Enzyme-linked immunosorbent assay for Clostridium difficile toxin A.

Antibodies against Clostridium difficile toxin A were purified by affinity chromatography from antiserum prepared against crude C. difficile toxin preparations. The affinity-purified antibody preparation was free of detectable amounts of antibodies to other C. difficile antigens, as demonstrated by crossed immunoelectrophoresis, and specifically neutralized the cytotoxicity of toxin A. An indirect enzyme-linked immunosorbent assay (ELISA) was subsequently developed using the antibody preparation for the specific detection of toxin A. The ELISA, which could detect 1 ng (5 ng/ml) of toxin A, was used to quantitate the toxin in the culture supernatant fluids of strains of C. difficile. The ELISA values for toxin A closely correlated with the toxin A and B cytotoxic titers of the supernatant fluids. In addition, toxin A was detected by ELISA in human fecal specimens from persons with antibiotic-associated colitis, demonstrating that this toxin is produced during C. difficile colitis.     

Enzyme-linked immunosorbent assay for chlamydial antibodies.

An enzyme-linked immunosorbent assay (ELISA) detected chlamydial antibodies in human sera. The assay antigen produced in cell cultures infected with Chlamydia psittaci was Formalin-fixed to microplates. Single convalescent-phase sera positive for chlamydial antibodies by a complement-fixation test were positive at even higher dilutions by ELISA. Paired sera with diagnostic rises in complement-fixing antibody showed seroconversion by ELISA also. Control sera from persons with no history of chlamydial infection were negative by both tests. Sera from patients with psittacosis or lymphogranuloma venereum were ELISA positive, indicating that the assay with the antigen used in this study is genus specific rather than species specific.     

Enzyme-linked immunosorbent assay for streptococcal M protein antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann, which uses antigen-coated tubes and enzyme-labeled anti-immunoglobulin, has been used for the detection of antibodies against streptococcal M protein. The antigen used in the assay was obtained by guanidine extraction of type M-12 streptococcal cell walls followed by hydroxyapatite chromatography. This antigen has the capacity to elicit bactericidal antibodies in rabbits. The results show that the ELISA is specific and highly sensitive for the detection of antibodies in rabbit and human antisera. Preliminary results suggest that, when M-12 antigen is used, the antibodies detected by ELISA are the same antibodies detected in the bactericidal test. The assay has been performed with human and rabbit sera. There was a 96% agreement between bactericidal and ELISA results with rabbit sera and 97.5% agreement with human sera. All bactericidal antibody-positive sera tested thus far yielded positive ELISA results.     

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.     

Enzyme-linked immunosorbent assay for the serodiagnosis of toxoplasmosis.

The enzyme-linked immunosorbent assay (ELISA) for toxoplasmosis proved as sensitive as the dye test and indirect haemagglutination (IHA). The reproducibility of end-point titres was better than that of the extinction values obtained from a single serum dilution. In a comparison of 152 sera, ELISA was found to correlate better with IHA than with the dye test. The use of ELISA for routine serology and population screening is discussed.     

Enzyme-linked immunosorbent assay to detect Shiga toxin of Shigella dysenteriae and related toxins.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga toxin. Four species of Shigella, Escherichia coli, and Vibro parahaemolyticus were tested for production of Shiga or Shiga-like toxin by ELISA and Vero cell bioassay. In the ELISA, most strains of S. dysenteriae and some strains of E. coli isolated from traveler's diarrhea were positive. These ELISA-positive strains were positive by Vero cell bioassay without exception. Some E. coli strains and most V. parahaemolyticus strains were toxic to Vero cells, although they were negative in the ELISA. Much of the cytotoxic activity was not neutralized by anti-Shiga toxin antiserum. The newly developed sandwich ELISA is specific and can be a substitute for the cumbersome Vero cell bioassay.     

Enzyme-linked immunosorbent assay for the phytotoxin cleistanthin A.

