Whole-bacterial cell enzyme-linked immunosorbent assay for Streptococcus sanguis fimbrial antigens

A whole-bacterial cell enzyme-linked immunosorbent assay (bactELISA) was developed for detecting fimbrial antigens on Streptococcus sanguis. In this assay, S. sanguis cells were directly adhered to polystyrene or polyvinyl via drying. Use of the assay indicated that consistently high and uniform optical densities could be obtained from well to well. In addition, radioactive assaying indicated increased adsorption to the polystyrene wells over polyvinyl, suggesting that polystyrene may prove superior in the gram-positive bactELISA. Use of the bactELISA may prove valuable to both the clinical and research laboratory involved in the study of bacterial cell surface components or in the evaluation of antisera directed against bacterial antigens, which are difficult to prepare as purified derivatives.     

A sandwich enzyme-linked immunosorbent assay for human interleukin-5

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for human interleukin-5 (hIL-5) using a combination of monoclonal anti-recombinant(r)-hIL-5 antibody and rabbit anti-r-hIL-5 IgG. Detection limit of this assay was estimated to be 7.8 pg/ml, which was about 10,000 times more sensitive than that of the bioassay using BCL1 cells of murine origin. This ELiSA was specific for hIL-5, showing no crossreactivity to recombinant human GM-CSF, IL-4, TNF-alpha, IFN-gamma and mouse IL-5 (mIL-5). The presence of 10% fetal calf serum did not interfere with the measurement of r-hIL-5. Coefficients of variation in intra-assay and interassay were 1.1-4.6% and 2.3-11.3%, respectively. These results indicate that this assay system can be quite useful in quantifying hIL-5 in various biological fluids.     

Measurement of antibody affinity for cell surface antigens using an enzyme-linked immunosorbent assay

We present a fast, simple, and accurate method to determine the affinity constants of antibodies that bind to cell surface antigens. This procedure utilizes intact cells and native, unmodified antibody in a conventional enzyme-linked immunosorbent assay. Target cells are incubated with serial dilutions of antibody and allowed to reach equilibrium. Cells are then pelleted by centrifugation, and aliquots of unbound antibody in the supernatant are added to a microtiter plate precoated with capture antibody and measured in a conventional enzyme-linked immunosorbent assay (ELISA). We measured the affinity constant of murine monoclonal antibody CLB-1H-gran2, which binds to K562 cells (a human erythroleukemia line), and compared the ELISA-based results to those obtained by flow cytometric determination of antibody affinity. The affinity constants obtained by the two methods are in good agreement. The affinity constant is calculated utilizing only the concentrations of bound and free antibody, so that the actual antigen concentration (or number of antigenic sites per cell) need not be known. However, the number of antibody molecules bound per cell can be estimated from the results.     

Interaction of staphylococcal protein A in enzyme-linked immunosorbent assays for detecting staphylococcal antigens

ELISA procedures for detecting staphylococcal antigens may be subject to interference by reactions between staphylococcal protein A (SPA) and IgG molecules. It was found that rabbit IgG reacted with SPA, both in the native state and after conjugation with peroxidase. Sheep IgG, however, did not react with SPA if conjugated with peroxidase. Peroxidase conjugated SPA reacted with rabbit IgG but not with sheep IgG. These results demonstrate that the source of IgG used in an ELISA system is of major importance to correct quantitation of staphylococcal antigens.     

Development of an enzyme-linked immunosorbent assay for studying Vibrio cholerae cell surface antigens.

An enzyme-linked immunosorbent assay for the measurement of antibodies directed against cell surface antigens of Vibrio cholerae (CSA ELISA) was developed. NaN3-killed whole cells of V. cholerae, adsorbed to polystyrene tubes, were used as immobilized antigens. The assay was capable of detecting antibodies directed against lipopolysaccharide and non-lipopolysaccharide surface antigens. In addition, the CSA ELISA was capable of detecting non-vibriocidal antibody. An antiserum raised in rabbits by immunization with live V. cholerae 1418 (Ogawa, El Tor) was capable of reacting with various heterologous strains of V. cholerae used as immobilized antigens. Therefore, common antigens shared by V. cholerae strains could be detected by using the CSA ELISA.     

Autoantibodies against liver cell membrane antigens detected by enzyme-linked immunosorbent assay in acute and chronic liver disease

An enzyme-linked immunosorbent assay was developed to detect serum antibody binding to liver membrane antigen derived from human hepatoma cell line SK-Hep-1. When we tested sera from 214 patients with this assay, IgM antibodies were detected in 100% of patients with acute type A, but not with type B or non-A, non-B hepatitis. IgM antibodies were also found in highest frequency (76%) and titer in patients with autoimmune chronic active hepatitis (CAH) among chronic liver disease groups. IgG antibodies occurred in over 50% of patients with acute type A hepatitis, type B chronic active liver disease (CALD), and autoimmune CAH. IgA antibodies were present in 43% of the patients with alcoholic liver disease, but were also seen in other patient groups. When freshly isolated rat hepatocytes were used as target cells, prevalences and titers similar to those obtained with SK-Hep-1 were found. The levels of serum membrane binding antibody were significantly reduced by the addition of human liver-specific membrane lipoprotein in all patient groups. In particular, IgM antibodies became negative in over 50% of patients with CALD (both type B and non-A, non-B) and autoimmune CAH, whereas in acute hepatitis over 50% lost their positivity for IgG antibody. These results indicate that circulating liver membrane binding autoantibodies are heterogeneous, occurring in hepatitis virus-induced acute and chronic liver disease as well as in autoimmune CAH.     

Development of an enzyme-linked immunosorbent assay for studying Pseudomonas aeruginosa cell surface antigens.

