radioimmunoassays it is the antigen that is labeled, and this type of immunoassay design will be used for illustrative purposes. Quantitation depends on the ability o f the unlabeled antigen (Ag) (the unknown) to inhibit binding of the radioactive antigen (Ag*) by antibody (Ab). The process is a simple competition in which Ag occupies a portion of the antibody combining sites, reducing the free Ab available to Ag*: Ag. A b ~ A g + Ab + A g * ~ A g * . A b In performing the assay, fixed concentrations of Ab and Ag* are incubated in the absence and presence o f the unknown samples containing Ag. When high sensitivity is needed the assay is carried out in the presence of only 2 S. A. Berson, and R. S. Yalow, Adv. Biol. Med. Phys. 6, 349 (1958). 3 G. C. Oliver, D. Brasfield, B. M. Parker, and C. W. Parker, J. Lab. Clin. Med. 68, 1002 (1966). 4 G. C. Oliver, B. M. Parker, D. L. Brasfield, and C. W. Parker, J. Clin. Invest. 47, 1035 (1968). 5 G. C. Oliver, B. M. Parker, and C. W. Parker, Am. J. Med. 51, 186 (1971). 6 S. Spector and C. W. Parker, Science 168, 1347(1970). 7 A. L. Steiner, D. M. Kipnis, R. Utiger, and C. W. Parker, Proc. Natl. Acad. Sci. U.S.A. 64, 367 (1969). 8 A. L. Steiner, C. W. Parker, and D. M. Kipnis, J. Biol. Chem. 247, 1106 (1972). 9 B. M. Jaffe, H. R. Behrman, and C. W. Parker, J. Clin. Invest. 52, 398 (1973). 10R. Roberts and C. W. Parker, this series, Vol. 74, p. 198.

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I00

80 z o p-

mE '-lz

60

40

20

0.1

0.2 '

'

' 0.5 ......

1.0

2'o.

'

's"".o

2b

'

' 50 . .

. . . .

100

UNLABELED CK(ng) FIG. 1. Representative standard inhibition curve with unlabeled MB isozyme of human creatine kinase (CK) taken from Roberts and Parker} ° shown on the abscissa, and percentage inhibition on the ordinate.

enough Ab to achieve substantial (40-50%) Ag* binding when no unlabeled Ag is present. Sensitivity may also be increased by preincubating the antibody with Ag before adding Ag*. After the Ag* has been added sufficient time is allowed for adequate Ag* binding and then the free and antibody-bound Ag* are separated, and one or the other is measured by radioactive counting. The concentration of Ag in an unknown sample is determined by finding out where the decrease of Ag* binding it produces falls on the standard Ag inhibition curve obtained by adding graded known quantities of Ag to the assay system (Fig. 1). For accurate quantitation the unlabeled Ag standard needs to be the same as the unknown, but the iodinated antigen and unlabeled antigen need not be identical. Antiserum Conditions for preparing antisera and radioactive markers suitable for immunoassay use are discussed elsewhere in this volume ([49], [50], and [54]), in previous volumes of this series (volumes 70 and 74), and in other sources, but since the quality of these reagents may be quite critical in determining the usefulness of an assay a few comments are necessary here. Obviously each antigen-antibody system has its own special requirements from the point of view of the sensitivity and specificity that are needed. If immunoassay sensitivity or specificity need to be maximized,

[53]

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the immunization of numerous animals under conditions which maximize antibody affinity with screening at multiple points during the immunization process may be needed to find the most suitable antiserum. While many excellent antisera are obtainable commercially, suitable sera for an investigator's individual needs may or may not be available, and the researcher must still validate antibody specificity in any case. Screening under the actual conditions that will eventually be used in the assay (in the presence of serum or tissue) may be helpful in selecting the most appropriate antiserum. Monoclonal antibodies are very useful for developing highly specific immunoassays, but their monospecificity may be a disadvantage from the point of view of immunoassay sensitivity. The sensitivity of an immunoassay depends in part on the spectrum of antigen epitopes that the antiserum recognizes. 1 The stability of antigen-antibody complexes is increased if the antigen as well as the antibody is operationally multivalent, permitting cross-linking and the formation of lattices. A protein-antiprotein reaction is probably best described by an overall avidity constant (Kay) which is affected in turn by the Ka values of the antibody combining sites for their individual epitopes as well as by the ability of the different antibodies to participate together in cooperative binding.l The effect of antigen valence on complex formation probably is the major explanation for the generally greater sensitivity of immunoassays for proteins and polypeptides than for low-molecular-weight (haptenic) antigens. As a rough rule of thumb, the practical sensitivity of an assay is equal to 1/Ka or 1~Kay. Ka values for antibody-hapten interactions usually range between 106 to 1010 liters mo1-1, whereas Kav values for antibody-protein interactions may be as high as 1012 to 1013 liters mole -~. To avoid the loss of multivalency with monoclonal antibodies in proteins which lack repetitive epitopes, mixtures of monoclonal antibodies recognizing different epitopes on the antigen may be very useful. However, if the affinities of the antibodies are not as high as in polyclonal hyperimmune sera, the monoclonal system may still not be optimal. While polyclonal antisera suitable for assay are likely to contain both high- and low-affinity antibodies, at the very dilute serum concentrations used in sensitive immunoassays, only the high-affinity antibodies are likely to be important in antigen binding. In screening antisera, agar gel diffusion and immunoelectrophoresis are of particular value in that they are simple to perform and provide information both on immunological cross-reactivity and on the presence or absence of multiple antigen-antibody systems. However, if a pure radioactive antigen is available, these analyses are just as easily made in the radioimmunoassay itself. Once antisera with intermediate or high titers have been identified, they are evaluated with regard to (1) sensitivity,

