INFETION
AND IMMUNITy,
May 1979,
p.
441-448
Vol. 24, No. 2
0019-9567/79/05-0441/08$02.00/0
Enzyme-Linked Immunosorbent Assay for Immunoglobulin G and Immnunoglobulin A Antibodies to Shigella flexneri Antigens DAVID F. KEREN' Department of Bacterial Diseases, The Walter Reed Army Institute of Research, Washington, D.C. 20012, and Department of Pathology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109' Received for publication 5 March 1979
An enzyme-linked immunosorbent assay has been developed to detect classspecific antibodies to Shigella flexneri lipopolysaccharide antigens. This enzymelinked immunosorbent assay system has been used to measure antibodies present in serum or intestinal secretions without further purification. It is considerably more sensitive than passive hemagglutination, allowing detection of as little as 1.3 ng of specific immunoglobulin G antibody per ml in immune sera. Optimal conditions for this assay are outlined in this report.
The enzyme-linked immunosorbent assay (ELISA) has been shown to be a highly sensitive technique capable of detecting antibodies to a wide variety of antigens (4-6, 8-10, 13, 18). This technique has the following advantages: the enzyme conjugates and the substrate reaction products are stable for long periods of time, results can be read visually or on a spectrophotometer, and ELISA lacks the potential radiation hazards of radioimmunoassay. In this report an ELISA technique is used to measure relative amounts of isotypically specific antibodies against the lipopolysaccharide (LPS) extracted from a hybrid of Shigella flexneri and Escherichia coli (Shigella X16). The ELISA technique used in this study is a modification of our earlier procedure which did not directly relate the absorbance units from the substrate reaction to specific antibody against Shigella X16 LPS (X16 LPS) in gravimetric terms (13, 18). Data from the present study (i) establish sensitivity and specificity of the technique, (ii) assess parameters affecting the ELISA system, and (iii) compare this ELISA to passive hemagglutination and quantitative precipitation by using immunoglobulin G (IgG) fractions from rabbit antisera to X16 LPS. MATERIALS AND METHODS Goat antisera. Goat anti-rabbit IgG (GARG) was obtained by immunizing a goat with 6.6 mg of Fc portion from rabbit IgG in complete Freund adjuvant. Serum was collected weekly and tested for GARG activity by gel diffusion. Specific GARG antibodies were absorbed onto rabbit IgG insolubilized by glutaraldehyde (3). After washing this IgG sorbent with saline, the GARG antibodies were eluted with 0.1 M glycine-HCl buffer, pH 2.8. Na2CO3 was added to
adjust the pH to 8.0. Goat anti-rabbit IgA (GARA) was obtained by immunizing a goat with 8 mg of rabbit IgA in complete Freund adjuvant. Sera were collected weekly and tested for GARA activity by gel diffusion. The globulin fraction was precipitated from serum by saturated ammonium sulfate as described by Herbert et al. (11) and dialyzed against three changes of 0.01 M phosphate buffer, pH 8.0. Antibodies cross-reacting with IgG were removed by passing this material over a 14ml column of Sepharose 4B covalently coupled to 24.5 mg of rabbit IgG. Specific antibodies to IgA were then absorbed onto a 10-ml column of Sepharose 4B coupled to 30 mg of rabbit IgA. These antibodies were eluted with 0.01 N NaOH in saline and quickly neutralized with 0.1 N HCI. GARG and GARA were concentrated by negativepressure dialysis to 3.9 and 7.1 mg/ml, respectively. They were conjugated to alkaline phosphatase (AP) (type VII, Sigma Chemical Co., St. Louis, Mo.) using the technique of Avrameas (2) as modified by Yardley et al. (18). Preparation of antigens for coating tubes. Rabbit IgG was prepared by using the third precipitate in 35% saturated (NH4)2S04 from rabbit serum (11). After the redissolved globulins were dialyzed against 0.01 M phosphate buffer, pH 8.0, they were applied to a diethylaminoethyl-cellulose column equilibrated with the same buffer. The proteins which passed through the column unimpeded were concentrated as the IgG standard. Rabbit IgA was prepared from intestinal secretions collected from chronically isolated ileal (Thiry-Vella) loops in rabbits (12). This fluid has been shown to contain mainly IgA with some IgG and little IgM (15). The fluid was clarified by centrifugation at 3,000 rpm for 15 min, and the globulin fraction was precipitated by slow addition of a saturated ammonium sulfate solution (neutralized to pH 7.8 by 1 N NaOH) until 50% saturation was achieved. After removal of the supernatant, the precipitate was dissolved in phos-
441
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INFECT. IMMUN.
