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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 1995, p. 177–181 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 2, No. 2

A Novel Method To Chemically Immobilize Antibody on Nylon and Its Application to the Rapid and Differential Detection of Two Vibrio parahaemolyticus Toxins in a Modified Enzyme-Linked Immunosorbent Assay TAKESHI HONDA,1* TOSHIO MIWATANI,2 YASUNORI YABUSHITA,3 NORIO KOIKE,3 AND KEISHI OKADA3 Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka 5651; Faculty of Health and Welfare Science, Okayama Prefectural University, Kuboki Sojya, Okayama 719-112; and Research and Development Center, Unitika Ltd., Uji-Kozakura, Uji, Kyoto 611,3 Japan Received 28 June 1994/Returned for modification 23 August 1994/Accepted 2 November 1994

A new method of chemically immobilizing antibody on nylon was developed. The method consists of serial treatments with HCl, polyethylene imine, and maleic anhydride methylvinyl ether copolymer, which resulted in the stable immobilization of sufficient amounts of antibodies on nylon. This principle was used to differentially detect two immunologically related but nonidentical hemolysins (thermostable direct hemolysin [TDH] and TDH-related hemolysin [TRH]) of Vibrio parahaemolyticus in a modified enzyme-linked immunosorbent assay with antibodies immobilized on nylon slips (NSIT). The results (dark purple color on nylon slips) were easily evaluated by the naked eye. The results with NSIT were compatible with those obtained by using DNA probes or a conventional bacterial culture test, not only with cultured specimens but also with clinical specimens (diarrheal stool samples). Furthermore, the NSIT differentially detected TDH and TRH in a single test. The antibody immobilization method developed here is applicable to various immunological detection methods and may improve their sensitivity and specificity. pends upon nonspecific physical adsorption of antibody for antigen capture, and the antigen-captured antibody may easily detach from the ELISA plate, resulting in difficulty in washing and rather low sensitivity. To overcome these problems, we developed a new method of chemically immobilizing antibody on an ELISA plate or polymeric sheet instead of using nonspecific physical adsorption to polymers. This improvement enabled visual detection of toxins more rapidly (within 1 h) and differential detection of several toxins in a single test. We applied this principle to the direct differential detection of the

The detection and identification of pathogenic organisms usually depend on classical tests such as phenotypic, biochemical, and morphological tests. However, knowledge of pathogenic mechanisms, especially virulence factors, has been accumulated, and the detection of virulence factors recently became absolutely necessary for the accurate identification of pathogens. For example, even when Escherichia coli is isolated from a patient with diarrhea, this does not necessarily mean that the diarrhea was caused by the E. coli, unless it is determined whether either heat-labile (LT) or heat-stable (ST) enterotoxins are produced (3). Vibrio parahaemolyticus is another example. Thermostable direct hemolysin (TDH) and TDHrelated hemolysin (TRH) are virulence factors of V. parahaemolyticus (5). Strains which produce TDH/TRH are mostly isolated from clinical specimens, and those which do not produce either are usually isolated from environmental sources, such as saltwater fish, shrimp, and shells (5). Thus, TDH and TRH or their genes have been considered markers of pathogenicity. The detection of virulence factors such as these has become increasingly important. There is also a need for a faster and more effective method of diagnosing gastrointestinal infections. Various techniques are available for detecting these virulence factors or their genes. These include biological assays and gene detection assays, such as DNA hybridization and PCR (2), as well as immunological assays (12). Each method has advantages and disadvantages. Immunological assays are the most convenient for clinical diagnostic laboratories. However, they also have several disadvantages. For example, an ordinary enzyme-linked immunosorbent assay (ELISA) de-

FIG. 1. Example of an NSIT result. The size of the slips can be estimated by comparison with the Eppendorf tube. Positive results (dark purple) are visible on slips A (immobilized MAb common to both TDH and TRH) and B (immobilized MAb specific to TDH), but not on slip C (immobilized MAb specific to TRH), indicating the presence of TDH in the specimen.