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linearity, and reproducibility of standard antigen inhibition curves; (2) susceptibility to nonspecific inhibition in tissue samples and buffer (see below); (3) reactivity with possible cross-reacting antigens. Usually the minimal quantity of antibody giving the desired level of radioactive antigen binding (generally 40 to 50% of the total radioactive antigen added) is used in the assay. Depending on the antibody and the immune system, this may represent an antiserum dilution of anywhere from 1 : 100 to 1 : 1,000,000. The adequacy of antibody binding must be verified in tissue extracts under the conditions in which the assay will be used. Where there is a generous margin of sensitivity in the system being assayed, reproducibility may be increased by using the same amount of tissue sample with more antibody, setting the sensitivity of the system at a lower working level. However, when assay sensitivity is already a problem, more tissue sample must also be used and problems of nonspecific interference or cross-reactivity may not be improved. As a rule, the antiserum is used without purification, particularly when high dilutions of antibody are employed. In certain situations purified y-globulin fractions are used in order to eliminate serum proteins with undesirable enzymatic or nonspecific binding activity. Almost all the binding activity in hyperimmune sera is in the IgG fraction, so conventional purification procedures for IgG, such as ammonium sulfate precipitation or chromatography on DEAE-cellulose or staphylococcal protein A agarose columns, can be used. Purification may also be desirable for monoclonal antibody preparations obtained from ascites tumors to remove interfering activities. Albumin and most enzymes in serum can be largely eliminated by ammonium sulfate precipitation at 1.6 M ammonium sulfate. The use of adsorption procedures to remove cross-reacting antibodies can be helpful in improving specificity. Adsorption with cross-reacting antigen coupled covalently to Sepharose, polyacrylamide, or agarose is almost always preferable to its use in solution since the equivalence point is likely to be missed and soluble antigen and antigen-antibody complexes may remain in the preparation. IgG antibodies can also be degraded to univalent fragments, but this process generally presents no advantages in terms of specificity and may be undesirable if the antigen is multivalent, since the functional avidity of reaction may be reduced. Antisera ordinarily can be stored at - 2 0 ° for at least 3 to 4 years with little or no detectible loss in immunologic reactivity. Storage is preferably done in small volumes so that antisera need not be repeatedly thawed and refrozen. Once antisera have been diluted, some degree of instability should be assumed even in the frozen state, and depending on the system fresh dilutions may need to be prepared frequently.

[53]

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Antigen (or Antibody) Marker A factor which places a practical limit on the sensitivity of an assay is the specific activity of the antigen or antibody marker. The lowest amount of antigen that can be measured is approximately equal to the quantity of marker that is needed for accurate detection. In their most sensitive forms radioactive and enzymatic immunoassays are approximately comparable in sensitivity. Regardless of which type of procedure is used, careful attention needs to be given to the conditions of iodination or conjugation to enzyme so that a maximal sensitivity is achieved without unacceptable losses of immune reactivity ([54] in this volume). Many radioimmunoassays involving radioactive antigens are carried out using about 8000-10,000 cpm of radioactive antigen, but larger or occasionally smaller amounts of radioactivity may be used. Generally, bound rather than free radioactivity is determined because the relative change when inhibitor is present is greater. If 8000 cpm of radioactive antigen is added to the sample and the binding is 40% complete, there will be about 3200 cpm of bound radioactivity in uninhibited samples which will have a coefficient of variation of less than 2% if samples are counted for 1 min. Although lower levels of total and bound radioactivity can be used, the gain in sensitivity is usually not that great, and the need for longer counting times or the greater statistical variation in counting if 1-min counts are used is a decided disadvantage. Thus, the quantity of radioactive antigen in the assay is fixed by the practical level of bound radioactivity that is required to discriminate between samples. In immunoassays with antigen-enzyme conjugates the criteria for how much marker antigen to use are much the same as those for iodinated antigens. The goal is to obtain about 40-50% binding in uninhibited samples with enough enzyme activity to detect decreases in binding down to about 5% or less of the original signal. In contrast to radioactive antigen, when antibodies labeled enzymatically or with radioactivity are used in "noncompetitive" radioimmunoassay systems much larger amounts of enzyme activity or 125Iare used. In this case the quantity of antibody should be capable of binding all of the unknown antigen in the sample. Tissue or Serum Sample All, or any portion, of the tissue or serum sample may be measured. Depending on the antigen-antibody system and the tissue, cell, or body fluid being studied, measurements may be made either without or with extraction and various degrees of partial purification. In any case, at-