KEREN
phate-buffered saline, pH 7.2 (PBS). After a second ammonium sulfate precipitation at 50% saturation, the protein was dissolved in 0.04 M phosphate buffer, pH 8.0, and dialyzed against three changes of this same buffer. Aggregates were removed by centrifugation at 3,500 rpm for 15 min in the cold. The protein was applied to a diethylaminoethyl-cellulose column equilibrated in 0.04 M phosphate buffer, pH 8.0. This column was washed extensively with 0.04 and 0.1 M phosphate buffers, pH 8.0. The protein eluted from the diethylaminoethyl with 0.2 M phosphate buffer was concentrated to 0.6 mg/ml by negative-pressure dialysis and shown to contain only IgA by gel diffusion and ELISA. This material was shown to be quantitatively equivalent in the ELISA to a standard IgA preparation (kindly provided by Stella M. Robertson from the laboratory of John J. Cebra) before use. X16 LPS and Pseudomonas aeruginosa LPS (PALPS) were prepared by the hot phenol-water method (17). These preparations contained less than 3.5% protein and from 3 to 5% nucleic acid. Rabbit antisera to X16 LPS. Sera from rabbits immunized intravenously with the parent strain of the S. flexneri-E. coli hybrid were kindly provided by S. B. Formal. IgG fractions of one antiserum (anti-X16) were prepared as described above for preparation of rabbit IgG. The fraction that passed through the diethylaminoethyl column with 0.01 M phosphate buffer (IgG anti-X16 fraction I) and the fraction which was eluted from the column with 0.04 M phosphate buffer (IgG anti-X16 fraction II) were shown to contain only IgG by gel diffusion and ELISA. Intestinal secretions (from Thiry-Vella loops) from several rabbits with previously demonstrated IgA antiX16 activity (13) were pooled, and the IgA fraction was prepared as described above. Passive hemagglutination. Sheep erythrocytes in Alsever solution were washed three times in 0.01 M PBS, pH 7.2. To determine optimal sensitization, various concentrations of X16 LPS from 10 ng/ml to 100 ,tg/ml were incubated with a 2% solution of sheep erythrocytes for 1 h at room temperature with occasional agitation. The sensitized sheep erythrocytes were washed three times with saline and diluted to a 1% suspension in PBS. Several twofold dilutions of serum were prepared with PBS. Into each well of a microtiter plate (Linbro Chemical Co., New Haven, Conn.) containing 25 ,ul of the serum, an equal volume of sensitized sheep erythrocytes was added. The microtiter plates were incubated for 1 h. The hemagglutination titer was the last well showing grossly visible hemagglutination. Sera with unsensitized sheep erythrocytes were run as controls. The optimum sensitizing dose was found to be 10 ag/ml. Quantitative precipitin assay. Both IgG antiX16 fractions I and II were studied for specific antibody content. The tests were performed in 0.5-ml polyvinyl tubes (Evergree- Scientific, Los Angeles, Calif.) by using 0.1 ml of fraction I or II with 0.1 ml of PBS (with 0.1 mg of Merthiolate per ml) containing 0, 1, 2, 4, 8, 16, 32, and 64 ,ug (by weight) of X16 LPS. These tubes were incubated at 37°C for 1 h and then at 4°C for 8 days with occasional agitation. The precipitates were sedimented by centrifugation at 11,600 rpm for 1 h on a Beckman Microfuge B (Beckman
Instruments, Inc., Palo Alto, Calif.). As the precipitates obtained with LPS antigen were gelatinous, the supernatants were removed to 0.01 ml and were carefully washed twice in 0.1 ml of ice-cold PBS each, followed by another centrifugation for 1 h. Protein content was determined by the method of Lowry et al. (14). All reactions were performed in duplicate. ELISA procedure. ELISA was performed as described by Engvall et al. (8-10). Disposable polystyrene tubes (12 by 75 mm; Falcon Plastics, Oxnard, Calif.) were incubated with 1 ml of solution containing antigen in 0.05 M carbonate buffer, pH 9.6 (coating buffer), for 3 h at 37°C except where otherwise noted. Tubes containing the antigen solution were covered with Parafilm (to prevent evaporation) and stored at 4°C until use. Immediately before testing, the antigen solution was removed and the tubes were washed four times (5 min with agitation per wash) with PBS containing 0.05% Tween 20 (Fisher Scientific Co., Pittsburgh, Pa.) and 0.02% sodium azide (PTA). The rabbit sera or IgG fraction of the sera to be tested were diluted 1:20 (or as stated) in PTA, and 0.5 ml was added to the tubes coated with antigen and to uncoated tubes (as a control for nonspecific adsorption). After the tubes were incubated at room temperature for 4 h (except where otherwise stated) on a horizontal rotary shaker, they were again washed four times with PTA, and 0.5 ml of AP conjugated to GARG (APGARG) or to GARA (AP-GARA) was added to the appropriate tubes. The reaction mixtures were incubated for 18 h (except where otherwise stated) at room temperature with agitation. After another series of four washes with PTA, 1 mg of the substrate, p-nitrophenyl phosphate disodium (Sigma Chemical Co.), in 1 ml of 0.05 M carbonate buffer (pH 9.8) containing 0.001 M magnesium chloride was added to each tube. The reaction was developed at room temperature until adsorbance at 400 nm was approximately 1.0 (the enzyme-substrate reaction is linear to this level [101), being stopped at 25, 50, or 100 min by the addition of 1 ml of 0.2 M NaOH. All results were extrapolated to 100 min (10). The optical densities of the uncoated tubes at 400 nm were subtracted from those of the coated tubes containing the same solutions. All reactions were performed in duplicate.
RESULTS
Optimal antigen coating of tubes. To determine the optimal X16 LPS concentration for coating tubes, varying concentrations of X16 LPS in coating buffer were incubated for 3 h at 37°C. These tubes were tested with a 1/104 dilution of the rabbit antisera to X16 LPS. Optimal sensitization was obtained at an antigen concentration of 10 ,ug/ml (Fig. 1). At higher concentrations, there was a decrease in the absorbance. The adsorption of the LPS occurs quickly, as no difference was observed between tubes coated for 5 min at 37°C and those coated for 24 h.
Specificity and optimal dilution of APGARG and AP-GARA. To determine the isotypic specificity of the AP-GARG and AP-
VOL. 24, 1979
ELISA FOR ANTIBODIES TO SHIGELLA ANTIGENS
6
443
a
c
5 E 0 0 0 0
a
A4 0
E
10-3 1id2 Clo
1O' 102
0
,sg LPS/ml
z
FIG. 1. Determination of optimal LPS concentration for coating tubes. Tubes were coated for 3 h at 370C with the concentrations of Shigella X16 LPS indicated on the abscissa, followed by incubation with antisera to Shigella X16 diluted 1/104. Enzymeanti-IgG conjugate was diluted 1:160. OD, Optical density.
