INFECTION AND IMMUNITY, Sept. 1978, p. 721-728 0019-9567/78/0021-0721$02.00/0 Copyright i 1978 American Society for Microbiology

Vol. 21, No. 3

Printed in U.S.A.

Parameters Affecting the Enzyme-Linked Immunosorbent Assay of Immunoglobulin G Antibody to a Rough Mutant of Salmonella Minnesota SCOTT C. BRUINS,'* IRWIN INGWER,2 MICHAEL L. ZECKEL,3 AND ARTHUR C. WHITE' Department of Internal Medicine, Indiana University School ofMedicine,' and the Veterans Administration hospital, Indianapolis, Indiana 46202; Memorial Sloan-Kettering Cancer Center, New York, New York 100212; and Internal Medicine, The Meridian Medical Group, Indianapolis, Indiana 462083

Received for publication 9 June 1978

An enzyme-linked immunosorbent assay was developed to detect rabbit immunoglobulin G antibodies against purified lipopolysaccharide of the rough Re mutant of Salmonella minnesota. The time necessary for each step was investigated, and this resulted in a test that could be completed in 1 working day. Serial dilutions of rabbit sera drawn throughout immunization with the Re mutant revealed an elimination of the prozone effect upon prolonged immunization. We interpret this to demonstrate an increase in immunoglobulin G affinity for the lipopolysaccharide on prolonged immunization. We propose that the enzymelinked immunosorbent assay prozone effect be investigated for use as a measure of antibody affinity.

An enzyme-linked immunosorbent assay (ELISA) has been developed for many antigenantibody systems, and it would seem that this new method will be applicable to almost any antigen-antibody system (2, 5). The advantages of ELISA are rapidly becoming well known (3). One of us had worked with an antigen-antibody system of particular interest for possible protection of humans from gram-negative bacteremia, i.e., the Salnonella Minnesota mutant 595 (Re) lipopolysaccharide (LPS) and its corresponding antibody (1, 7). Since protection of animals from bacteremia by active and passive immunization with this mutant has been demonstrated but the height of the classical hemagglutination titers or even immunodiffusion titers did not seem to correlate well with demonstrated passive protection, new serological tests were needed. We therefore set out to develop a solid-phase test for the S. Minnesota mutant 595 (Re) LPS-antibody system to investigate the new information that it could provide. MATERIALS AND METHODS Antigens. The S. Minnesota Re mutant 595 was

obtained from Otto Westphal, Freiburg, West Germany. This organism was grown in Casamino Acids broth supplemented with glucose and yeast extract, precipitated with acetone, washed with acetone, and dried. The LPS was prepared from the rough mutant by the phenol-chloroform-petroleum ether extraction technique of Galanos et al. (6). Antisera. Approximately 1.7- to 2.0-kg New Zealand white rabbits were used for immunization. The

dried, whole Re bacilli were suspended in 0.3% saline, and this was injected into the ear veins of the rabbits. They received 0.2 mg on day 0, 0.4 mg on day 3, 0.8 mg on day 7, 1.6 mg on day 10, 3.2 mg on day 14, 6.4 mg on day 17, and 1.6 mg on days 25, 37, 51, 66, and 79. Sera were collected on days 0, 7, 14, 22, 30, 44, 57, 65, 72, and 86. Indirect hemagglutination Re LPS was saponified with 0.25 N NaOH for 30 min at 370C and then neutralized with HCl. This was used at a concentration of 100 ,g/ml to sensitize a 10% suspension of human type 0 erythrocytes for 30 min at 370C (7). Antibody titrations were carried out in round-bottomed wells of microtiter plates (Cook Engineering Co., Alexandria, Va.) with equal volumes of a 0.5% suspension of sensitized erythrocytes and of the serum dilutions. These plates were incubated at 370C for 1 h and then overnight at 40C and read for hemagglutination. Immunodiffusion. Double-diffusion precipitin tests in agar were done with the apparatus as described by Crowle (4). Re LPS (1 mg/ml of distilled water) was placed in the center well, and serial twofold dilutions of the antisera were placed in the outer wells. These were incubated for 48 h at room temperature before reading. Quantitative precipitation. The amount of specific Re antibodies in the sera was measured by the quantitative precipitation method of Engvall and Perlman (5)..