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tempts to minimize proteolysis by the use of protease inhibitors and low temperatures are very frequently desirable. Sample sizes that bring the level of immunoinhibitory activity within the middle range of the standard curve are chosen. When the level of antigen in a tissue is variable, it is desirable to divide tissue extracts into several portions and assay small and large aliquots in order to ensure that the inhibitory activity will fall somewhere in the most sensitive region of the standard curve. Measurements at several sample levels have the added advantage of providing information on whether inhibition curves with endogenous tissue antigens parallel standard antigen inhibition curves in buffer. Such parallelism is a necessary, but not sufficient, condition for establishing that the inhibitory activity in tissue samples is truly antigen specific. Cross-reacting antigens may give parallel or nonparallel inhibition curves. Partially purified tissue samples may also be studied as a further means of validating the assay (see below). If sensitivity is a problem, it is sometimes possible to concentrate antigen in an extract by specific or nonspecific adsorption. 11 Samples can be passed through columns of Sepharose-coupled antibody and then eluted with 6 M guanidine. Up to 500-fold increases in immunoassay sensitivity with apparent recoveries of 85 to 95% were estimated by using this approach. This and similar selective concentration procedures provide a powerful approach to the quantitation of substances too dilute to be measured by routine methods. However, a number of important pitfalls should be kept in mind. They include a failure to obtain quantitative adsorption or elution of antigen, or inadvertent concentration of cross-reacting or nonspecific interfering substances as the antigen is being concentrated. Incubation Conditions In choosing the assay conditions considerations such as the stability and physiochemical properties of the antigen, the assay sensitivity that is needed, the anticipated time and cost of the assay, and the experience of the investigator may each be important. Assays are often conducted in a final volume of 0.15 to 0.5 ml. However, Ciabattoni recommends using volumes of 2.0-2.5 ml to permit more precise aliquoting and a greater dilution of reagents in the assay. 12Dilution has the added advantage that it often helps eliminate nonspecific binding effects in the assay. Obviously, the avidity of the antibody for antigen is an important consideration here. tl B. D. Weintraub, Biochem. Biophys. Res. Commun. 39, 83 (1970). ~z G. Ciabattoni, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 181. Springer-Verlag, Berlin, 1987.

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It therefore seems desirable that the incubation volume be optimized for the antigen-antibody system being studied. Commonly used buffers for dilution include phosphate, borate, or Tris-buffered saline. Usually, the choice of the buffer is not important. Nonetheless, a careful examination of the effect of buffer, pH, ionic strength, and divalent cations should always be made in a new immunoassay system in order to maximize sensitivity and anticipate unexpected sources of interference in the assay. Although assays are usually carried out at neutrality, doing so is not always optimal.~'13 Nonspecific adherence of antigens and haptens (especially hydrophobic haptens) to glass and plastic tubes or pipets may markedly influence measured activity in the immunoassay. With some proteins and polypeptides [adrenocorticotropic hormone (ACTH) and parathormone, for example], nonspecific binding is reduced if plastic tubes are used. The addition of protein to the medium minimizes nonspecific adsorption and also helps avoid denaturation of highly diluted antigens and antibodies. Therefore, assays involving iodinated antigens are generally carried out in proteincontaining buffers. Bovine serum albumin, gelatin, lysozyme, and ovalbumin are commonly used, usually at final concentrations of 1 to 5 mg/ml. In some systems diluted whole serum or proteins present in the sample itself are just as satisfactory. However, even though added proteins are often beneficial, they should not be used indiscriminately without making an evaluation for possible adverse effects. For example, contaminating enzymes may degrade the marker. Possible additives, apart from buffer and protein, include enzyme inhibitors and chelating agents. In assays lasting longer than 3 days, a bacteriostatic agent, such as sodium azide, 0.1 to 0.2%, may be incorporated into the medium to help avoid microbial growth. Assay conditions that are used in immunoassays vary widely with total incubation times extending from a few minutes to as long as 6 days and should be optimized for the immune system in question. An initial relatively short incubation (5 to 60 min) at 37° or room temperature helps accelerate immune complex formation; however, since almost all assays are completed in the cold, unless the assay must be completed rapidly, it seems preferable to initiate the assay at low temperatures. Cold temperatures should also be maintained during washing. Highly sensitive assays involving complex antigens usually require 24 hr or longer. Too short an incubation time is undesirable because of possible disequilibrium when antibody-bound and free antigen are separated. Depending on whether an equilibrium or nonequilibrium assay is being used, the radioactive antigen 13 j. A. Fischer, U. Binswanger, and F. M. Dietrich, J. Clin. Invest. 54, 1382 (1974).

