Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.     

Improved GM1-enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

Previously described GM1 ganglioside enzyme-linked immunosorbent assays (GM1-ELISA) for the detection of Escherichia coli heat-labile enterotoxin (LT) showed sensitivity equal to the Y-1 adrenal cell assay when anti-LT (a reagent not commercially available) was used. However, when antitoxin to immunologically related (commercially available) cholera toxin was substituted, a marked loss in sensitivity occurred. We modified the GM1-ELISA that employed anti-cholera toxin to make it comparable in sensitivity to the Y-1 adrenal cell assay. When five media commonly used for LT production were compared, Mundell's Casamino Acids medium was shown to be significantly superior. Lincomycin (45 micrograms/ml) added to E. coli cultures significantly increased net optical densities in the GM1-ELISA, a direct measure of the amount of LT. Treatment of broth cultures or bacterial cell pellets with polymyxin B or extension of culture time to 48 h also significantly increased net optical density by allowing enhanced release of periplasmic LT. A major innovation involved the direct culture of E. coli strains in GM1-coated wells of microtiter plates followed by ELISA. This direct culture method GM1-ELISA (DCM-GM1-ELISA) saved not only assay time, but also materials and reagents. The net optical densities that result from this assay allow the test to be read visually without a spectrophotometer. Three independent observers read plates with E. coli tested by DCM-GM1-ELISA. Thirty-four of 35 adrenal cell-positive strains (97% sensitivity) and 30 of 30 LT-negative control E. coli strains (100% specificity) were identified by all three observers reading coded plates. The DCM-GM1-ELISA provides a simple, practical and efficient assay for LT for less sophisticated laboratories.     

GM1 ganglioside enzyme-linked immunosorbent assay for detection of heat-labile enterotoxin produced by human and porcine Escherichia coli strains.

Human and porcine enterotoxigenic strains of Escherichia coli were cultivated in tryptone-yeast extract medium or brain heart infusion broth and tested for production of heat-labile enterotoxin by the GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) and the Y1 adrenal cell assay. When testing for enterotoxigenicity by the GM1-ELISA technique, homologous antisera for human and porcine heat-labile enterotoxins had to be used to detect enterotoxigenic strains of human and porcine origin, respectively. This observation indicates a serological difference between the heat-labile enterotoxins produced by human and porcine strains. Furthermore, brain heart infusion broth was found to have an inhibitory effect on detection of enterotoxin both in the GM1-ELISA and in a toxin-binding modification of the Y1 adrenal cell test, but not in the conventional adrenal cell assay.     

Microtiter ganglioside enzyme-linked immunosorbent assay for vibrio and Escherichia coli heat-labile enterotoxins and antitoxin.

We have developed a microtiter enzyme-linked immunosorbent assay method for detecting the heat-labile enterotoxins of Vibrio cholerae and Escherichia coli using GM1 ganglioside as the base coat. This method compares favorably with a similar assay using anticholera toxin as the base coat, and with the Y1 adrenal cell assay. The assay should be useful in detecting enterotoxin production in E. coli and vibrios (including non-agglutinating Vibrio), in quantitating the toxin, and in determining binding properties of enterotoxins to ganglioside. The assay can also be used to quantitate antibodies which block the attachment of the toxin to the ganglioside.     

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect pure native Escherichia coli heat-stable toxin (ST) and to identify ST-producing strains among clinical isolates was determined. Two synthetically produced ST preparations were used to raise hyperimmune antisera in rabbits and goats: ST(S), which has the same antigenicity as native ST; and ST(C), which is 15-fold more immunogenic. These antisera were used in the double-sandwich technique as either crude double-species antisera or pure single-species antibody. The sensitivity of the assay was increased by using either a purer antibody preparation or the antiserum to the more potent immunogen; the assay in which pure antibody to ST(C) was used was 2,857-fold more sensitive in detecting ST than the assay in which crude antiserum to ST(S) was used. The minimum amount of ST detectable by the ST(C) ELISA was 140 pg/ml, which was an amount 285-fold smaller than that detectable by the suckling mouse assay. Among 50 human E. coli isolates examined by both the ST(C) ELISA and an ELISA for heat-labile toxin (LT), which had a sensitivity of 290 pg/ml for LT, the respective toxins were consistently identified in broth cultures of 10 LT+ and ST-, 15 LT+ and ST+, and 10 LT- and ST+ strains, and there were no false-positive responses. The ST(C) ELISA also detected ST in all of seven ST - producing E. coli strains tested of human origin, which had been shown elsewhere by DNA hybridization probes to have ST-coding genes of either human or porcine origin, and in all of three ST-producing E. coi strains tested of porcine origin. These results indicate that the sensitivity of the ST(C) ELISA is the same as that of previously described LT ELISAs. The concomitant use of both ST and LT ELISAs provides a rapid, simple, and sensitive method for identifying among clinical isolates enterotoxigenic strains of E. coli which produce either toxin.     

