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Adaptation of Enzyme-Linked Immunosorbent Assay to the Avian Systemt STANLEY S. SLAGHT, TSU-JU THOMAS YANG,* AND LOUIS VAN DER HEIDE Department of Pathobiology, University of Connecticut, Storrs, Connecticut 06268 Received for publication 15 August 1979

A microplate enzyme-linked immunosorbent assay was developed to detect chicken anti-reovirus antibodies. Studies of the parameters which affect the outcome of the assay with avian serum revealed two aspects for a successful assay. First, enzyme-antibody conjugates prepared by the periodate oxidation technique were found to have retained far more immunological activity than conjugates produced by a glutaraldehyde cross-linking. Second, the results indicated an unusually high affinity of chicken immunoglobulin for the microplate plastic which was mostly eliminated by a pretreatment technique with fixed fetal calf serum. The enzyme-linked immunosorbent assay compared favorably with the latex passive agglutination test, yielding a titration endpoint of 1:51,000, or approximately 1,300 times more sensitive than the latex passive agglutination assay. The assay proved not only to be sensitive to less than 1 ng of specific antibody, but also to have low to moderate variance and high reliability.

According to Schuurs and van Weemen (13), the number of publications concerning various enzyme immunoassays has doubled every year since 1971 and, in consideration of the diversity of applications that these techniques offer, their usefulness as a research or clinical tool has been immense. Accordingly, our attention was drawn to these methods as a possible alternative to the radioimmunoassay, especially since our need was for an intermittent assay in the study of avian viruses, tumors, and consequent immunological responses. The method originated by Engvall and Perlmann (3) and adapted for microplate multiple assays by Voller et al. (16) was the logical first choice as a proven technique. This technique has been used successfully by many workers in determining antibody responses to many biological agents, including bacteria (1), mycoplasma (7), viruses (12, 20), and parasites (11, 18). Furthermore, assays have been conducted utilizing sera from many mammalian species such as rabbits (1), calves (19), humans (12), swine (11), mice (7), and rats (10). Unfortunately, in our hands, the use of this methodology, which involves the direct binding of antigen to a plastic surface, has resulted in no useful information when the assay is being conducted with avian antisera. To those of us working with an avian system, this result might not have been unexpected,

since avian antibodies are known to exhibit generally lower affinity constants than the mammalian type (17) and require unusually high salt concentrations (10 times normal) or a low pH (pH 5.5) for the commonplace precipitin assay


Recently, however, we briefly presented one solution to the avian enzyme immunoassay problem which allows for determination of avian antibodies against a reovirus antigen without the need for instrumentation (14). This study evaluates the parameters of success of that avian enzyme-linked immunosorbent assay (ELISA) and determines its reliability and highest sensitivity, using reovirus strain S-1133 as a model.

t Scientific contribution no. 763, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, Conn. 698

MATERIALS AND METHODS Reovirus antigen. Viral antigen preparations of reovirus S-1133 were identical to those previously described, using chicken embryo fibroblast (CEF) cultures and media without the use of avian serum (14). Antisera. The anti-reovirus antiserum (HIS-1133) was the same as that previously described (14), being a hyperimmune serum produced by intramuscular injections of specific-pathogen-free (SPF) chickens (Spafas, Inc., Norwich, Conn.) with tissue culture virus preparations and Freund incomplete adjuvant. Reovirus-negative control sera were obtained from uninoculated SPF chickens as pools of sera from 3-weekold (SPF-3) or 16-week-old (SPF-16) chickens. Enzyme conjugates. Rabbit anti-chicken immunoglobulin conjugated with horseradish peroxidase was prepared by the technique of Nakane and Kawaoi (9). Briefly, 5 ml of horseradish peroxidase (type VI,