* Corresponding author. Mailing address: Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka 565, Japan. Phone: 06-879-8276. Fax: 06-879-8277. 177

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FIG. 2. Schematic representation of the chemical immobilization of MAbs on nylon membranes and detailed conditions. This procedure allows the binding of multiple MAbs onto the nylon. PEI, polyethylene imine; DCC, N,N9-dicyclohexylcarbodiimide.

immunologically related but nonidentical TDH and TRH toxins not only in culture supernatant but also in diarrheal stool samples to rapidly diagnose V. parahaemolyticus gastroenteritis. MATERIALS AND METHODS Polymeric materials. Nylon (nylon 6), polyethylenevinyl-acetate copolymer, polyurethane, and polyvinyl chloride, purchased from Unitika Co., DuPont Mitsui Polychemical Co., Thermedics Co., and Tigers Polymer Co., respectively, were used as antibody coat carriers (slips). These slips measured 8 mm in length by 2 mm in width by 0.2 mm thick (Fig. 1). Purification of TDH and TRH toxins. TDH and TRH were purified from cultured clinical isolates of Kanagawa phenomenon-positive and -negative V. parahaemolyticus strains, respectively, as described previously (6, 8). Bacterial strains, culture, and DNA probe hybridization test. A total of 102

strains of V. parahaemolyticus were isolated from patients with traveler’s diarrhea at the Osaka Airport Quarantine Station (72 clinical strains) and from imported frozen seafoods at the Kobe Quarantine Station (30 environmental strains). Each test strain was cultured as described before (6, 8). DNA probe hybridization for identifying the genes (tdh and trh) encoding TDH and TRH was performed as described before (15). Antibodies. Polyclonal antibodies (PAb) against purified TDH or TRH were developed in rabbits as described before (6, 8). Immunoglobulins were partially purified by ammonium sulfate fractionation (50% saturation). Monoclonal antibodies (MAbs) against both TDH and TRH were prepared as described before (4, 7). MAbs that reacted with only either TDH (MAb 1-24) or TRH (MAb 21G) and with both TDH and TRH (common MAb; MAb 2A) were screened by ELISA with TDH- and TRH-coated ELISA plates as described before (4, 7). Immobilization of antibody. The following procedures which have been used previously for immobilization of enzymes (14) were used with some modifications for immobilizing the antibodies on individual polymeric materials. Polyethylenevinyl-acetate slips were immersed in 15% (wt/vol) NaOH in 80% (vol/vol)

VOL. 2, 1995

ELISA WITH CHEMICALLY IMMOBILIZED ANTIBODY

TABLE 1. NSIT procedure for differential detection of TDH and TRHa Step

Procedure

1 ...................Dip the tip of nylon slips on which MAbs have been immobilized in test samples (TDH or TRH) at 378C for 15 min. 2 ...................Wash the slips three times in phosphate-buffered saline (PBS)–0.05% Tween 20 in an Eppendorf tube for about 1 min. 3 ...................Dip the slips in a 500-fold diluted mixture of anti-TDH and anti-TRH rabbit sera at 378C for 15 min. 4 ...................Wash the slips three times with PBS-Tween 20 for about 1 min. 5 ...................Dip the slips in 500-fold diluted alkaline phosphataselabeled anti-rabbit IgG (Organon Teknika Co., Durham, N.C.) at 378C for 15 min. 6 ...................Wash the slips three times with PBS-Tween 20 for about 1 min. 7 ...................Incubate the slips in substrate (0.25 mM nitroblue tetrazolium plus 5-bromo-4-chloro-3-indolylphosphate) at 378C for 10 min. a

All steps can be completed within 60 min.