Labeled antibody. Goat antirabbit immunoglobulin G (IgG) serum was chromatographed on Sephadex G200, and the IgG fraction was collected and concentrated to 5 mg/ml. This goat antirabbit IgG was labeled with "I by the chloramine-T method as described by McConahey and Dixon (8). Some of the same batch of GAR was labeled with alkaline phosphatase as described by Engvall and Perlman (5). 721

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ELISA. The first ELISA method tried was the method of Engvall and Perlman (5). Solid-phase tests. The solid-phase assay was performed in polystyrene tubes (12 by 77 mm; Falcon Plastics, Oxnard, Calif.) in the following order at 370C. In step 1, the insides of the tubes were coated by 0.2 ml of a solution of Re LPS in distilled water with 1 Al of triethylamine per ml to solubilize the LPS. This solution was then aspirated, and the tubes were washed three times with distilled water. In step 2, the tubes were then filled with 0.5 ml of a bovine serum albumin (BSA) solution in 0.05 M tris(hydroxymethyl)aminomethane buffer adjusted to a pH of 9.8 with HCL Then, this fluid was aspirated, and the tubes were washed three times with distilled water. In step 3, 0.2 ml of rabbit sera diluted in a buffer consisting of 0.1 M saline, 0.1 M phosphate, and BSA (pH 7.2) was placed in the bottom of the tube. This fluid was then aspirated, and the tubes were again washed three times with distilled water. In step 4, 0.2 ml of a diluted, labeled IgG fraction of goat anti-rabbit IgG sera was placed in the tubes to detect any rabbit IgG attached to the Re LPS immobilized on the plastic tube. After this was aspirated, the tubes were washed seven times with distilled water. For step 5, in the case of the solidphase radioimmunoassay (SPRIA), the tubes were then counted in the "I channel of a Searle gamma counter. In the case of the ELISA, 0.2 ml of p-nitrophenyl phosphate (1 mg/ml) in 0.05 M sodium carbonate and 0.001 M magnesium chloride buffer (pH 9.8) was added to the tubes. After incubation at 370C, this reaction was stopped by addition of 1.0 ml of 1.0 M NaOH, and the absorbance of the solutions at 400 nm was read on a spectrophotometer (Gilford model 240). All tubes were done in duplicate, and the values were averaged. Controls including each serum variation were done in tubes with no antigen coating, and antigen-coated tubes were run with serum diluent only.

RESULTS The results of the SPRIA and ELISA were essentially the same in all experiments in which both labels were used. Due to the great advantage of the ELISA test, we continued only with this test once we had convinced ourselves that the two methods gave the same results within experimental error. We found that the Re LPS bound to the polystyrene tubes when it was dissolved in distilled water but not when an attempt to suspend it in saline was made. Less variability in the test results occurred when the Re LPS was dispersed with 1 1d of triethylamine per ml of distilled water. The concentration of the Re LPS necessary to obtain adequate test results was 0.1 to 0.5 mg/ml as shown in Fig. 1. This held for both the SPRIA and ELISA. Adequate antigen binding occurred very quickly, but we chose 1 h as a routine coating time (Fig. 2). The next step in the SPRIA test had previously been standardized in this laboratory with

EFFECT OF ANTIGEN CONCENTRATION

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LOG2 (12.5/SERUM DILUTION) FIG. 1. Effect of antigen concentration. Serial dilutions of rabbit sera from day 30 of immunization were used, with specified concentrations of Re LPS to coat the tubes.