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may be added at the same time as the sample or later in the assay. When assay sensitivity needs to be maximized, it is desirable to determine the effect of preincubating nonradioactive antigen with antibody before adding Ag*. The delayed addition of radioactive antigen gives the unlabeled antigen an opportunity to bind to the antibody first. While some assays are not appreciably improved in regard to their sensitivity by the delayed addition of radioactive antigen, others are quite substantially affected. |4 Separation

Systems

At the completion of the incubation, bound and free Ag* generally must be separated. Methods for separation of free and bound Ag* have included the use of a second antibody, salt or organic solvent precipitation, adsorption onto insolubilized staphylococcal protein A, charcoal or another nonspecific adsorbent, electrophoresis, gel filtration, or two-phase systems in which the antigen or the antibody is attached to a solid phase. With certain exceptions, each of the above procedures is applicable to a large number of different antigen-antibody systems. A variety of factors enter into the selection of a system, including the rapidity and sensitivity required in the immunoassay, possible unusual physiochemical properties of the antigen, the affinity, antibody class, or subclass and specificity of available antisera, the skill of the technical personnel, and the usual number of samples to be processed. Often the separation method that is chosen is based as much on the previous experience of the individual investigator as the peculiarities of the particular antigen-antibody system being studied. Nonetheless, an investigator who is setting up an immunoassay for the first time should carefully review any published results in the same antigen-antibody system, both for possible useful technical details and any concrete evidence that one separation system is preferable to another. Double-antibody immunoprecipitation is probably the most broadly applicable of the separation systems. 15'~6Obviously enough second antibody should be used to separate all of the first antibody. Each new batch of second antibody must be verified by titration in the assay. The use of a second antibody is not practical when the first antibody has a low titer because large amounts of both antibodies, particularly the second antibody, will then be needed. If a monoclonal IgG antibody is used in a double-antibody system the effectiveness of the second antibody as an immunoprecipitant for the Ig class or IgG subclass of the monoclonal 14 K. Ichihara, T. Yamamoto, M. Azukizawa, and K. Miyai, Clin. Chim. Acta 98, 87 (1979). ~5 R. D. Utiger, M. L. Parker, and W. H. Daughaday, J. Clin. Invest. 41, 254 (1962). 16 A. R. Midgley, Jr. and M. R. Hepburn, this series, Vol. 70, p. 266.

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antibody must be established and the use of an IgG subclass-specific second antibody may be desirable. Relatively prolonged incubation times are sometimes needed in double-antibody systems to maximize the precipitation reaction. However, the use of accelerators of precipitation such as polyethylene glycol may permit the assay to be completed within 5 to 60 min. 17.18 Single- or double-antibody immunoassays may be performed in solidphase systems. One approach involves antibodies that are noncovalently adsorbed to the walls of a microtiter plate or test tube. Denaturation can be a problem and can decrease the sensitivity of such assays. Nonetheless this type of assay is very frequently used. The nonspecific adsorption step is usually done at pH 9-10. Alternatively, the antibody is attached covalently to disks or beads which are used in suspension. In other variants the antigen is insolubilized instead of the antibody. Some solid-phase immunoassays appear to be particularly sensitive to the exact nature of the complexes formed between antigen and antibody. Although a number of solid-phase immunoassay systems have been shown to give highly satisfactory results, others are less practical due to delayed equilibration times, problems with reproducibility, or high nonspecific binding blanks. Factors affecting the kinetics of antigen-antibody reactions in solid-phase systems have been reviewed. 19 The procedure of Nash et al., which utilizes antibody coupled to commercial cross-linked polyacrylamide beads (Immunobeads, Bio-Rad) using a water-soluble carbodiimide, is rapid and reproducible.2° The assay is performed in microtiter plates in a volume of approximately 100/zl. At the completion of the incubation the beads are transferred to glass fiber filter strips on a microharvester, washed, and counted. This procedure lends itself readily to the rapid processing of many samples. The beads can be aliquoted and stored frozen at - 8 0 ° for extended periods prior to use in the assay.