GARA, various concentrations of rabbit IgG and IgA in coating buffer were used to coat polystyrene tubes. These tubes were washed four times with PTA, and 0.5 ml of AP-GARG (diluted 1: 640 in PTA) or AP-GARA (diluted 1:320 in PTA) was added to the appropriate tubes and incubated for 18 h at room temperature. After another four washes with PTA, the substrate reaction was carried out as described above. Tubes coated with solutions containing as little as 62 ng of rabbit IgG or IgA per ml gave detectable reactions with AP-GARG and APGARA, respectively (Fig. 2). No cross-reactivity was detectable with tubes coated by solutions containing up to 10 ,Ag of immunoglobulin per ml. The working dilution for each batch of APGARG and AP-GARA was determined by reacting tubes coated by solutions containing 1 tLg of immunoglobulin per ml with various dilutions of the phosphatase-conjugated reagents. The dilution which would give an increase in optical density at 400 nm of 5 to 10 was chosen (H.
Carlsson, personal communication). Kinetics of primary antibody binding. The rate of binding of rabbit anti-X16 to X16 LPS was investigated by using tubes coated with solution containing 10 jig of X16 LPS per ml. Rabbit antisera to X16 diluted to 1/104, 1/5 x 104, and 1/105 were used to determine if the kinetics of primary antibody binding was related to antibody concentration. The antisera were allowed to react with the antigen-coated tubes for varying periods of time. Thereafter, the tubes were washed and AP-GARG was allowed to react for 18 h, followed by the substrate reaction. The primary antibody was completely bound
0
1 ---J.L-S
A,
-
0.007 0.015 0.031 0.062 0.125 0.25 0.5 1 jlg/ml in Coating Solution 6
k g. i
ii{ I I
10
k--10
5
4
c0 6
2
1
.
0L-
A----O
0.017 0.015 0.031 0.062 0.125 0.25 0.5 1 10 jug/ml in Coating Solution FIG. 2. (a) Specificity of enzyme-conjugated antiIgG (AP-GARG). AP-GARG diluted 1:640 gave a typical reaction curve with tubes coated with IgG (0) (concentrations of coating solution on abscissa), but not with tubes coated with IgA (-). (b) Specificity of enzyme-conjugated anti-IgA (AP-GARA). APGARA diluted 1:320 gave a typical reaction curve with tubes coated with IgA (0), but not with tubes coated with IgG (0). O.D., Optical density.
after 4 h of incubation with coated tubes, regardless ofthe concentration of the antisera (Fig. 3). No increase in reactivity was seen when incubation was extended to 24 h.
444
INFECT. IMMUN.
KEREN 4.5
4.0 3.5
3.0 C
.E
082.5 E C!
0 0
2.0
c0 1.0
7 8 4 5 6 2 3 WITH ANTISERUM HOURS OF INCUBATION FIG. 3. Kinetics of the binding ofprimary antibodies to tubes coated with a solution containing 10 pg of LPS. Antisera to X16 LPS diluted 1/104 (0), 1/5 x 104 (0), and i/10i (x) were incubated in the LPS-coated tubes for the time indicated on the abscissa and thereafter for 18 h with AP-GARG diluted 1:160. OD, Optical density.
Kinetics of binding of AP conjugate (secondary antibody). The rate of binding of APGARG was investigated by using tubes coated by a solution containing 10 jig of X16 LPS per ml reacted with one of two concentrations of primary antibody (1/10' and 1/105). The primary antibodies were allowed to react for 4 h. AP-GARG (diluted 1:160 in PTA) was allowed to react for varying periods of time. Reactions were begun at intervals which allowed for the substrate reactions to be carried out simultaneously. By 4 h the conjugate reaction was complete for both concentrations of primary antibody used (Fig. 4). No increase in reactivity was found with incubations of AP-GARG as long as 28 h. For practical purposes, incubations with AP-GARG in the present test system are usually carried out overnight for 18 h. Reproducibility. Using the optimum conditions described above, 10 replicate samples of serum from a rabbit inmunized with Shigella
X16 were assayed. A dilution of 1/104 gave a mean of 1.632 ± 0.037 (standard error of the mean), whereas dilutions of 1/105 gave a mean of 0.169 ± 0.012 (standard error). Eight replicate samples of IgA anti-X16 diluted 1/40 yielded a mean of 2.225 ± 0.045 (standard error). Quantitative precipitin test. To determine the sensitivity of the present ELISA test, the amount of precipitating antibody in standard rabbit sera to X16 LPS was determined by the quantitative precipitin test. The rabbit sera had some IgM activity against X16 LPS as demon-
strated by hemagglutination studies (Table 1). The hemagglutination titer of the serum was 1: 5,120 without 2-mercaptoethanol, but the addition of 2-mercaptoethanol reduced this titer to 1:640. Therefore, since the present ELISA system detects isotypically specific antibodies to X16 LPS, it was necessary to purify IgG fractions of this serum. Both IgG anti-X16 fractions I and II had titers of 1:640 regardless of the presence
VOL. 24, 1979
ELISA FOR ANTIBODIES TO SHIGELLA ANTIGENS
445