Enzyme-linked immunosorbent assay is reported for the estimation of cleistanthin A, a major constituent of the toxic plant Cleistanthus collinus. Rabbit antibodies were obtained by immunisation with cleistanthin A hemisuccinate-BSA conjugate and the ELISA developed thereupon could detect cleistanthin A at as low a concentration as 3 ng/ml. Cross-reactivity studies with structural analogs as well as with other phytotoxins and drugs of common occurrence established the suitability of the ELISA to specifically monitor the C. collinus marker molecules in emergency clinical and forensic cases. The simplicity and specificity make the ELISA superior to the other available techniques.     

Enzyme-linked immunosorbent assay for group B streptococcal antibodies.

We report here on the development of enzyme-linked immunosorbent assays (ELISAs) for antibodies to types II and III group B streptococci. Streptococcal antigens were prepared by trichloroacetic acid extraction and fractional alcohol precipitation. Microtiter wells were coated with antigen in 0.1 M carbonate buffer at pH 9.6. Lyophilization was found to be an essential step for efficient binding of the streptococcal antigens. After incubation with antibody-containing rabbit serum, bound antibody was detected with peroxidase-labeled goat anti-rabbit immunoglobulin G. Optimal antigen concentrations were 200 micrograms/ml for type II and 100 micrograms/ml for type III. An ELISA is also described that uses intact bacteria as antigen. Hyperimmune rabbit serum reacted at a titer in excess of 512 against trichloroacetic acid-soluble antigen and 4,096 against whole bacteria. Sera from human subjects were also tested. Most human sera contained antibody which bound to intact bacteria but not to trichloroacetic acid-solubilized streptococcal antigens. Antibody titers in human sera against intact bacteria correlated very well with opsonic antibody activity measured in a chemiluminescence assay.     

Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A.

An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.     

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.     

Enzyme-linked immunosorbent assay for detection of measles antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of measles immunoglobulin G antibody (MEASELISA). This assay was found to be comparable to the measles hemagglutination inhibition (HAI) test. Approximately 500 sera from three centers were tested by MEASELISA and the HAI test. MEASELISA demonstrated values of greater than 99% for sensitivity, specificity, and accuracy. Values were very precise, with a mean coefficient of variation of 5.4%. MEASELISA values were shown by linear regression analysis to increase as HAI titers increased. A coefficient of determination of 1.00 was obtained from test center three. MEASELISA values were found to be linearly related (r2 greater than 0.97) to MEASELISA titers, thus enabling quantitation of measles antibody from a single value. Also, data are presented that show MEASELISA to be equivalent to complement fixation for evaluating paired sera for the presence of a significant increase in antibody levels to measles virus.     

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.     

Enzyme-linked immunosorbent assay for diagnosis of brucellosis

The performance of an ELISA for detection of total antibodies to Bruceila spp. was compared with that of the Rose Bengal, standard agglutination and Coombs test in the diagnosis of brucellosis. Sera tested were from 208 patients from whom Brucella melitensis had been isolated, 177 patients with significant results in at least two conventional tests, and 107 patients with fever from whom no Brucella spp. had been isolated and in whom all conventional tests were negative. ELISA was the most sensitive test (97 %), showing greater specificity (96 %) and good predictive positive and negative values (98 % and 94 % respecitvely). ELISA was the only positive test in 6 % of patients in whom brucellosis had been confirmed by culture.     

Enzyme-linked immunosorbent assay for immunological diagnosis of human tularemia.

The enzyme-linked immunosorbent assay (ELISA) was applied for immunological diagnosis of human tularemia, using lipopolysaccharide from Francisella tularensis as antigen. Sera collected from patients, healthy individuals, and vaccinated volunteers were investigated for antibodies against F. tularensis by ELISA and tube agglutination. In ELISA all sera were titrated with a polyspecific anti-immunoglobulin enzyme conjugate. A limited number of consecutive sera from individual patients were also investigated for immunoglobulin G (IgG) and IgM antibodies by means of immunoglobulin class-specific conjugates. On an average ELISA was more than 10-fold as sensitive as tube agglutination. Two weeks after onset of disease, sera from patients had significantly higher titers in ELISA than sera from healthy controls. High titers persisted after more than 2 years. Significant amounts of both IgG and IgM antibodies were present within 1 to 2 weeks after infection. The antibody activity increased during the first month, without any significant change of the relation between IgG and IgM titers. After 2.5 years the IgG/IgM titer ratio of sera from patients was significantly increased. Within 6 weeks after vaccination sera from about half of the vaccinees had significantly elevated titers in ELISA. Titers observed after vaccination were generally lower than those found after infection. An elevated ELISA titer can be of diagnostic importance by the end of the first week of illness. A significant increase of titer in consecutive serum samples indicates a diagnosis of tularemia. Determination of IgG and IgM antibodies may be of value in determing whether a positive titer of a single serum sample is of longstanding or recent origin.     