Solid-Phase Immunoassay of Human IgA with Polyacrylamide-Antibody Beads 1. In 96-well microtiter plates, add the following: 0.01 ml sample or standard 0.05 ml 125I-labeled human IgA (25,000 cpm) in barbital-buffered sai7 A. A. Ansari, L. M. Bahunguna, and H. V. Malling, J. Immunol. Methods 26, 203 (1979). x8 W. H. C. Walker, Clin. Chem. 23, 384 (1977). 19 M. Stenberg and H. Nygren, J. Immunol. Methods 113, 3 (1988). 2o G. S. Nash, M. V. Selden, M. G. Beale, and R. P. MacDermott, J. Immunol. Methods 49, 261 (1982).

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line (BBS) with 10% fetal calf serum (FCS): The BBS is freshly prepared from 5 x BBS [5,5-diethylbarbituric acid (2875 g), sodium 5,5-diethylbarbiturate (1.875 g), and NaC1 (42.5 g), which are dissolved in 250 ml hot water; CaCI2 • 2H20 (0.110g) and MgCI2 • 6H20 (0.508 g) are added and the volume is brought to 1 liter with water] 0.05 ml of a suspension (60-120 /zg/ml) of rabbit anti-human IgA beads (Bio-Rad, Richmond, CA) 2. Incubate overnight at 25° 3. Resuspend beads on a microtiter plate shaker (Bellco, Vineland, N J). 4. Transfer to glass filter strips on a Microharvester under vacuum (Bellco). 5. Wash 10 times with about 0.2 ml (filling the wells) of 5% FCS in BBS. 6. Remove strips with forceps and count. In general, adsorption assays involving charcoal are based on differences in size or charge of free and bound antigen affecting solid-phase binding. They usually work well with relatively small peptides. They provide rapid measurements but are sensitive to the protein content of the medium and, depending on the number of samples processed, may be unusually subject to intraassay variation. Salt precipitation with ammonium sulfate is a reliable, rapid, and inexpensive method in appropriate systems, but is applicable only to radioiodinated antigens that are soluble in 40 to 50% ammonium sulfate. Polyethylene glycol is also useful for separating antigen-antibody complexes. The charcoal, ammonium sulfate, and polyethylene glycol methods are reviewed in an earlier volume of this s e r i e s . 21'22 Once the separation of bound and free antigen has been initiated, nonequilibrium conditions are established and, depending on the conditions of washing and the particular antigen-antibody system, significant amounts of previously complexed antigen may dissociate. This is not necessarily a major disadvantage since relatively low-affinity interactions involving cross-reacting antigens may be particularly subject to reversal. Some investigators have proposed the use of nonequilibrium assays in which the ability of unknown antigen to displace radiolabeled antigen from preformed antigen-antibody complexes is measured. 23 While such nonequilibrium assays are potentially useful because of their simplicity and decreased dependence on sample volume, they also may be subject to 21 W. D. Odell, this series, Vol. 70, p. 274. 22 T. Chard, this series, Vol. 70, p. 280. 23 F. Cocola, A. Orlandini, G. Barbarulli, P. Tarli, and P. Neff, Anal. Biochem. 99, 121 (1979).

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seemingly minor variations in assay time and temperature, and special care may be needed to minimize intraassay variation. Washing solutions usually contain the buffer and nonspecific protein inhibitor used in the original incubation mixture. The use of cold washing solutions is almost always desirable. Regardless of the procedure used, separation of free and bound antigens should be performed as rapidly and reproducibly as possible, and for this reason it may be unwise to process too many samples simultaneously. As already noted, solid-phase assays may facilitate the rapid handling of large numbers of samples.

Representative Immunoassays

Immunoassay Using the Charcoal Method The procedure of Walsh and Wong for secretin will be used for illustration: All pipetting procedures are carried out in an ice bath. Samples, standards, antiserum, and labeled secretin are diluted in 0.1 M sodium acetate, pH 4.5, containing 2% serum bovine albumin, 2500 klU aprotinin (FBA Pharmaceuticals, New York, NY) per milliliter buffer, and 0.02 M EDTA. 24 Standards are prepared first by diluting the standards to contain 1000, 100, and 10 pg/ml. Each of these standards is pipetted in amounts of 200, 100, 50, and 20/A, producing 10 different concentrations with two points of overlap. Standards and serum samples or other unknowns are diluted to contribute a volume of 1 ml to the reaction mixture. For assays of serum specimens, it is desirable to add charcoal-stripped serum to the standard samples to correct for nonspecific interference by serum protein. Aliquots of unknown serum samples (200 and 50/.d) are diluted to 1 ml with the standard buffer to give a final concentration of 1/10 and 1/40 in the reaction mixture. To each assay tube is added 2000 cpm of labeled secretin plus diluted antibody (predetermined to bind 50% of the label) to give a final volume of 2 ml. The nonspecific binding controls contain the diluted label and standard buffer instead of antibody. Tubes are incubated for 24-72 hr at 4 °. Separation of bound and free labeled peptide is performed with dextran-coated charcoal. The separation mixture contains 20 mg activated charcoal (Mallinckrodt, Paris, KY), 20 mg dextran T-70 (Pharmacia, Piscataway, NJ), and 20/xl 5% bovine serum albumin in a final volume of 0.2 ml. Tubes should be kept on ice during the separation procedure. After thorough mixing, the tubes are centrifuged at 3000 rpm (-2000 g) for 10-15 min and the supernatant solutions are removed by 24 j. H. Walsh and H. C. Wong, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 315. Springer-Verlag, Berlin, 1987.