of 2-mercaptoethanol (Table 1). In addition, by gel diffusion and ELISA these fractions were shown to contain only IgG. The results of the .s 3.5 quantitative precipitin test on the IgG anti-X16 8 3.0 fractions are shown in Fig. 5, and the specific E 2.5 antibody contents are noted in Table 1. o 2.0 . Sensitivity and isotypic specificity. The 1.5 quantitative precipitin test results were compared to those obtained when several dilutions 1.0 v '-o of the rabbit sera and the IgG anti-X16 fractions .5 were analyzed by ELISA (Fig. 6a). 0 The level of 0.5 optical density unit at 400 nm 34 2 56 7 8 28 was arbitrarily chosen as the lower limit of senHOURS OF INCUBATION WITH AP '-GARG sitivity of the present ELISA system, since at FIG. 4. Kinetics of the binding of sect this level the results are approximately five body (AP-GARG). Tubes coated with a d,ohain antwcons- times greater than the usual background level taking 10 pg ofLPS per ml were reacted awt (which varied between 0.08 and 0.13). The senantisera to X16 diluted 1/104 (0) or 1/ (0) sitivity for the present ELISA test for IgG antithen with AP-GARG (diluted 1:160) fl or the indicated on the abscissa. O.D., Optical density. X16 fraction I was 1.3 ng/ml, and for the IgG anti-X16 fraction II it was 3.5 ng/ml (Table 1). TABLE 1. Sensitivity ofELIS'A Although insufficient purified IgA anti-X16 was obtained to perform a quantitative precipiHemaggluti- Specific Serum di- Antbody tin test, ELISA was performed to determine the nation titer Source of antilution t(ng) of the AP-GARA and AP-GARG respecificity 0.5 anti-X16 No 2- With body give 0 under the conditions of the assay. VirA EUSA agents MEb 2-ME (mg/ml) ELIS tually no cross-reaction occurs between the IgA ND Serum ND` 1/6 x 105 5,120 640 anti-X16-purified fraction and AP-GARG or Fraction I 640 640 0.26 1/2 x o5 3 3 with the IgG-purified fractions and AP-GARA Fraction II 640 640 0.35 W 3.5 1/1 x 6a and b). ~(Fig. ° Determined by quantitative precipitation. Antigenic specificity of EITISA. To deterb 2-ME, 2-Mercaptoethanol. mine whether antibodies to unrelated gram-neg'ND, Not done. s
-
-
-
-
itfo tiaes
35
30
C2 25tS 0. CL h.
%.
,20
5
.
.
.
.
10 I5 20 25 30 35 40 45 50 55 60 65 pg Lipopolysoccharide Added
FIG. 5. Quantitative precipitation curves ofShigella X16 LPS with two IgG fractions: 0.01 M IgG anti-X16 fraction I (0); 0.04 M IgG anti-X16 fraction II (0).
446
INFECT. IMMUN.
KEREN
a 5
c
AI
8 E
E c
3
§ so 2
1
an
0
No
Antisera
-Loq Dilution
4
c
b
3
0
E 2
-
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0o
E5 2
0o
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1
2 3 4 -Log Dilution
5
6
FIG. 6. Titration of specific antisera against Shigella X16 by ELISA. Tubes coated with a solution containing 10 pg of LPS and reacted with the indicated dilutions (on abscissa) of rabbit antiserum to Shigella X16 (A), IgG anti-X16 fraction I (-) and IgG anti-X16 fraction II (-), IgA anti-X16 (5), or with buffer only (0). Tubes were then incubated with either AP-GARG diluted 1:320 (a) or with AP-GARA diluted 1:320 (b). O.D., Optical density.
ative bacteria would cross-react in the present ELISA system, intestinal secretions from two rabbits which had been immunized in isolated ileal Thiry-Vella loops with Shigella X16 only or with Shigella X16 and P. aeruginosa were studied. Assays for IgG and IgA anti-X16 LPS
were performed as described above, using a 1:20 dilution of loop fluid in PTA. Assays for IgG and IgA anti-PA-LPS were performed on tubes coated by solutions containing 10 ,Rg of PA-LPS per ml in coating buffer. A 1:20 dilution of loop fluid was also used in the PA-LPS assays. Rabbit 1 (Table 2) immunized with Shigella X16 only had a readily detectable IgA response to X16 LPS; rabbit 2, which received both organisms, had strong IgA anti-PA-LPS on days when no reaction was present to X16 LPS. In addition, absorption studies were performed to insure that the ELISA reactions represented specific responses to the LPS coating the tube walls. IgA anti-X16 was mixed with either X16 LPS or PA-LPS and then reacted in the ELISA system using tubes coated with X16 LPS. The IgA anti-X16 activity was completely removed by preincubation with 1 mg of X16 LPS per ml, whereas no activity was lost by preincubation with 1 mg of PA-LPS per ml (Table 3). IgG anti-X16 activity could also be removed by specific LPS (data not shown). Effect of competition between specific IgG and IgA antibodies for antigen. To determine whether interference of binding by one class of specific antibody occurs when large amounts of another class of specific antibody are present, various dilutions of purified IgA antiX16 were mixed with several dilutions of IgG anti-X16. Each mixture reacted with tubes coated with X16 LPS and then with either APGARA (1:320 in PTA) or AP-GARG (1:640 in PTA). All studies yielded similar results; data from one such study are shown in Table 4. No decrease in IgA anti-X16 activity was seen in the specimen diluted 1:40 even when mixed with strongly reactive IgG anti-X16. Although the 1: 20 dilution of IgA anti-X16 had a slightly deTABLE 2. Antigenic specificity of ELISA
OD4e/100 mina Rabbit no.
Antigen given
Day
ELISA IgA ELISA IgA anti-Shianti-P. gella X16 aeruginosa 1 Shigella X16 0
AND IMMUNITy,
May 1979,
p.