Evaluation of a competitive enzyme-linked immunosorbent assay for porcine Escherichia coli heat-stable enterotoxin.

A competitive enzyme-linked immunosorbent assay (ELISA) was compared with the conventional suckling mouse assay for the identification of heat-stable enterotoxin (STa) in samples from piglets suffering from diarrhea. A total of 110 Escherichia coli isolates, 22 primary cultures, and 26 fecal samples from piglets up to 8 weeks of age with diarrhea were compared in parallel by both assays. Of the 110 isolates tested, all gave consistent results by the ELISA and the suckling mouse assay; 60 strains were negative and 50 strains produced STa by both tests. Identical results were obtained when 22 primary agar cultures were screened for STa production by both methods; 6 were found to produce STa, while 16 did not. When 26 fecal samples were tested for the presence of STa, 10 were negative and 12 were positive by both assays. One of the remaining four samples gave questionable positive results by both the suckling mouse assay and the ELISA, but E. coli isolated from this sample gave positive results by both tests. The remaining three samples were negative by the suckling mouse assay, but gave questionable positive results by the ELISA. E. coli isolates from these three samples were always negative by both assays. The ELISA used in this study provides a reliable and convenient method for diagnosing STa-producing enterotoxigenic E. coli of porcine origin.     

Microtiter solid-phase radioimmunoassay for detection of Escherichia coli heat-labile enterotoxin.

The development of a microtiter solid-phase radioimmunoassay for the detection of Vibrio cholerae enterotoxin and heat-labile Escherichia coli enterotoxin is described. The test is based on the immunological similarity between V. cholerae toxin and E. coli heat-labile toxin. The assay is easy to perform, quantitative, and at least as sensitive and specific as the Y-1 adrenal cell system.     

Rapid GM1-enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin

A modified, quicker and simpler GM1-enzyme-linked immunosorbent assay procedure for detection of Escherichia coli heat-labile enterotoxin (LT) has been developed with the intent that it should be useful in less well-equipped laboratories. The method, which makes use of stable reagents including commercially available horseradish peroxidase immunoglobulin conjugate, can detect LT in overnight cultures within 1 working day (8 h), and the tests can be read with the naked eye. This GM1-horseradish peroxidase-enzyme-linked immunosorbent assay shows excellent quantitative and qualitative correlation with the conventional GM1-enzyme-linked immunosorbent assay. When 100 human E. coli strains were analyzed blindly and in parallel by the two methods, LT production was identified in 50 out of 50 LT-positive strains and in 0 out of 50 LT-negative strains by either method.     

A competitive immunosorbent assay for the detection of heat-stable enterotoxin of Escherichia coli

A competitive ELISA procedure for the detection of Escherichia coli heat stable enterotoxin (ST) with monoclonal antibody has been developed. This test is 10 times more sensitive than the suckling-mouse bioassay and it is specific, simple and cheap. A set of 882 strains of E. coli isolated from man were tested both by ST-ELISA and suckling-mouse bioassay, the latter serving as the reference method. Positive results in both tests were obtained with 152 strains. The remaining strains gave negative results in both tests, with the exception of two strains, known to be ST producers, that gave negative results in the suckling-mouse assay, but gave positive results by the ELISA method.     

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II.

The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII.     

Modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli.

Reactivity to Trypanosoma cruzi antigens and autoreactivity to heart antigens were evaluated in 27 patients with Chagasic cardiomyopathy (group I), 52 patients without evidence of cardiac dysfunction (group II), and 36 selected controls, either healthy patients or patients with other heart diseases (group III). The in vitro lymphoblastogenesis response to T. cruzi antigens was found to be high in groups I and II and low in group III. The mean stimulation index to T. cruzi antigens, in fact, tended to be highest in group I, suggesting a more intense immune response in patients with Chagasic cardiomyopathy. The proportion of individuals with reactivity to heart antigens was 28.6% in group I, 25% in group II, and 0% in group III. The finding of an equal percentage of reactivity to heart antigens in groups I and II was unexpected, as a higher incidence of positive reactions in group I was predicted. Consequently, it is thought that this finding and its relevance to the pathogenic process of Chagasic cardiomyopathy need to be carefully assessed in a longitudinal study.     