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pouring off into a separate tube. Both the pellet containing the free secretin and the supernatant containing the antibody-bound secretin are counted in a gamma scintillation spectrometer for a minimum of 2 min. Double-Antibody Immunoprecipitation Assay The procedure of Roberts et al. 25 for the rapid determination of creatine kinase (CK) isoenzymes (MM, MB, BB) will be used for illustration. 1. To 0.120-0.165 ml buffer (50 mM Tris-HC1, 20 mM 2-mercaptoethanol, 20 mM EDTA, 2 mg/ml BSA, 0.2% sodium azide final pH 8.5) add 0.01 ml of the first antibody (rabbit anti-MB CK), 1 : 10,000, and 0.005-0.170 ml MB standard or the sample 2. Incubate 15 min at 22° 3. Add 0.02 ml 125I-labeled BB CK (25,000 cpm). 4. Incubate 60 min at 22 °. 5. Add 0.02 ml of the second antibody [excess anti-rabbit IgG (goat)]. 6. Incubate 15 min at 22°. 7. Centrifuge 15 min (3500 g at 4°), remove supernatant by aspiration, and count pellet. Solid-Phase Immunoenzyme Analysis: Enzyme-Linked Immunosorbent Assay (ELISA) The procedure of Katnik et al., 26 in which a polystyrene microtiter plate is coated with a haptoglobin (Hp) and used to screen for monoclonal Hp antibodies in hybridoma cultures, is representative of a large number of solid-phase immunoenzyme procedures. The wells of polystyrene microtiter plates (Plastomed, Poland) are coated with 200/zl of human haptoglobin Hp (200 ng) in 0.1 M carbonate/bicarbonate buffer, pH 9.2, at 37° for 1 hr and at 4° overnight. The plates are washed four times with 250 tzl of casein buffer (154 mM NaCI, 0.5% casein, 10 mM Tris-HC1, 0.02% thimerosal, pH 7.6) and unoccupied binding sites are blocked by casein for 2 hr at 37°. Fifty microliters of supernatant from mouse hybridoma cultures being screened for anti-Hp antibody cultures and 150/xl of casein buffer are added and the plates are incubated at 37° for 3 hr with gentle shaking. After rinsing three times at 5-min intervals with casein buffer, the goat antimouse IgG-horseradish peroxidase conjugate (IgG-HRP) (diluted 2000fold with casein buffer) is added (60 ng/well) and the plates incubated for 3 hr at 37°. Excess conjugate is then thoroughly removed by washing, and z5 R. Roberts, B. E. Sobel, and C. W. Parker, Science 194, 855 (1976). 26 I. Katnik, M. Podgorska, and W. Dobryszycka, J. lmmunol. Methods 102, 279 (1987).

[5 3]

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peroxidase fixed to the wells is detected by the addition of o-phenylenediamine. 26The reaction is followed by increases in absorbancy at 492 nm and comparison with a standard peroxidase reaction. The incubation time for the antigen-antibody reaction can be shortened to 30 min if 2% polyethylene glycol (Mr 6000) is present.

Assay Controls, Variance, and Repeatability Replicate samples, preferably in triplicate or quadruplicate, should always be analyzed, ideally using at least two different dilutions of the unknown sample. Insofar as possible each sample should be treated identically. The assay must be rigidly standardized in terms of total reaction volume, buffer content, quantity of radioactive antigen, and duration and temperature of incubation. Every assay should include a full antigen standard curve. In an immunoassay of any size, binding controls and antigen standards should be interspersed at the beginning, middle, and end of the assay to detect any systematic variations in the assay related to the number of samples involved. As a part of every assay, several calibration or control measurements must be made, including (1) determination of total antigen radioactivity added to the assay, (2) determination of nonspecific marker binding (counts present when samples containing the antigen marker but no antibody are processed), (3) standard antigen inhibition curves in buffer (and sometimes in tissue extracts as well) (4) extraction or reagent blank controls, especially if new extraction procedures or reagents are being utilized, (5) possibly, incubation controls for damage to the immunoreactivity of the radioactive antigen when tissue extracts are present. The standard curve helps identify day-to-day variations in the assay due to deterioration of the radioactive antigen or an incorrect dilution of antibody. Ideally, incubation mixtures containing standard and unknown antigen should be identical in every respect. Sometimes adsorbants such as charcoal or insolubilized antibody can be used to prepare antigendepleted serum or tissue samples. If completely antigen-free tissue samples are not available (and they often are not), the best approach is to carry out standard curves both in buffer and in tissue extracts. However, this approach is not completely satisfactory, since the standard curve in tissue is superimposed on the background of tissue antigen. The standard curve should cover a broad (at least 1000-fold) range of antigen concentrations, extending from minimal to complete inhibition of radioactive antigen binding. While 2-fold dilutions are normally used in the working part of the standard curve, a narrower dilution span may be desirable in the most important parts of the curve, particularly when standard curves are non-