Enzyme-linked immunosorbent assay for detection of Streptococcus pneumoniae antigen.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Streptococcus pneumoniae polysaccharide antigen in cerebrospinal fluid and serum. Sensitivity and specificity were determined for purified antigen preparations. Specificity was also evaluated in the rabbit meningitis model, and the sensitivity was compared to counterimmunoelectrophoresis, using the infected rabbits' cerebrospinal fluid and serum. The ELISA was a specific technique for detecting S. pneumoniae antigen. ELISA was 25 times more sensitive than counterimmunoelectrophoresis for purified antigen and resulted in an increased positivity of the cerebrospinal fluid and serum from infected rabbits. ELISA should prove very useful in the diagnosis of pneumococcal infections.     

Enzyme-linked immunosorbent assay for detection of streptolysin O antibodies.

An enzyme-linked immumosorbent assay (ELISA), based upon the detection of streptolysin O antibodies in human sera, was developed. Disposable polystyrene tubes, sensitized with streptolysin O antigen, were used as the test vehicles. Corresponding antibodies, present in test sera, were detected by binding of the antibodies to goat anti-human immunoglobulin G conjugated to horseradish peroxidase. Demonstration of bound conjugate was accomplished by monitoring peroxidase activity spectrophotometrically at 450 nm, using 5-aminosalicylic acid as the indicator. A total of 97 human sera, previously analyzed by means of the anti-streptolysin O titration technique, were evaluated with the ELISA procedure. A direct quantitative relationship, found to be statistically significant, was demonstrated between Todd units and absorbance values obtained with ELISA.     

Enzyme-linked immunosorbent assay for circulating anti-glomerular basement membrane antibodies.

An enzyme-linked immunosorbent assay for the quantitative determination of anti-glomerular basement membrane antibodies in human sera, which is both sensitive and reproducible, is described. The test detected circulating antibodies in each of seven patients with active anti-glomerular basement membrane disease, whilst sera from 42 patients, with a variety of other glomerulonephropathies, were negative by the test. It has also been possible to demonstrate a good correlation between the levels of circulating anti-glomerular basement membrane antibodies and the clinical course of disease in one patient with Goodpasture's syndrome.     

Enzyme-linked immunosorbent assay for diagnosis of chronic Q fever.

From 1982 through 1987 we diagnosed 13 chronic Q fever cases. Clinically these patients presented a culture-negative endocarditis, and all but two had high complement-fixing antibody titers to Coxiella burnetii phase I (reciprocal titer above 200). With the enzyme-linked immunosorbent assay (ELISA), titers of immunoglobulin G (IgG) to phases I and II of C. burnetii averaged 158,000 and 69,900, respectively, whereas they reached 300 and 3,200 in acute Q fever cases. Similarly, IgA to both phases of C. burnetii and IgM to phase I were consistently higher during chronic than acute Q fever. The serological follow-up of one patient with chronic Q fever over a 4-year period showed a good correlation between the titers of IgG and IgM antibody titers detected by ELISA and indirect fluorescent-antibody test (IFA) to both phases of C. burnetii. Few discrepancies appeared with IgA. Shortly after initiation of antibiotic treatment, a slow and steady decrease of the antibody titers to C. burnetii phases I and II was observed. The complement fixation, IFA, and ELISA tests showed the same type of antibody response. The ELISA proved to be an excellent diagnostic test for chronic Q fever. It distinguished negative from positive reactions clearly, and results were highly reproducible. The reading is objective, and the test is simple to perform and more sensitive than the IFA and complement fixation tests. The ELISA is recommended for serologic evaluation of patients with chronic Q fever.