441-448
Vol. 24, No. 2
0019-9567/79/05-0441/08$02.00/0
Enzyme-Linked Immunosorbent Assay for Immunoglobulin G and Immnunoglobulin A Antibodies to Shigella flexneri Antigens DAVID F. KEREN' Department of Bacterial Diseases, The Walter Reed Army Institute of Research, Washington, D.C. 20012, and Department of Pathology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109' Received for publication 5 March 1979
An enzyme-linked immunosorbent assay has been developed to detect classspecific antibodies to Shigella flexneri lipopolysaccharide antigens. This enzymelinked immunosorbent assay system has been used to measure antibodies present in serum or intestinal secretions without further purification. It is considerably more sensitive than passive hemagglutination, allowing detection of as little as 1.3 ng of specific immunoglobulin G antibody per ml in immune sera. Optimal conditions for this assay are outlined in this report.
The enzyme-linked immunosorbent assay (ELISA) has been shown to be a highly sensitive technique capable of detecting antibodies to a wide variety of antigens (4-6, 8-10, 13, 18). This technique has the following advantages: the enzyme conjugates and the substrate reaction products are stable for long periods of time, results can be read visually or on a spectrophotometer, and ELISA lacks the potential radiation hazards of radioimmunoassay. In this report an ELISA technique is used to measure relative amounts of isotypically specific antibodies against the lipopolysaccharide (LPS) extracted from a hybrid of Shigella flexneri and Escherichia coli (Shigella X16). The ELISA technique used in this study is a modification of our earlier procedure which did not directly relate the absorbance units from the substrate reaction to specific antibody against Shigella X16 LPS (X16 LPS) in gravimetric terms (13, 18). Data from the present study (i) establish sensitivity and specificity of the technique, (ii) assess parameters affecting the ELISA system, and (iii) compare this ELISA to passive hemagglutination and quantitative precipitation by using immunoglobulin G (IgG) fractions from rabbit antisera to X16 LPS. MATERIALS AND METHODS Goat antisera. Goat anti-rabbit IgG (GARG) was obtained by immunizing a goat with 6.6 mg of Fc portion from rabbit IgG in complete Freund adjuvant. Serum was collected weekly and tested for GARG activity by gel diffusion. Specific GARG antibodies were absorbed onto rabbit IgG insolubilized by glutaraldehyde (3). After washing this IgG sorbent with saline, the GARG antibodies were eluted with 0.1 M glycine-HCl buffer, pH 2.8. Na2CO3 was added to
adjust the pH to 8.0. Goat anti-rabbit IgA (GARA) was obtained by immunizing a goat with 8 mg of rabbit IgA in complete Freund adjuvant. Sera were collected weekly and tested for GARA activity by gel diffusion. The globulin fraction was precipitated from serum by saturated ammonium sulfate as described by Herbert et al. (11) and dialyzed against three changes of 0.01 M phosphate buffer, pH 8.0. Antibodies cross-reacting with IgG were removed by passing this material over a 14ml column of Sepharose 4B covalently coupled to 24.5 mg of rabbit IgG. Specific antibodies to IgA were then absorbed onto a 10-ml column of Sepharose 4B coupled to 30 mg of rabbit IgA. These antibodies were eluted with 0.01 N NaOH in saline and quickly neutralized with 0.1 N HCI. GARG and GARA were concentrated by negativepressure dialysis to 3.9 and 7.1 mg/ml, respectively. They were conjugated to alkaline phosphatase (AP) (type VII, Sigma Chemical Co., St. Louis, Mo.) using the technique of Avrameas (2) as modified by Yardley et al. (18). Preparation of antigens for coating tubes. Rabbit IgG was prepared by using the third precipitate in 35% saturated (NH4)2S04 from rabbit serum (11). After the redissolved globulins were dialyzed against 0.01 M phosphate buffer, pH 8.0, they were applied to a diethylaminoethyl-cellulose column equilibrated with the same buffer. The proteins which passed through the column unimpeded were concentrated as the IgG standard. Rabbit IgA was prepared from intestinal secretions collected from chronically isolated ileal (Thiry-Vella) loops in rabbits (12). This fluid has been shown to contain mainly IgA with some IgG and little IgM (15). The fluid was clarified by centrifugation at 3,000 rpm for 15 min, and the globulin fraction was precipitated by slow addition of a saturated ammonium sulfate solution (neutralized to pH 7.8 by 1 N NaOH) until 50% saturation was achieved. After removal of the supernatant, the precipitate was dissolved in phos-
441
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INFECT. IMMUN.