VOL. 10, 1979


Sigma Chemical Co., St. Louis, Mo.), dissolved in 1 ml of 0.3 M NaHCO3 (pH 8.1), was reacted at room temperature with 0.1 ml of a 1% (vol/vol) solution (in ethanol) of 1-fluoro-2,4-dinitrobenzene (Eastman Organic Chemicals, Rochester, N.Y.) for 1 h. The carbohydrate moiety of the enzyme was then oxidized for 30 min by the addition of 1 ml of 0.08 M NaIO4. The oxidation reaction was stopped by the addition of 1 ml of 0.16 M ethylene glycol, which was allowed to stand for 1 h before dialysis at 4°C against four 250-ml changes of 0.01 M Na2CO3 (adjusted to pH 9.5 with NaHCO3). The activated enzyme was then incubated with gentle mixing for 3 h at room temperature with purified inmmunoglobulin at a ratio of enzyme to immunoglobulin of 1:1.5. The product was then dialyzed extensively against phosphate-buffered saline and tested for conjugation and enzyme activity after separation on a Sephadex G-200 column (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). The conjugate was stored at -20°C with the addition of 10 ng of bovine serum albumin per ml. Enzyme conjugates of alkaline phosphatase were also prepared with the same rabbit anti-chicken immunoglobulin as described above and with a high-titer (greater than 1:800 by latex passive agglutination) chicken anti-Marek's disease virus globulin. This avian antiserum globulin, rather than the chicken anti-reovirus, was used as a test of the conjugate procedure since the latter exhibits considerably lower reactivity (14; see below). The conjugation method for the alkaline phosphatase labeling was that described by Voller et al. (16). ELISA procedures. Initially, the procedure of Engvall and Perlmann (3) as modified by Voller et al. (16) was followed. However, for reasons indicated below, all data concerning the reovirus system were derived from the method previously described by Slaght et al. (14), except for the replacement of normal serum with an uninfected CEF preparation for use as a negative control (for explanation, see below). The ELISA procedure follows the basic format of Saunders and Clinard (11), but was adapted to increase its sensitivity. Polystyrene microtiter plates (Cooke Engineering Co. [Dynatech Corp.], Alexandria, Va.; catalog no. 1829A) were used as the plastic substrate. The plates were initially coated with 0.05 ml of 2.5% heatinactivated fetal calf serum (FCS) in distilled water and allowed to dry in a dust-free environment. Then 0.05 ml of a 2% glutaraldehyde solution in phosphatebuffered saline, pH 7.4, was added and allowed to react for 30 min, after which the plates were washed four times in running distilled water and shaken dry. The cell culture virus preparation or the CEF control was diluted 1:50 in distilled water, and 0.05 ml of the dilution was added to each well and allowed to dry at room temperature. The plates were washed as described above. Dilutions (1.5-fold) of the antisera were made with 0.5 M NaCl containing 1% Tween 80, pH 7.5 (adjusted with K2HPO4), and 0.05 ml of the dilution was allowed to react in the wells for 1 h at room temperature. The plates were washed rapidly twice in running distilled water, followed by a 1-min incubation in 0.5 M NaCl containing 0.5% Tween 80, pH 7.5. This sequence was repeated two times.