methanol at 508C for 2 h, washed with water, heated in 2% (vol/vol) amino-acetal in 0.5 N HCl at 588C for 5 h, washed and dried, soaked in 1% (wt/vol) maleic anhydride methylvinyl ether copolymer (MAMEC) in water-free acetone at 258C for 2.5 h, and dried. Polyurethane slips were heated in water at 808C for 6 h, washed with distilled water, dried, treated in 1% (wt/vol) MAMEC in water-free acetone at 258C for 2.5 h, washed with acetone, and then dried. Polyvinyl chloride slips were immersed in 1% (wt/vol) MAMEC in water-free acetone at 258C for 2.5 h, washed with acetone, and then dried. MAMEC was introduced onto nylon slips as described in Fig. 2. After MAMEC was introduced onto various polymeric slips, MAbs (protein concentration, 2 mg/ml) suspended in phosphate-buffered saline were immobilized on the slips at 258C overnight. Rabbit ileal loop test. The ileal loop test with live V. parahaemolyticus strains was performed as described by Twedt et al. (11). The loops were challenged with live cells (about 108/ml) of three V. parahaemolyticus strains (a TDH-producing strain, T4750; a TRH-producing strain, TH4037; and a TDH- and TRH-producing strain, TH3766) suspended in brain heart infusion broth supplemented with 0.5% NaCl (4). After a 14-h incubation, the accumulated intestinal fluids were centrifuged (15,000 3 g for 30 min), and the supernatant was used. Diarrheal stool samples. A total of 60 stool samples from patients with traveler’s diarrhea were collected at the Osaka Airport Quarantine Station. V. parahaemolyticus was isolated from 10 specimens by conventional culture. Nine of these were TDH producers, and one produced both TDH and TRH (13).

RESULTS Polymeric materials for immobilization of antibody. We immobilized rabbit antibodies on various polymeric materials, including nylon sheets. The amount of immobilized immunoglobulin (antibody) was measured by using anti-rabbit immunoglobulin labeled with alkaline phosphatase (plus substrate for color development). Polyurethane, ethylene vinyl acetate, nylon, and polyvinyl chloride were tested. We found that MAMEC treatment of all four materials significantly increased the degree of antibody immobilization (Fig. 2). Nylon (optical density at 405 nm [OD405] in the ELISA, ^2.0) was the best, followed by polyvinyl chloride (ELISA value, 1.0 % OD , 2.0), polyurethane (ELISA value, 0.2 % OD , 1.0), and polyethylenevinyl-acetate (ELISA value, 0.2 % OD , 1.0). We therefore selected MAMEC-treated nylon for further study. Test procedure and differential detection of purified toxins. The quickest and most satisfactory results were obtained by using the procedure shown in Table 1. The slips can be handled by fingers or tweezers. It is possible to test more than one sample at a time, and all reagents can be used simultaneously for both TDH and TRH detection. All procedures can be completed within 60 min after receipt of samples.

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TABLE 2. Differential detection of TDH and TRH by NSIT MAb

Degree of positive staininga at hemolysin concn (ng/ml)

Hemolysin 100

10

1

0

2A

TDH TRH

111 111

111 111

11 11

2 2

1-24

TDH TRH

111 2

111 2

11 2

2 2

21G

TDH TRH

2 111

2 111

2 11

2 2

a 111, 11, and 1 represent marked, moderate, and weak staining, respectively.