5% BSA for 1 h to cover the remaining protein binding sites on the tube to eliminate nonspecific reactivity. We found that this worked well. However, the second step as originally described by Engvall and Perlman (5) for the ELISA system resulted in inability to detect Re LPS, probably because the Tween 20 detergent removed the Re LPS from the polystyrene walls. Therefore, the SPRIA method of using BSA to block protein absorption sites on the polystyrene tubes and washing with distilled water was used and found to work well. To conserve material and ease handling of the fluids, 5, 1, and 0.5% BSA concentrations were evaluated, and the variance of the results was quite similar with these BSA concentrations unless very low dilutions of antisera were used, i.e., a 1:10 dilution of antisera. Thus, we standardized this step using 0.5% BSA. Other buffers were tried, including 0.15 M saline-0.01 M phosphate (pH 7.2) and 0.1 M saline-0.1 M phosphate (pH 7.2) with poor results. The third step involves the primary antibody (PAb) attaching to the immobilized antigen. We again evaluated buffers and found the 0.1 M saline-0.1 M phosphate (pH 7.2) buffer to be as

ELISA TO ANTIBODIES OF ROUGH MUTANTS

VOL. 21, 1978 TIME

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FIG. 2. Time dependence of antigen binding. A 0.5mg amount of Re LPS per cm3 of distilled water was incubated for varying times, and then the standard ELISA was completed with hyperimmume rabbit sera. Controls were tubes without Re LPS added.

good or better than other buffers tried. Using a hyperimmune sera pool of rabbits bled on day 86 of immunization, we investigated the time course of binding of the PAb (Fig. 3), being sure that the labeled antibody step and the antigen were not limiting. A majority of the binding had occurred by 1 h. A 1-h incubation for this step was chosen as our standard assay procedure so that the whole test could be completed in 1 day. Investigation of the antibody dilutions to be used revealed very surprising results (Fig. 4). The sera from early immunization (days 14, 22, and 30) showed a definite prozone effect, with elimination of this later during immunization. This prozone phenomenon at day 30 and elimination of it by day 60 were also found in a crude LPS-antibody system of Pseudomonas maltophilia (unpublished data). The fourth step, in which the alkaline phosphatase-labeled IgG fraction of goat antibody to rabbit IgG (secondary antibody [SAb]) attached to PAb, was also evaluated by using various buffers, as was the third step. Again, the 0.1 M saline-0.1 M phosphate (pH 7.2) buffer was found to be as good or better than other buffers tried in our system. The variation of test results with dilution of the SAb label can be seen in Fig.

60

180 240 120 TIME (MINUTES)

FIG. 3. Time dependence of PAb attachment. Hyperimmune rabbit sera at specified dilutions were incubated for the indicated times.

5. To conserve labeled SAb, a 1:1,000 dilution (5 ,ug of protein per ml) of the SAb was chosen as a standard test dilution. The time course of one of many preparations of the SAb label attaching to PAb is shown in Fig. 6. The plateau effect (Fig. 6) at 240 min was due to limitation of PAb, as was shown in another experiment by elimination of the plateau when the PAb was incubated for 2 h. To determine the validity of ELISA run with the standard SAb dilution and a 1-h incubation time, we plotted a standard serum titration curve using varying times of SAb incubation (Fig. 7), and demonstrated similar curves outside the plateau region. Therefore, any plateau region is investigated to determine the particular step that is limiting. At higher dilutions of PAb, standardized ELISA values are proportional to values that would be obtained by increasing the time of SAb or its concentration. A standard 1-h incubation time was chosen so as to complete the test in 1 day. The fifth step of the ELISA was addition of an indicator, p-nitrophenyl phosphate. Alkaline phosphatase converted the colorless p-nitrophenyl phosphate to yellow p-nitrophenol with an absorption maximum at 400 unm. We investigated varying the volume of this solution to try to get more color change in the same amount of time. However, 0.2 ml later diluted to 1.2 ml by