The peroxidase-anti-chicken immunoglobulin conjugate was diluted 1:400 in 0.5 M NaCl containing 1% Tween 80, pH 7.5, and 0.05 ml of the dilution was added to the wells and allowed to incubate for 1 h at room temperature. Washing was done as described for antisera. The presence of conjugated enzyme was determined by adding a 0.05-ml portion of freshly made substrate (0.01% H202 and 0.6 mg of O-phenylenediamine per ml in 0.1 M phosphate-buffered saline at pH 7.0). The plates were allowed to react for 1 h in the dark before the reaction was stopped with 0.03 ml of 4 N H2SO4. Readings to determine optical density were taken at a 460-nm wavelength with a Gilford model 250 spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio) with microcuvettes. Agar gel precipitation. Agar gel precipitation tests for precipitin activity were conducted by either the microplate or the template microtechniques described by Crowle (2). Buffers for the mammalian immunoglobulin or conjugates utilize isotonic salts, whereas those for the avian system require salts 10fold higher in ionic strength. Latex passive agglutination. As a means of comparing the ELISA results, the anti-reovirus antiserum was tested for agglutination against reovirus antigencoated latex particles. A volume of 0.15 ml of a latex (0.81-,um diameter; Difco Laboratories, Detroit, Mich.) dilution (1:30 in 0.05 M sodium borate-0.15 M NaCl, pH 8.2) was mixed with 0.15 ml of an antigen dilution or normal CEF control. After 15 min, 0.07 ml of serial dilutions of antisera was added and mixed thoroughly. The results were read after an overnight incubation (16 h) at room temperature. The titer expressed is the last dilution showing macroscopic agglutination. Immunoglobulin quantitation. With the method described by Heide and Schwick (5), precipitation of chicken immunoglobulin was carried out at sodium sulfate concentrations of 18, 15, and 15% (wt/vol). The protein content of the purified immunoglobulin was determined by the highly sensitive spectrophotometric technique of Kalb and Bernlohr (8). For this determination, a bovine serum albumin standard was run as a control check, and the protein content was determined by the following equation: protein content = 183 OD230- 75.8 OD260, where OD230 and OD260 are the optical densities at 230 and 260 nm, respectively.

RESULTS Determination of ELISA conditions for avian antisera. (i) Preparation of enzyme conjugates. Initially, we expected that the technique developed by Engvall and Perlmann (3) would be satisfactory for our needs in an avian system. However, we immediately encountered problems. First, the conjugation technique using glutaraldehyde to couple the alkaline phosphatase to the rabbit anti-chicken immunoglobulin resulted in a product without measurable immunological activity. For example, when the unconjugated antibody and the conjugated antibody were tested in a micro-agar gel precipitation assay, a titer of 1:32 was obtained for the




unconjugated immunoglobulin, but there was no detectable reaction with the conjugate. Similar results were obtained with the alkaline phosphatase-conjugated chicken anti-Marek's immunoglobulin. Here, the conjugated and free forms of the immunoglobulin were tested in the more sensitive latex passive agglutination test at equivalent immunoglobulin concentrations. The results with the unconjugated antibody showed an endpoint of 1:320, whereas the conjugate remained negative for agglutination. Although it is possible that some activity may have remained, a loss of antibody activity of greater than 95% was considered unacceptable. Based on a paper by Nakane and Kawaoi (9), which reported the conjugation of horseradish peroxidase to sheep anti-rabbit immunoglobulin G (IgG) without signiflcant loss of either immunological or enzymatic activity, we were successful in producing a rabbit anti-chicken immunoglobulin horseradish peroxidase conjugate which maintained approximately 50% of its original activity as measured by agar gel precipitation. (ii) Determination of a suitable binding substrate. After several attempts at adapting the procedure of Voller et al. (16), using the reovirus antigen bound to the plastic for the detection of avian anti-reovirus antibody, it became apparent that the binding of the avian anti-reovirus antiserum was irrespective of the bound viral antigen or normal control. Furthermore, negative sera collected from SPF birds bound as well as the positive serum. To prevent apparent nonspecific attachment, we tried several treatments, including the use of different plastic sources of polyvinyl chloride or polysty-