GM1 erythroimmunoassay for detection and titration of Escherichia coli heat-labile enterotoxin.

A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it uses are stable, in particular, tanned and sensitized sheep erythrocytes; GM1 ganglioside is commercially available; erythro-adsorption can be read with the naked eye; the test can be completed in 1 day; and as little as 4 ng of V. cholerae toxin or LT per ml can be detected accurately. The GM1 ganglioside erythroimmunoassay showed good quantitative and qualitative correlation with the Vero cell assay and the conventional GM1 enzyme-linked immunosorbent assay. The GM1 ganglioside erythroimmunoassay was somewhat less sensitive than the GM1 enzyme-linked immunosorbent assay but more sensitive than the Vero cell assay. Results obtained for 12 LT-positive and 138 LT-negative E. coli strains correlated with results obtained with GM1 enzyme-linked immunosorbent and Vero cell assays.     

Factors influencing heat-labile Escherichia coli enterotoxin activity.

In this study, conditions for production, detection, and storage of heat-labile Escherichia coli enterotoxin (LT) in culture filtrates from E. coli H-10407 were defined by using the adrenal tumor cell assay system. An enriched medium containing 0.6% yeast extract, 2% Casamino Acids, and 0.25% glucose buffered at pH 8.5 produced the highest LT activity of the various test media. In E. coli strain H-10407, LT activity was markedly decreased if the initial pH of the culture media was reduced to pH 7.5 or less. In contrast to E. coli P-263, if strain H-10407 was grown in the presence of mitomycin C there was no increase in LT production. Crude-culture filtrates containing LT can be stored at 4 degrees C for several days without an appreciable loss of activity; however, for long-term storage lyophilization or freezing at -70 degrees C is recommended.     

Evaluation of three simple and rapid immunological tests for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli.

Three simple immunological tests, the modified Elek (Biken) test, the modified staphylococcal coagglutination test, and the rapid GM1-horseradish peroxidase-enzyme-linked immunosorbent assay have been evaluated for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli. Of the 100 coded E. coli strains tested, 94 gave consistent results with all the three immunological tests; a discrepancy was observed in only 6 strains. Identical results were obtained when the Biken test was conducted with complete and incomplete Biken kits (Meguro Institute Ltd., Osaka, Japan). All three immunological tests evaluated in this study were found to be sensitive and simple and can be easily adopted by any laboratory for detection of heat-labile enterotoxins of enterotoxigenic E. coli strains.     

Properties of homogeneous heat-labile enterotoxin from Escherichia coli.

Recently, the heat-labile enterotoxin (LT) of Escherichia coli has been purified to homogeneity and partially characterized (Clements and Finkelstein, Infect. Immun. 24:760-769, 1979). This study extends our observations on the physicochemical properties of LT. The toxin has an isoelectric point of pH 8.0, as compared with choleragen and choleragenoid, which have isoelectric points of pH 6.75 and 7.75, respectively. Sedimentation equilibrium measurements established an approximate molecular weight for LT of 91,440. LT had an even more marked affinity than choleragen for agarose-containing matrixes in gel filtration. Of several mono- and disaccharides tested, only galactose and lactose were highly efficient in removing 125I-labeled LT from agarose-containing columns. LT dissociated into subunits (designated A and B) during gel filtration in the presence of 5 M guanidine. These subunits were immunologically distinct and possessed unique and shared antigenic determinants to the corresponding A and B subunits of choleragen. During gel filtration of LT at pH 6.5 and room temperature, a spontaneously occurring toxoid of LT, analogous to choleragenoid, was discovered and designated "coligenoid." This product contains only the B subunits of the toxin. A partial amino acid sequence of the B subunit of LT revealed a remarkable homology to the primary structure of cholera toxin B. Within the first 20 amino acids of the two chains, only 5 differ, and these differences may be attributable to single base substitutions.     