Antibody immobilization on the nylon slips was designed to be like that shown in Fig. 1 (we designated this test NSIT for nylon slip immunotest). MAbs specific to either TDH or TRH or common to TDH and TRH were immobilized on the slips. Only the tip of the brush slips was dipped in samples (either bacterial culture or diarrheal stool samples directly). Further steps were done in an Eppendorf tube, as summarized in Table 1. When samples contained TDH, a dark purple color developed on the tip of the brush slips (A and B in Fig. 1) that had immobilized MAb specific for TDH (B in Fig. 1) or the MAb common to TDH and TRH (A in Fig. 1). The sensitivity and specificity of the NSIT prepared as described above were examined with purified TDH and TRH. As summarized in Table 2, the NSIT detected as little as 1 ng of either TDH or TRH per ml, as visualized by the naked eye. It also simultaneously differentiated between TDH and TRH in a single test. Comparison of results obtained by NSIT and DNA hybridization. A total of 102 strains of V. parahaemolyticus were tested to determine whether they possess tdh and/or trh or produce TDH and/or TRH by means of DNA probe hybridization and NSIT, respectively. The NSIT was performed with supernatants obtained from a culture in SSP broth for 18 h at 378C. The results are compared in Table 3. Only a few discrepancies were found between the two assays. Thus, the NSIT was sufficiently sensitive (63 of 66 [95.5%]) and perfectly specific (100%). Application of NSIT for detection of TDH and TRH in stool specimens. Furthermore, we used the NSIT test to the detection and identification of TDH and TRH in intestinal fluids in an animal model (rabbit ileal loop test) challenged with various V. parahaemolyticus strains. We found that the NSIT detected and differentiated TDH and TRH in the intestinal fluids. When the TDH-producing strain was used for the challenge, only the TDH-specific MAb (and common MAb)-immobilized brush slip became positive when dipped in the intestinal fluids (n 5 3), whereas only TRH was detected in fluids induced by

TABLE 3. Comparison of NSIT and DNA probe hybridization test for differential detection of TDH and TRH of V. parahaemolyticus Method

a

NSIT DNA probe test a

TDH/TRH or tdh/trh detected (no. of samples)

No. of strains tested

102 102

TDH

TRH

TDH and TRH

Neither

53 54

6 6

4 6

39 36

Sensitivity (63 of 66), 95.5%; specificity, 100.0%.

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TABLE 4. Direct, differential detection of TDH and TRH of V. parahaemolyticus in diarrheal stool samples by NSIT Isolation of V. parahaemolyticus (no. of samples)

Genes possessed by the isolates

Toxin detected by NSIT

No. of strains isolateda

Positive (10)

tdh tdh tdh/trh

TDH TDH/TRH TDH

8* 1 1

Negative (50)

NTb NT NT

None TDH TDH/TRH

46** 2 2

a The following enteropathogens were recovered from the indicated number of cases: *, Salmonella spp. and Vibrio cholerae non-O1 (one case) and Plesiomonas shigelloides (one case); **, Salmonella spp. (seven cases), Shigella spp. (two cases), and P. shigelloides (four cases). b NT, not tested.

challenge with the TRH-producing V. parahaemolyticus strain (n 5 3). Moreover, both TDH and TRH were detected in intestinal fluids (n 5 3) induced by challenge with a TDH- and TRH-producing V. parahaemolyticus strain. These data prove that V. parahaemolyticus can produce TDH and TRH in vivo (intestinal) and also indicate that the NSIT is applicable to the direct differential detection of TDH and TRH in human diarrheal stool samples. We then tested the NSIT on clinical specimens. A total of 60 diarrheal stool samples were examined by NSIT, and the results are compared with those of conventional culture in Table 4. Eight of nine stool samples from which TDH-producing V. parahaemolyticus was isolated by conventional culture were positive for TDH by NSIT (sensitivity, eight of nine [88.9%]). Four of 50 diarrheal stool samples from which no V. parahaemolyticus was isolated were positive for either TDH or TDH and TRH by NSIT (specificity, 46 of 50 [92.0%]). DISCUSSION One basic problem of conventional ELISA is that the antibody for antigen capture is nonspecifically and physically adsorbed onto ELISA plates, and thus the antigen captured by the antibody may easily detach from the ELISA plates during subsequent processing. To improve this, we developed a novel method for chemically immobilizing antibody onto polymeric materials, especially nylon membranes (slips). Two chemical means of immobilizing antibody on ELISA plates have so far been reported. One method (9) uses hydrazide-treated plates treated with glutaraldehyde to introduce aldehyde residues, followed by NaCNBH2 with proteins (antibodies). Another (10) uses alkylamine plates and succinic anhydride to introduce COOH residues, followed by ethyl-3-carbodiimide HCl with proteins (antibodies). These two methods were used for antibody immobilization on nylon slips, and we found that the two methods gave a lower sensitivity (detection limit, 10 ng of TDH per ml) than the method described here. Our method can theoretically introduce more chemical hands (maleic anhydride residues) than those previously developed (Fig. 2). Moreover, antibody suspended in phosphate-buffered saline (pH 7.4) can be covalently bound without any further chemicals onto nylon slips with maleic anhydride residues, and thus our method provides milder conditions than those developed previously. Thus, we believe that our antibody immobilization technique is better than those described previously because of the increased degree of antibody immobilization and the decreased chemical damage to the antibody. This enabled solid