plates, post-antigen saturation binding with various proteins (bovine serum albumin, ovalbumin, and FCS), the inclusion of a 1% protein solution together with the avian serum, alteration of the pH of the binding medium, and the use of different salts and detergents. None of the treatments was able to prevent the nonspecific attachment while still allowing the specific antigen-antibody binding. It was found, however, that the modification of the method of Saunders and Clinard (11), which was originally developed to increase the binding of antigens by a pretreatment of the plastic support, could effectively prevent most of the nonspecific binding of avian immunoglobulin. A comparison of the binding of avian immunoglobulin to treated and untreated plastic is shown in Table 1. The experiment was conducted as a standard ELISA, but without the use of antigen. Two control sera, as well as the anti-reovirus HIS-1133 serum, or the rabbit conjugate alone was tested. The results indicated in all cases that the pretreatment with glutaraldehyde-fixed FCS reduced the total nonspecific binding of the avian immunoglobulin and the conjugate by 60 to 80%, while reducing the level of nonspecific binding by avian immunoglobulin by 63 to 99%. The minimal amount of total binding exhibited by the SPF-3 serum to the plastic substrate, as well as the almost complete elimination of this nonspecific binding to the treated plates, led us to determine the possible relationship between serum immunoglobulin levels and the "nonspecific" attachment of avian sera to plastic surfaces. Total immunoglobulin levels for SPF-3, SPF-16, and HIS-1133 sera, as rene

TABLE 1. Binding capacity of chicken sera to FCS-treated and untreated plastic surfacesa Avian binding' (total less conTotal binding'' Binding agent jugate) Chicken serum






SPF-3 (2.3 mg/ml) SPF-16 (6.2 mg/ml) HIS-1133 (7.1 mg/ml)
























499 198 60 NAd NA NA The plastic surfaces, treated or untreated with FCS, as described in the text, were compared for their capacity to bind nonspecifically either normal chicken sera (SPF-3 and SPF-16), anti-reovirus immune serum (HIS-1133), horseradish peroxidase-conjugated rabbit anti-chicken immunoglobulin, or all of these. The experiment was conducted as standard ELISA but in the absence of antigen. 'Values within parentheses are the levels of total immunoglobulin for the sera, as determined by salt fractionation, described in the text. Values indicated, except where otherwise stated, are given as the optical density at 460 nm x 1,000 of the enzyme substrate reaction, as given in the text for ELISA. dNA, Not applicable. +



VOL. 10, 1979


determined by sodium sulfate fractionation, were 2.3, 6.2, and 7.1 mg/ml, respectively. Although the FCS pretreatment procedure was largely successful in preventing nonspecific binding by avian sera, it is apparent that some immunoglobulin still binds and that this binding is dependent upon serum immunoglobulin levels. Since the immunoglobulin levels of control and experimental test sera are not likely to be identical, the use of negative sera as a control in ELISA has been replaced by a normal CEF preparation which, except for the virus, is otherwise equivalent to the viral antigen preparation. A serum is tested for reactivity against viral antigen and its control, i.e., CEF. Therefore, variabilities due to differences in immunoglobulin levels of test sera are eliminated. Sensitivity and precision of ELISA. Figure 1 shows the mean values of the results of four determinations of the HIS-1133 serum titer against reovirus S-1133. All points expressed, except for the highest dilution of 1:204,800 (log = 5.31), are significantly higher than those of controls at the confidence limits of greater than 95% by the t statistical evaluation of a twosample test. Although the precision of the assay as measured by an average coefficient of variation of less than 10% is reasonably good, less variation could be achieved by the use of a particulate-free antigen, unlike that used here. Nevertheless, the assay is quite reproducible, as shown by the datum points for HIS-1133 serum assayed 1 month earlier. The determination of the sensitivity of the assay can be compared relative to other assays or in more absolute terms. As previously mentioned (14), the precipitin activity of this serum was only barely perceptible, but a titration of this serum by the more sensitive latex passive agglutination test established an endpoint titer of 1:40. Relative to this latex passive agglutination assay, the present ELISA technique gave an approxiimate 1,300-fold increase in detection ability, having a titer of 1:51,000 (log = 4.71; Fig. 1). On an absolute basis, only an approximation of its sensitivity can be given, since the level of specifically reactive immunoglobulins in the sample is unknown. Levels of specifically reactive immunoglobulins of less than 10% have been reported for hyperimmunized chickens (6) and, if this level is used for computation, the ELISA technique described here shows a sensitivity limit of approximately 0.7 ng of specific antibody. This level of detection agrees well with that found by Yolken et al. for the detection of a mammalian IgG by the ELISA technique (20).