Differential detection of cholera enterotoxin and Escherichia coli heat-labile enterotoxin by enzyme-linked immunosorbent assays with antibodies specific to the two toxins

Enzyme-linked immunosorbent assays (ELISAs) with antibodies specific to either cholera enterotoxin (CT) of Vibrio cholerae or heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli were developed to detect LT and CT, respectively. With these ELISA systems, LT and CT could be detected only with the respective specific antibody. Both antibody ELISA and ganglioside ELISA were used for differential detection of LT and CT, but the former method seemed to be more specific. By this ELISA, as little as 0.1 ng of purified LT or CT could be detected per ml. The type of toxins in fluids in intestinal loops of experimental animals challenged with living cells of either V. cholerae or LT-producing E. coli was identified correctly by this ELISA. These results suggest that the ELISA systems reported here could be used to detect and differentiate CT and LT in unknown samples; it could also be used for assaying toxins in stool specimens for the diagnosis of diarrhea due to V. cholerae or LT-producing E. coli directly, without or before bacterial isolation.     

Urea-induced release of heat-labile enterotoxin from Escherichia coli.

Urea induces the release of heat-labile enterotoxin (LT) from cells of LT-producing Escherichia coli strains. Optimal conditions were defined by using the checkerboard immunoblotting system. LT release was highest when E. coli cells were incubated in 8 M urea, pH 8.0, at 37 degrees C in a water bath for 30 min. Urea was more effective than polymyxin B in inducing the release of LT antigen from E. coli; the activity of LT from urea-treated cells was seven times that of LT from polymyxin B-treated cells. Urea also increased the antigenic and biological reactivities of purified LT. This procedure is potentially applicable for the detection of LT-producing E. coli strains in the clinical laboratory.     

Recombinant fusion protein for simple detection of Escherichia coli heat-stable enterotoxin by GM1 enzyme-linked immunosorbent assay.

A recombinant gene fusion protein composed of an Escherichia coli heat-stable enterotoxin (STa) peptide epitope fused to the amino end of the cholera toxin B subunit was used to detect STa produced by clinical isolates of enterotoxigenic E. coli (STa-ETEC) by a single monoclonal antibody-based inhibition GM1 enzyme-linked immunosorbent assay. In this test, 100% sensitivity and 100% specificity were observed for use of the recombinant protein in either its purified form or as crude Vibrio cholerae culture supernatants in detection of STa-ETEC.     

Adaptation of the staphylococcal coagglutination technique for detection of heat-labile enterotoxin of Escherichia coli.

Protein A-containing staphylococci coated with specific antiserum were tested for heat-labile enterotoxin of Escherichia coli. The immunological cross-reactivity of E. coli heat-labile enterotoxin with Vibrio cholerae toxin (choleragen) was the basis for sensitizing stabilized suspensions of the Cowan I strain of Staphylococcus aureus with anticholeragen. Unconcentrated culture supernatant fluid containing E. coli heat-labile enterotoxin produced macroscopic agglutination when mixed with sensitized staphylococci in capillary tubes. A total of 15 toxigenic and 61 nontoxigenic isolates were tested by the staphylococcal coagglutination technique in a coded fashion and found to be in agreement with previous results of the Chinese hamster ovary cell assay and the passive immune hemolysis test. The staphylococcal coagglutination technique is simple, relatively inexpensive to perform, and requires the immunoglobulin fraction of anticholeragen as the only specific reagent. The staphylococcal coagglutination technique appears to have potential for routine use in diagnostic microbiology laboratories.     

Direct serological assay for the heat-labile enterotoxin of Escherichia coli, using passive immune hemolysis.

Sheep erythrocytes sensitized with the heat-labile enterotoxin (LT) of Escherichia coli exhibited passive immune hemolysis (PIH) when exposed to specific antitoxin and complement. Thus, PIH serves as the basis for an in vitro serological assay for LT that is sufficiently specific and sensitive to differentiate LT-positive and LT-negative E. coli isolates. The PIH assay for E. coli LT has been performed with the standard Microtiter system and also by a tube method employing the spectrophotometric determination of hemoglobin release. The spectrophotometric method enhances the sensitivity, accuracy, and objectivity of the PIH assay. The increased sensitivity of the spectrophotometric method also facilitates the identification of LT-positive cultures employing polymyxin "mini-extracts" of whole overnight (18 h) broth cultures of 2.0% Casamino Acid-0.6% yeast extract-salts medium rather than mini-extracts of cells derived from 3.5-h subcultures. Thus, large numbers of E. coli isolates can be individually tested for LT in less than 24 h after broth inoculation by a rapid in vitro assay which requires anti-LT serum as the only specific reagent.