KEREN
phate-buffered saline, pH 7.2 (PBS). After a second ammonium sulfate precipitation at 50% saturation, the protein was dissolved in 0.04 M phosphate buffer, pH 8.0, and dialyzed against three changes of this same buffer. Aggregates were removed by centrifugation at 3,500 rpm for 15 min in the cold. The protein was applied to a diethylaminoethyl-cellulose column equilibrated in 0.04 M phosphate buffer, pH 8.0. This column was washed extensively with 0.04 and 0.1 M phosphate buffers, pH 8.0. The protein eluted from the diethylaminoethyl with 0.2 M phosphate buffer was concentrated to 0.6 mg/ml by negative-pressure dialysis and shown to contain only IgA by gel diffusion and ELISA. This material was shown to be quantitatively equivalent in the ELISA to a standard IgA preparation (kindly provided by Stella M. Robertson from the laboratory of John J. Cebra) before use. X16 LPS and Pseudomonas aeruginosa LPS (PALPS) were prepared by the hot phenol-water method (17). These preparations contained less than 3.5% protein and from 3 to 5% nucleic acid. Rabbit antisera to X16 LPS. Sera from rabbits immunized intravenously with the parent strain of the S. flexneri-E. coli hybrid were kindly provided by S. B. Formal. IgG fractions of one antiserum (anti-X16) were prepared as described above for preparation of rabbit IgG. The fraction that passed through the diethylaminoethyl column with 0.01 M phosphate buffer (IgG anti-X16 fraction I) and the fraction which was eluted from the column with 0.04 M phosphate buffer (IgG anti-X16 fraction II) were shown to contain only IgG by gel diffusion and ELISA. Intestinal secretions (from Thiry-Vella loops) from several rabbits with previously demonstrated IgA antiX16 activity (13) were pooled, and the IgA fraction was prepared as described above. Passive hemagglutination. Sheep erythrocytes in Alsever solution were washed three times in 0.01 M PBS, pH 7.2. To determine optimal sensitization, various concentrations of X16 LPS from 10 ng/ml to 100 ,tg/ml were incubated with a 2% solution of sheep erythrocytes for 1 h at room temperature with occasional agitation. The sensitized sheep erythrocytes were washed three times with saline and diluted to a 1% suspension in PBS. Several twofold dilutions of serum were prepared with PBS. Into each well of a microtiter plate (Linbro Chemical Co., New Haven, Conn.) containing 25 ,ul of the serum, an equal volume of sensitized sheep erythrocytes was added. The microtiter plates were incubated for 1 h. The hemagglutination titer was the last well showing grossly visible hemagglutination. Sera with unsensitized sheep erythrocytes were run as controls. The optimum sensitizing dose was found to be 10 ag/ml. Quantitative precipitin assay. Both IgG antiX16 fractions I and II were studied for specific antibody content. The tests were performed in 0.5-ml polyvinyl tubes (Evergree- Scientific, Los Angeles, Calif.) by using 0.1 ml of fraction I or II with 0.1 ml of PBS (with 0.1 mg of Merthiolate per ml) containing 0, 1, 2, 4, 8, 16, 32, and 64 ,ug (by weight) of X16 LPS. These tubes were incubated at 37°C for 1 h and then at 4°C for 8 days with occasional agitation. The precipitates were sedimented by centrifugation at 11,600 rpm for 1 h on a Beckman Microfuge B (Beckman
Instruments, Inc., Palo Alto, Calif.). As the precipitates obtained with LPS antigen were gelatinous, the supernatants were removed to 0.01 ml and were carefully washed twice in 0.1 ml of ice-cold PBS each, followed by another centrifugation for 1 h. Protein content was determined by the method of Lowry et al. (14). All reactions were performed in duplicate. ELISA procedure. ELISA was performed as described by Engvall et al. (8-10). Disposable polystyrene tubes (12 by 75 mm; Falcon Plastics, Oxnard, Calif.) were incubated with 1 ml of solution containing antigen in 0.05 M carbonate buffer, pH 9.6 (coating buffer), for 3 h at 37°C except where otherwise noted. Tubes containing the antigen solution were covered with Parafilm (to prevent evaporation) and stored at 4°C until use. Immediately before testing, the antigen solution was removed and the tubes were washed four times (5 min with agitation per wash) with PBS containing 0.05% Tween 20 (Fisher Scientific Co., Pittsburgh, Pa.) and 0.02% sodium azide (PTA). The rabbit sera or IgG fraction of the sera to be tested were diluted 1:20 (or as stated) in PTA, and 0.5 ml was added to the tubes coated with antigen and to uncoated tubes (as a control for nonspecific adsorption). After the tubes were incubated at room temperature for 4 h (except where otherwise stated) on a horizontal rotary shaker, they were again washed four times with PTA, and 0.5 ml of AP conjugated to GARG (APGARG) or to GARA (AP-GARA) was added to the appropriate tubes. The reaction mixtures were incubated for 18 h (except where otherwise stated) at room temperature with agitation. After another series of four washes with PTA, 1 mg of the substrate, p-nitrophenyl phosphate disodium (Sigma Chemical Co.), in 1 ml of 0.05 M carbonate buffer (pH 9.8) containing 0.001 M magnesium chloride was added to each tube. The reaction was developed at room temperature until adsorbance at 400 nm was approximately 1.0 (the enzyme-substrate reaction is linear to this level [101), being stopped at 25, 50, or 100 min by the addition of 1 ml of 0.2 M NaOH. All results were extrapolated to 100 min (10). The optical densities of the uncoated tubes at 400 nm were subtracted from those of the coated tubes containing the same solutions. All reactions were performed in duplicate.
RESULTS
Optimal antigen coating of tubes. To determine the optimal X16 LPS concentration for coating tubes, varying concentrations of X16 LPS in coating buffer were incubated for 3 h at 37°C. These tubes were tested with a 1/104 dilution of the rabbit antisera to X16 LPS. Optimal sensitization was obtained at an antigen concentration of 10 ,ug/ml (Fig. 1). At higher concentrations, there was a decrease in the absorbance. The adsorption of the LPS occurs quickly, as no difference was observed between tubes coated for 5 min at 37°C and those coated for 24 h.
Specificity and optimal dilution of APGARG and AP-GARA. To determine the isotypic specificity of the AP-GARG and AP-
VOL. 24, 1979
ELISA FOR ANTIBODIES TO SHIGELLA ANTIGENS
6
443
a
c
5 E 0 0 0 0
a
A4 0
E
10-3 1id2 Clo
1O' 102
0
,sg LPS/ml
z
FIG. 1. Determination of optimal LPS concentration for coating tubes. Tubes were coated for 3 h at 370C with the concentrations of Shigella X16 LPS indicated on the abscissa, followed by incubation with antisera to Shigella X16 diluted 1/104. Enzymeanti-IgG conjugate was diluted 1:160. OD, Optical density.