immobilization of antibody, which allowed vigorous washing to remove nonspecific binding materials and gave visible results (clear antibody-antigen reaction) on the membranes. A further benefit is that the slip with immobilized antibody can be stored even in a dried condition (data not shown). In this study, the slips with chemically immobilized antibodies not only detected TDH and TRH but also differentiated these two toxins. This chemical antibody immobilization principle can also be applied to conventional ELISA to increase the sensitivity (data not shown). However, we designed the NSIT as shown in Fig. 1 to enable differential detection of pleural toxins by a single test and reading of the result by the naked eye. If the number of slips (and antibodies) is increased, many toxins can be differentiated by a single test. Further studies along these lines are planned. Moreover, all NSIT procedures can be completed within 1 h after specimens are obtained. These features allow more rapid and simple differential detection of toxins than conventional ELISA. In conclusion, we have developed a novel chemical means of immobilizing antibody, which enabled the high-affinity binding of sufficient antibody onto polymeric materials, especially nylon. This technique will be widely applicable for many immunological tests, such as ELISA and latex agglutination. We applied this technique to the differential detection of the TDH and TRH toxins of V. parahaemolyticus in a modified ELISA with nylon slips (NSIT), and the results revealed a sensitivity of 88.9% and specificity of 92.0% in the direct and differential detection of TDH and TRH in diarrheal stool samples. The reason for a few discrepancies between the results of NSIT and culture will require additional experiments to be clarified, but the following three reasons came to mind: (i) the presence of the tdh or trh gene does not directly mean production of TDH or TRH (5), and thus the NSIT gave false-negative results for TRH detection in a sample from which trh-possessing V. parahaemolyticus was isolated; and false-positive results may occur because (ii) too few bacteria are present in the stool sample for recovering V. parahaemolyticus, which may result from ingestion of antibiotics, or (iii) other, unknown bacteria may produce immunologically cross-reactive toxins or materials. The latter is an interesting possibility, and further study along this line is planned. The detection of toxins by NSIT was simple (several toxins were differentially detected by a single test and the result can be read by the naked eye) and rapid (all procedures were completed within 1 h). Thus, we believe that the NSIT will be widely applicable to the rapid identification of various infectious agents. ACKNOWLEDGMENT This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Allen, S. D. 1992. Rapid methods and recent topics in clinical microbiology in the U.S.A. JARMAN 4:7–24. 2. Echeverria, P., O. Sethabutr, and O. Serichantalergs. 1993. Modern diagnosis (with molecular tests) of acute infectious diarrhea. Gastroenterol. Clin. North Am. 22:661–682. 3. Honda, T. 1992. Enteropathogenic Escherichia coli that cause food poisoning—current status of diarrheagenic E. coli. Asian Med. J. 35:259–267. 4. Honda, T., M. A. Abad-Lapuebla, Y. Ni, K. Yamamoto, and T. Miwatani. 1980. Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus. J. Gen. Microbiol. 137:253–259. 5. Honda, T., and T. Iida. 1992. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Rev. Med. Microbiol. 4:106–113.

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