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linear. Since dilutions of standard antigen are generally very marked, they must be made accurately, since there is no way of directly verifying the antigen concentration once the dilutions have been made. The instability of highly diluted standard solutions can also be a problem and, as noted, the inclusion of protein in the medium is usually desirable to minimize denaturation. Problems in interpretation of course arise when the standard antigen itself is impure or was obtained from a heterologous species. Interassay variation can be a major problem. Large-scale collaborative studies in which immunoassay results have been compared in different laboratories indicate that the major source of immunoassay variation is interassay variation, which may be due to a variety of factors such as fluctuations in the quality of the radioiodinated antigen marker or unstable or improperly prepared antigen standards. 27'28 Many different ways have been suggested for treating radioimmunoassay data statistically. Frequently, laboratories use a logit-log transformation of the data to linearize the standard binding curve and permit easy comparisons among assays performed on different d a y s . 28-3° This method of calculation has been shown to be applicable to a large number of antigen-antibody systems. In general, there appears to be no real advantage to the use of more complex mathematical analyses, but in about 5-10% of assays four-parameter logistic models may have to be u s e d . 31 Regardless of what mathematical transformation is used it should be recognized that results are not as precise at the extreme ends of the binding curve. The complete automation of radioimmunoassays has been described (see, e.g., Ref. 32), although the procedures are relatively complex and expensive, and are practical only in laboratories performing large numbers of assays .32 Validation of Immunoassay Regardless of the immunoassay system that is used, the operational specificity and sensitivity of the system must be rigorously evaluated under the conditions in which it will be used. It cannot be assumed that immunoassays that give sensitive and reproducible results in buffer free of 27 W. M., Hunter and I. McKenzie, J. Endocrinol. 79, 49P (1978). 28 C. W. Parker, Annu. Reo. Pharmacol. Toxicol. 21, 113 (1981). 29 D. Rodbard, Clin. Chem. 20, 1255 (1974). 3o D. Rodbard, and J. E. Lewald, Acta Endocrinol. (Copenhagen) 64, 79 (1970). 31 D. Rodbard, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 193. Springer-Verlag, Berlin, 1987. 32 G. Brooker, W. L. Terasaki, and M. G. Price, Science 194, 270 (1976).

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serum or tissue proteins will necessarily give valid results in biological samples, l To a certain extent each new antigen-antibody system and each new tissue is an individual problem. For example, antiserum which specifically measures thromboxane B2 in serum cannot be used in urine without sample fractionation because of the presence of at least 20 thromboxane B2 metabolites. 33 Tissue and blood samples may contain interfering molecules that degrade the unknown antigen or radiolabeled antigen marker, competitively bind the antigen, decrease antigen binding nonspecifically, exhibit expected or unexpected immunologic cross-reactivity, affect counting efficiency (primarily in assays involving/3 particle emitters), or interfere with the separation of bound and free Ag*. Antigenic reactivity may be lost either before or during the immunoassay. Careful attention must be given to the adequacy of recovery of the unknown substance from biologic samples. Samples in which the antigen is extensively degraded as it is processed obviously give misleading resuits. Storage of samples at - 7 0 ° may be critical for adequate preservation of immune reactivity. In some systems insoluble debris in the tissue sample may be detrimental. Special problems may be observed with partially insoluble antigens as may be the case with recombinant proteins produced in organisms such as Escherichia coli. Falsely high or low immunoassay results may be obtained with these preparations. There may be effects on the assay due to alterations in pH or the presence of salts, organic solvents or detergents which affect antigen-antibody binding. These types of interference may be particularly misleading if samples that have not been processed identically are compared. Because effects such as these are especially common in crude tissue extracts and inhibition of radioactive antigen binding is normally interpreted as a high immunoassay value, during purification the amount of a protein may appear to decrease disproportionately to the actual losses that are occurring. Divalent cations, chelating agents, substrates, coenzymes, anticoagulants, protease inhibitors, antibacterial agents, and reducing agents also may affect immune reactivity depending on the antigen and the particular epitope(s) that is being recognized. While these changes may be minimized by an intelligent choice of reaction conditions, careful controls are needed. Nonspecific interference in the assay is particularly prone to occur when unusually small amounts of antigen are being measured. It is usually unwise to push the sensitivity of the system to its extreme limits. Analysis of the immunoassay system is greatly facilitated if the major forms of the antigen, its metabolites, and possible cross-reacting proteins 33 C. Patrono, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 213. Springer-Verlag, Berlin, 1987.