GARA, various concentrations of rabbit IgG and IgA in coating buffer were used to coat polystyrene tubes. These tubes were washed four times with PTA, and 0.5 ml of AP-GARG (diluted 1: 640 in PTA) or AP-GARA (diluted 1:320 in PTA) was added to the appropriate tubes and incubated for 18 h at room temperature. After another four washes with PTA, the substrate reaction was carried out as described above. Tubes coated with solutions containing as little as 62 ng of rabbit IgG or IgA per ml gave detectable reactions with AP-GARG and APGARA, respectively (Fig. 2). No cross-reactivity was detectable with tubes coated by solutions containing up to 10 ,Ag of immunoglobulin per ml. The working dilution for each batch of APGARG and AP-GARA was determined by reacting tubes coated by solutions containing 1 tLg of immunoglobulin per ml with various dilutions of the phosphatase-conjugated reagents. The dilution which would give an increase in optical density at 400 nm of 5 to 10 was chosen (H.
Carlsson, personal communication). Kinetics of primary antibody binding. The rate of binding of rabbit anti-X16 to X16 LPS was investigated by using tubes coated with solution containing 10 jig of X16 LPS per ml. Rabbit antisera to X16 diluted to 1/104, 1/5 x 104, and 1/105 were used to determine if the kinetics of primary antibody binding was related to antibody concentration. The antisera were allowed to react with the antigen-coated tubes for varying periods of time. Thereafter, the tubes were washed and AP-GARG was allowed to react for 18 h, followed by the substrate reaction. The primary antibody was completely bound
0
1 ---J.L-S
A,
-
0.007 0.015 0.031 0.062 0.125 0.25 0.5 1 jlg/ml in Coating Solution 6
k g. i
ii{ I I
10
k--10
5
4
c0 6
2
1
.
0L-
A----O
0.017 0.015 0.031 0.062 0.125 0.25 0.5 1 10 jug/ml in Coating Solution FIG. 2. (a) Specificity of enzyme-conjugated antiIgG (AP-GARG). AP-GARG diluted 1:640 gave a typical reaction curve with tubes coated with IgG (0) (concentrations of coating solution on abscissa), but not with tubes coated with IgA (-). (b) Specificity of enzyme-conjugated anti-IgA (AP-GARA). APGARA diluted 1:320 gave a typical reaction curve with tubes coated with IgA (0), but not with tubes coated with IgG (0). O.D., Optical density.
after 4 h of incubation with coated tubes, regardless ofthe concentration of the antisera (Fig. 3). No increase in reactivity was seen when incubation was extended to 24 h.
444
INFECT. IMMUN.
KEREN 4.5
4.0 3.5
3.0 C
.E
082.5 E C!
0 0
2.0
c0 1.0
7 8 4 5 6 2 3 WITH ANTISERUM HOURS OF INCUBATION FIG. 3. Kinetics of the binding ofprimary antibodies to tubes coated with a solution containing 10 pg of LPS. Antisera to X16 LPS diluted 1/104 (0), 1/5 x 104 (0), and i/10i (x) were incubated in the LPS-coated tubes for the time indicated on the abscissa and thereafter for 18 h with AP-GARG diluted 1:160. OD, Optical density.
Kinetics of binding of AP conjugate (secondary antibody). The rate of binding of APGARG was investigated by using tubes coated by a solution containing 10 jig of X16 LPS per ml reacted with one of two concentrations of primary antibody (1/10' and 1/105). The primary antibodies were allowed to react for 4 h. AP-GARG (diluted 1:160 in PTA) was allowed to react for varying periods of time. Reactions were begun at intervals which allowed for the substrate reactions to be carried out simultaneously. By 4 h the conjugate reaction was complete for both concentrations of primary antibody used (Fig. 4). No increase in reactivity was found with incubations of AP-GARG as long as 28 h. For practical purposes, incubations with AP-GARG in the present test system are usually carried out overnight for 18 h. Reproducibility. Using the optimum conditions described above, 10 replicate samples of serum from a rabbit inmunized with Shigella
X16 were assayed. A dilution of 1/104 gave a mean of 1.632 ± 0.037 (standard error of the mean), whereas dilutions of 1/105 gave a mean of 0.169 ± 0.012 (standard error). Eight replicate samples of IgA anti-X16 diluted 1/40 yielded a mean of 2.225 ± 0.045 (standard error). Quantitative precipitin test. To determine the sensitivity of the present ELISA test, the amount of precipitating antibody in standard rabbit sera to X16 LPS was determined by the quantitative precipitin test. The rabbit sera had some IgM activity against X16 LPS as demon-
strated by hemagglutination studies (Table 1). The hemagglutination titer of the serum was 1: 5,120 without 2-mercaptoethanol, but the addition of 2-mercaptoethanol reduced this titer to 1:640. Therefore, since the present ELISA system detects isotypically specific antibodies to X16 LPS, it was necessary to purify IgG fractions of this serum. Both IgG anti-X16 fractions I and II had titers of 1:640 regardless of the presence
VOL. 24, 1979
ELISA FOR ANTIBODIES TO SHIGELLA ANTIGENS
445