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BRUINS ET AL. TIME DEPENDENCE OF IMMUNIZATION TRESPONSE

EFFECT OF SECONDARY ANTIBODY CONCENTRATION

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FIG. 4. Time dependence of immunization response. Serial twofold dilutions of antisera were pooled from 11 rabbits immunized with Re organisms. Sera were drawn on the day specified. Controls were tubes without Re LPS coating.

sodium hydroxide gave as reliable and almost as great absorbance readings as 1 ml of thep-nitrophenyl phosphate solution which was stopped by 0.2 ml of 1 N sodium hydroxide. Therefore, 0.2 ml of p-nitrophenyl phosphate was chosen for the standard test. The relation of absorbance readings to time of thep-nitrophenyl phosphate incubation is shown in Fig. 8. A standard incubation time of 90 min was chosen so as to be able to complete the test in 1 day. The results of the classical hemagglutination and immunodiffusion tests gave the expected results and are compared with the ELISA and SPRIA done with a 1:100 serum dilution (Fig. 9). The precipitable Re antibody from day 30 sera from a representative rabbit was 1.24 mg/ml, and that from day 65 sera was 1.27 mg/ml. DISCUSSION The ELISA test has become very popular (10), in part because of the ease with which a laboratory can set up this test without additional expensive equipment. The greater stability of the enzymes as compared with radioactive tracers is valuable in being able to test sera or antibody preparations immediately when they

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LOG2 (1000/ SAb DILUTION) FIG. 5. Effect of SAb concentration. Standard ELISA was used with designated SAb dilutions; 0 equals 5 jg of goat IgG anti-rabbit IgG per ml. Controls were tubes without Re LPS coating.

become available, thus saving time. This test seems very flexible in that it can be set up to screen large numbers of sera at a set dilution or can be used to investigate the properties of a single sera. As has been the case of many ELISA systems, the original coating of the tubes occurs very rapidly, usually well under 60 min. However, contrary to some suggestion (2), modifications may be necessary for antigens that are not classical proteins or polysaccharides due to their unique physical characteristics. The high concentrations of this lipophilic LPS necessary to coat the tubes was somewhat at variance with experience with the LPSs from smooth organisms. Certainly the unsuccessful experience with the Tween 20 detergent washing procedures is not the general experience with protein and polysaccharide antigens but may be expected with any lipid antigens, such as lipid A and other LPS from rough mutants. Because the solid-phase tests, SPRIA and ELISA, generate a quantitative number for each serum dilution, they may provide more information than previous serological procedures. Because we were used to dealing with titers, we tried to relate the quantitative absorbance value

ELISA TO ANTIBODIES OF ROUGH MUTANTS

VOL. 21, 1978

at a set serum dilution with a titer, i.e., the highest serum dilution that just gave a positive test. It quickly became apparent that the time during immunization was important in that immune sera from days 14 to 30 did not give the same relationship as did sera drawn 60 to 90 days after the start of immunization. We also noted that the total precipitable Re antibody protein in the sera from days 30 and 65 was about the same. However, this same amount of antibody protein gave higher IgG ELISA absorbance values on day 65 than on day 30 even though the hemagglutination and immunodiffusion test did not change significantly. Therefore, we performed the ELISA again over a greater range of serum dilutions on sera obtained during immunization of 11 rabbits. We found a previously reported prozone at low serum dilutions in the first month of immunization but found that this disappeared by day 44 (Fig. 4). The convergence of the IgG ELISA absorbance values at higher serum dilutions seemed to correlate with the stable immunodiffusion titers and precipitable antibody protein measurements between days 30 and 65, indicating that the IgG ELISA absorbance values at high dilutions were a measurement of the number of antibody molecules available to attach to the antigen. Therefore, we were left with a situation in which

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FIG. 6. Time dependence of SAb attachment. Standard ELISA conditions were used, with a hyperinmune rabbit sera pool at 1:200 dilution, and the SAb at 1:1,000, for indicated times of SAb incubation. Controls were tubes without Re LPS coating.

EFFECT OF SAb INCUBATION TIME ON PAb DILUTION CURVE I

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