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are already known, if procedures for their chromatographic separation are already established, if the purified proteins are available in reasonable quantities for labeling and as immunoassay standards, and if the amount of antigenic reactivity in tissue samples is sufficient in quantity and stability to permit its chromatographic behavior to be analyzed in some detail under representative assay conditions. Frequently, part of the purpose of the as say is to distinguish the antigen from structurally related proteins which may be immunologically crossreactive. Initially it is often necessary to physically separate the individual species and determine the contribution of each to overall immunologic reactivity. The antigen itself is often heterogeneous. Many hormones and enzymes are secreted in multiple molecular forms and exhibit immunologic cross-reactivity with structurally related proteins present at comparable or higher concentrations. Moreover, the protein of interest is frequently partially degraded by proteolysis and the fragments may or may not react significantly with the antiserum. Under these circumstances antisera recognizing different epitopes may give considerably different results. Procedures of established value in the validation of immunoassay resuits include the following: 1. Parallel studies in another assay system using a different principle of measurement (for example, a functional analysis such as measurement of enzymatic activity or a radioreceptor assay) over the full range of concentrations of interest 2. The use of internal standards (the addition of known amounts of purified unlabeled antigen to tissue samples) to see if the expected increase in measured Ag concentration is demonstrated. Superimposition of dilution curves in a linear plot of the data over at least a 100-fold range of concentrations should be seen 3. The use of enzymes or other proteins that can be expected to selectively alter the reactivity of the antigen in the assay 8 4. Comparison of the slopes of the Ag dose-inhibition curves in the unknown sample and the standard 5. Demonstration that the immunoreactivity in the tissue samples comigrates With the antigen in question through a series of chromatographic purification steps or is removed under conditions in which the antigen is selectively adsorbed; evaluation of the yield of antigenic reactivity after fractionation both in the expected region and elsewhere in the chromatograph; wherever possible, identification of cross-reacting proteins migrating outside the major antigen region and a direct analysis of their absolute reactivities in the immunoassay if the purified proteins are

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available. Chromatographic verification is especially strong if several chromatographic systems are used 6. Use of small amounts of radioactive antigen to monitor its recovery during tissue extraction and purification. Instability of the radioactive antigen usually means that the unknown antigen is also unstable 7. Determination of the stability of the radioactive antigen during the immunoassay 8. Evaluation of samples in which the results of the assay are expected to be markedly positive or negative (for example, in animals in which the organ producing the protein has been surgically removed) or tissues subjected to a known pharmacologic stimulator or inhibitor 9. Exchange of samples between different laboratories making the same measurements 10. The use of antisera which may emphasize different epitopes in the same laboratory 11. Serial analyses of stored samples to determine how much decomposition may normally be occurring prior to the assay 12. Control measurements in the presence of pathologically altered tissue or blood samples Obviously the use of a number of these criteria in combination is stronger than any single criterion alone.

Strategies to Reduce Immunologic Cross-Reactivity Although cross-reactivity presents few if any difficulties in some immunoassays, it often places substantial limitations on the interpretation of the data and may be a major source of error. Thus, it is useful at this point to discuss some general types of antigenic cross-reactivity and consider possible practical solutions. For illustrative purposes the different kinds of immunologic cross-reactivity involving proteins fall into three major categories. One type is represented by two proteins, one with determinants A and B and the second with the identical determinant A and an unrelated determinant C. In this case, an antibody directed solely toward B should detect protein AB in the presence of protein AC with no demonstrable cross-reactivity. The problem, then, is to obtain an effective anti-B antiserum, not contaminated by anti-A antibody. The second type of cross-reactivity is illustrated by two proteins, AB and A'C. Here the only determinants that resemble one another are structurally similar, but not identical. In this case the presence of anti-A' antibodies may or may not be detrimental in measurements of AB, depending on the degree of similarity between A and A', although the use of a monospecific anti-B

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serum may still be preferable. A third type of cross-reactivity is exhibited by proteins AB, A'B, or AB'. In this case, there is no non-cross-reacting determinant and it may be difficult or impossible to obtain a completely specific antiserum, although the inherent cross-reactivity between A and A' or B and B' may be reduced to a minimum by the proper selection of an antiserum. When a cross-reacting antiserum must be used, chromatographic fractionation of the sample or use of a less cross-reactive radioactive antigen marker may eliminate or minimize the problem. Simultaneous immunoassays in two cross-reacting systems occasionally are useful, but have to be interpreted with caution. Acknowledgment The author wouldlike to thank Mrs. CarolynDavinroyfor her expert secretarialassistance.