of 2-mercaptoethanol (Table 1). In addition, by gel diffusion and ELISA these fractions were shown to contain only IgG. The results of the .s 3.5 quantitative precipitin test on the IgG anti-X16 8 3.0 fractions are shown in Fig. 5, and the specific E 2.5 antibody contents are noted in Table 1. o 2.0 . Sensitivity and isotypic specificity. The 1.5 quantitative precipitin test results were compared to those obtained when several dilutions 1.0 v '-o of the rabbit sera and the IgG anti-X16 fractions .5 were analyzed by ELISA (Fig. 6a). 0 The level of 0.5 optical density unit at 400 nm 34 2 56 7 8 28 was arbitrarily chosen as the lower limit of senHOURS OF INCUBATION WITH AP '-GARG sitivity of the present ELISA system, since at FIG. 4. Kinetics of the binding of sect this level the results are approximately five body (AP-GARG). Tubes coated with a d,ohain antwcons- times greater than the usual background level taking 10 pg ofLPS per ml were reacted awt (which varied between 0.08 and 0.13). The senantisera to X16 diluted 1/104 (0) or 1/ (0) sitivity for the present ELISA test for IgG antithen with AP-GARG (diluted 1:160) fl or the indicated on the abscissa. O.D., Optical density. X16 fraction I was 1.3 ng/ml, and for the IgG anti-X16 fraction II it was 3.5 ng/ml (Table 1). TABLE 1. Sensitivity ofELIS'A Although insufficient purified IgA anti-X16 was obtained to perform a quantitative precipiHemaggluti- Specific Serum di- Antbody tin test, ELISA was performed to determine the nation titer Source of antilution t(ng) of the AP-GARA and AP-GARG respecificity 0.5 anti-X16 No 2- With body give 0 under the conditions of the assay. VirA EUSA agents MEb 2-ME (mg/ml) ELIS tually no cross-reaction occurs between the IgA ND Serum ND` 1/6 x 105 5,120 640 anti-X16-purified fraction and AP-GARG or Fraction I 640 640 0.26 1/2 x o5 3 3 with the IgG-purified fractions and AP-GARA Fraction II 640 640 0.35 W 3.5 1/1 x 6a and b). ~(Fig. ° Determined by quantitative precipitation. Antigenic specificity of EITISA. To deterb 2-ME, 2-Mercaptoethanol. mine whether antibodies to unrelated gram-neg'ND, Not done. s
-
-
-
-
itfo tiaes
35
30
C2 25tS 0. CL h.
%.
,20
5
.
.
.
.
10 I5 20 25 30 35 40 45 50 55 60 65 pg Lipopolysoccharide Added
FIG. 5. Quantitative precipitation curves ofShigella X16 LPS with two IgG fractions: 0.01 M IgG anti-X16 fraction I (0); 0.04 M IgG anti-X16 fraction II (0).
446
INFECT. IMMUN.
KEREN
a 5
c
AI
8 E
E c
3
§ so 2
1
an
0
No
Antisera
-Loq Dilution
4
c
b
3
0
E 2
-
CD CD c
0o
E5 2
0o
0
1
2 3 4 -Log Dilution
5
6
FIG. 6. Titration of specific antisera against Shigella X16 by ELISA. Tubes coated with a solution containing 10 pg of LPS and reacted with the indicated dilutions (on abscissa) of rabbit antiserum to Shigella X16 (A), IgG anti-X16 fraction I (-) and IgG anti-X16 fraction II (-), IgA anti-X16 (5), or with buffer only (0). Tubes were then incubated with either AP-GARG diluted 1:320 (a) or with AP-GARA diluted 1:320 (b). O.D., Optical density.
ative bacteria would cross-react in the present ELISA system, intestinal secretions from two rabbits which had been immunized in isolated ileal Thiry-Vella loops with Shigella X16 only or with Shigella X16 and P. aeruginosa were studied. Assays for IgG and IgA anti-X16 LPS
were performed as described above, using a 1:20 dilution of loop fluid in PTA. Assays for IgG and IgA anti-PA-LPS were performed on tubes coated by solutions containing 10 ,Rg of PA-LPS per ml in coating buffer. A 1:20 dilution of loop fluid was also used in the PA-LPS assays. Rabbit 1 (Table 2) immunized with Shigella X16 only had a readily detectable IgA response to X16 LPS; rabbit 2, which received both organisms, had strong IgA anti-PA-LPS on days when no reaction was present to X16 LPS. In addition, absorption studies were performed to insure that the ELISA reactions represented specific responses to the LPS coating the tube walls. IgA anti-X16 was mixed with either X16 LPS or PA-LPS and then reacted in the ELISA system using tubes coated with X16 LPS. The IgA anti-X16 activity was completely removed by preincubation with 1 mg of X16 LPS per ml, whereas no activity was lost by preincubation with 1 mg of PA-LPS per ml (Table 3). IgG anti-X16 activity could also be removed by specific LPS (data not shown). Effect of competition between specific IgG and IgA antibodies for antigen. To determine whether interference of binding by one class of specific antibody occurs when large amounts of another class of specific antibody are present, various dilutions of purified IgA antiX16 were mixed with several dilutions of IgG anti-X16. Each mixture reacted with tubes coated with X16 LPS and then with either APGARA (1:320 in PTA) or AP-GARG (1:640 in PTA). All studies yielded similar results; data from one such study are shown in Table 4. No decrease in IgA anti-X16 activity was seen in the specimen diluted 1:40 even when mixed with strongly reactive IgG anti-X16. Although the 1: 20 dilution of IgA anti-X16 had a slightly deTABLE 2. Antigenic specificity of ELISA
OD4e/100 mina Rabbit no.
Antigen given
Day
ELISA IgA ELISA IgA anti-Shianti-P. gella X16 aeruginosa 1 Shigella X16 0