0 -O HIS- 1133 vs. S-1133 Anligen O------O HIS- 1133 vs. CE F Control

8 a


log / serum dilution

FIG. 1. ELISA titration of anti-reovirus HIS-1133 serum against a reovirus S-1133 CEF preparation and a normal CEF control preparation. The arrow indicates the significant endpoint as determined by the t statistical evaluation. Point spread bars indicate the standard deviation. A, Mean of values of the ELISA assay performed 1 month earlier. OD460, Optical density at 460 nm.

ulins does not always allow for ready adaptation of even common immunochemical techniques developed for mammalian systems. The unusually high affinity expressed here by avian immunoglobulins for the plastic substrate remains an unsolved problem. In this regard, it is interesting to note that this nonspecific binding to untreated plastic is maximal at a pH of 5.5 (Slaght, unpublished observations). This pH is somewhat below the reported pl for chicken IgG, but is close to the pH required for immunoprecipitation of avian IgG in an isotonic buffered solution (4). Although this anomalous behavior of avian immunoglobulins to maximally bind plastic surfaces at pH 5.5 cannot be explained at this point, the data from Table 1 suggest that all immunoglobulins do not bind equally. The level of nonspecific binding to FCS-treated plates is related generally to the immunoglobulin levels of the serum. The proportionality of immunoglobulin levels to binding seems to occur only with SPF16 and HIS-1133 sera. The level of nonspecific binding by SPF-3 serum is disproportionately small. This discrepancy, however, can be attribDISCUSSION uted to the nature of the immunoglobulin isoavian of nature immunoglob- types present in the SPF-3 serum pool as comThe capricious



pared with the others. Birds 3 weeks of age (as were those from which SPF-3 was taken) exhibit not only low levels of total immunoglobulin, but also very low levels of IgG and an inversely high proportion of IgM (15). Thus, it would appear likely that the low binding expressed by the SPF-3 serum pool is due to its extremely low IgG level. The procedure described here, although not completely satisfactory in eliminating the unique high-affinity attachment to plastic by avian immunoglobulin, is more than sufficient to allow for the detection of less than 1 ng of specifically bound antibody, and without the equipment or technical expertise required for the radioimmunoassay. This technique, however, as well as other ELISA procedures, must utilize antigen preparations which are free from immunoglobulins of the species being tested, whether in free or complexed forms. The presence of such immunoglobulins would probably raise the background readings sufficiently to obscure any specific immune reaction. Thus, the tissues used as a source of antigen are best grown in vitro, without the use of (as in this case) avian serum. Unfortunately, this reaction also eliminates the use of crude preparations from inoculated chicken embryos, due to the presence of maternal antibodies in the egg. Although the technique as described is a considerable improvement in sensitivity over other techniques, such as the agar gel precipitation test, and allows for detection at a much earlier stage of infection due to its high sensitivity, it is not presently known whether the unusually high-affinity binding of avian immunoglobulins to plastic will interfere in an inhibition mode of the ELISA technique to allow for the quantitation of unknown levels of antigen. Much more work needs to be done in this area for avian immunochemical assays. ACKNOWLEDGMENT We acknowledge Patricia Timmins for manuscript preparation.

LITERATURE CITED 1. Bruins, S. C., I. Ingwer, M. L. Zeckel, and A. C. White. 1978. Parameters affecting the enzyme-linked immunosorbent assay of immunoglobulin G antibody to a rough mutant of Salmonella minnesota. Infect. Immun. 21:721-728. 2. Crowle, A. J. 1973. Immunodiffusion, 2nd ed. Academic

Press Inc., New York. 3. Engvall, E., and P. Perlmann. 1972. Enzyme-linked

immunosorbent assay, ELISA. III. Quantitation of spe-

J. CLIN. MICROBIOL. cific antibodies by enzyme-linked anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109:129-135. 4. Gallagher, J. S., and E. W. Voss, Jr. 1970. Immune precipitation of purified chicken antibody at low pH.

Immunochemistry 7:771-785. Heide, K., and H. G. Schwick. 1973. Salt fractionation of immunoglobulins, p. 6.1-6.11. In D. M. Weir (ed.), Handbook of experimental immunology, 2nd ed., vol. 1. J. B. Lippincott Co., Philadelphia. 6. Hoffmeister, M. J., and E. W. Voss, Jr. 1974. Binding of E-DNP-L-lysine exceeding two moles by purified chicken IgG anti-DNP antibody. Immunochemistry 11: 5.



Horowitz, S. A., and G. H. Cassell. 1978. Detection of antibodies to Mycoplasma pulmonis by an enzymelinked immunosorbent assay. Infect. Immun. 22:161170.


Kalb, V. K., Jr., and R. W. Bernlohr. 1977. A new spectrophotometric assay for protein in cell extracts.


Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labelled antibody. A new method of conjugation. J. His-

Anal. Biochem. 82:362-371.

tochem. Cytochem. 22:1084-1091. 10. Payment, P., and J.-P. Descoteaux. 1978. Enzymelinked immunosorbent assay for the detection of antibodies to pneumonia virus of mice in rat sera. Lab. Anim. Sci. 28:676-679. 11. Saunders, G. C., and E. H. Clinard. 1976. Rapid micromethod of screening for antibodies to disease agents using the indirect enzyme-labeled antibody test. J. Clin.

Microbiol. 3:604-608. 12. Schmitz, H., H.-W. Doerr, D. Kampa, and A. Vogt. 1977. Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus. J. Clin. Microbiol. 5:629-634. 13. Schuurs, A. H. W. M., and B. K. van Weemen. 1977. Enzyme-immunoassay. Clin. Chim. Acta 81:1-40. 14. Slaght, S. S., T.-J. T. Yang, L. van der Heide, and T. N. Fredrickson. 1978. An enzyme-linked immunosorbent assay (ELISA) for detecting chicken anti-reovirus antibody at high sensitivity. Avian Dis. 22: 802-805. 15. van Meter, R., R. A. Good, and M. D. Cooper. 1969. Ontogeny of circulating immunoglobulins in normal, bursectomized and irradiated chickens. J. Immunol.


16. Voller, A., D. Bidwell, and A. Bartlett. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections, p. 506-512. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. 17. Voss, E. W., Jr., and R. M. Watt. 1977. Comparison of the microenvironment of chicken and rabbit antibody

active sites. Adv. Exp. Med. Biol. 88:391-401. 18. Walls, K. W., S. L. Bullock, and D. K. English. 1977. Use of the enzyme-linked immunosorbent assay (ELISA) and its microadaptation for the serodiagnosis of toxoplasmosis. J. Clin. Microbiol. 5:273-277. 19. Yolken, R. H., B. Barbour, R. G. Wyatt, A. R. Kalica, A. Z. Kapikian, and R. M. Chanock. 1978. Enzymelinked immunosorbent assay for identification of rotaviruses from different animal species. Science 201:259262. 20. Yolken, R. H., R. G. Wyatt, H. W. Kim, A. Z. Kapikian, and R. M. Chanock. 1978. Immunological response to infection with human reovirus-like agent: measurement of anti-human reovirus-like agent immunoglobulin G and M levels by the method of enzymelinked immunosorbent assay. Infect. Immun. 19:540546.

Adaptation of enzyme-linked immunosorbent assay to the avian system.

Vol. 10, No. 5 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1979, p. 698-702 0095-1137/79/11-0698/05$02.00/0 Adaptation of Enzyme-Linked Immunosorbent As...
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