An enzyme-linked immunosorbent assay for the measurement of antibodies directed against Pseudomonas aeruginosa cell surface antigens was developed. Formalin-killed whole cells of P. aeruginosa, adsorbed to polystyrene acrylic copolymer cuvettes, were used as immobilized antigens. Antisera to P. aeruginosa mucoid strain 144M and to its spontaneous nonmucoid derivative, 144NM, were raised in rabbits by immunization with Formalin-killed bacteria. By using this enzyme-linked immunosorbent assay, anti-144M serum was found to have a ca. 10-fold-higher antibody titer to 144M than did anti-144NM serum, suggesting that 144M may have either immunogenic determinants not present on 144NM or perhaps simply more antigenic determinants. In contrast, anti-144M and anti-144NM immune sera were found to have nearly identical antibody titers to 144NM, suggesting that these strains share many determinants. Anti-P. aeruginosa immune serum was found to contain Pseudomonas-specific antibodies as well as antibodies which cross-reacted with other gram-negative bacteria. Finally, absorption studies demonstrated that this assay can detect both LPS and non-LPS surface-exposed antigenic determinants. Thus, this whole bacterial cell enzyme-linked immunosorbent assay should prove useful in monitoring patient sera and secretions for potentially protective immunoglobulins directed at P. aeruginosa cell surface antigens.     

A sensitive enzyme-linked immunosorbent assay for human interleukin-8.

In order to quantify human interleukin-8 (IL-8), which is chemotactic for T cells and basophils as well as neutrophils, we developed an enzyme-linked immunosorbent assay (ELISA). Since binding inhibition tests indicated that three monoclonal antibodies (mAbs; BS-1, WS-4, WS-6) blocked the binding of 125I-labelled IL-8 to neutrophils, we tested an ELISA using these mAbs as primary antibodies, rabbit anti-IL-8 Ab as the secondary antibody, and alkaline phosphatase-labelled goat anti-rabbit Ab as the conjugate. Among the three mAbs tested, WS-4 was the most sensitive with a detection limit of 16 pg/ml. Several other cytokines, including monocyte chemotactic and activating factor (MCAF), which is structurally related to IL-8, showed no cross-reactivity in this system, indicating that this ELISA is specific for IL-8. The coefficients of variation for the intra- and interassays were below 10%. Furthermore, this ELISA also detected natural IL-8 (including both 72 and 77 amino acid forms) produced by cultured human cells and cell lines stimulated with IL-1, suggesting that this system will be useful in the detection of natural IL-8 in various body fluids.     

Development of a competitive enzyme-linked immunosorbent assay for detecting cytomegalovirus antibody

A competitive enzyme-linked immunosorbent assay (ELISA) for detecting cytomegalovirus (CMV) antibody was developed. The competitive ELISA was five times more sensitive than the complement fixation test (CFT) and twice as sensitive as indirect ELISA. Testing of paired sera from cardiac transplant patients taken before and after transplantation showed good correlation between results of competitive and indirect ELISA and CFT. The competitive ELISA was more successful than CFT or indirect ELISA in detecting passively acquired antibody, but detection of CMV antibody by competitive ELISA immediately after primary CMV infection was unreliable, possibly because of the high affinity of the monoclonal antibody chosen for the horseradish peroxidase conjugate. However, competitive ELISA may well prove to be more suitable than indirect ELISA for detecting CMV antibody in blood donations.     

A sensitive enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigens

A sandwich ELISA for the detection of herpes simplex virus (HSV) antigens was developed using sheep anti-HSV F(ab')2 fragments for capture and an indirect antibody system for detection. Current detection limits are 0.5 ng protein for HSV1 and 1.5 ng protein for HSV2. This compares to a single HSV1-infected Vero-cell in a background of 10(6) non-infected cells or 10 plaque forming units (PFU) of HSV1 in culture supernatants as determined in separate experiments. Limiting dilution experiments show that one PFU of HSV1 can be detected after overnight culture in both supernatant and cell extracts. The use of F(ab')2 for capture completely eliminated binding of Staphylococcus aureus. No cross-reactivity was observed with other human herpes viruses. When evaluated with 245 random 'left-overs' of genital swab specimens in transport medium the test showed a sensitivity and specificity of 77.2 and 97.8%, respectively, with respect to virus isolation in culture. In a preliminary study on 16 direct ELISA swab-specimens extracted in 0.5 ml ELISA sample buffer both sensitivity and specificity were 100% with respect to culture. In both clinical series there was a proportional relationship between the ELISA value and the estimated amount of infectious virus in the specimen.     

Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test.     

A reverse-type sandwich enzyme-linked immunosorbent assay for detecting antibodies to Borna disease virus.

To investigate whether there is an epidemiological correlation between Borna disease virus (BDV) infection and human neuropsychiatric diseases, we established a reverse-type sandwich enzyme-linked immunosorbent assay (RS-ELISA) for detecting specific antibodies to BDV. In this assay, microplate wells were coated dispersely with BDV p40 antigen, followed by the addition of test samples at a low dilution and then the biotinylated p40. A preformed complex of streptavidin and horseradish peroxidase-conjugated biotin and an enzyme substrate were used to measure the captured biotinylated p40. Theoretically, RS-ELISA should specifically detect anti-BDV antibodies without nonspecific signals; such signals possibly occur in conventional serological assays. Additionally, the RS-ELISA could be applied under the same protocols to test samples from a variety of animals. By using anti-BDV rat and rabbit sera, the assay was standardized so that it had high specificity and sensitivity. When we used the RS-ELISA to determine the presence of anti-BDV antibodies in plasma from 70 patients with chronic schizophrenia as well as 40 healthy individuals in the Tokyo area of Japan, no plasma sample was found to possess specific antibodies to BDV p40, indicating no association between BDV infection and the disease in our testing population. A negative reaction was also shown for the sera that had previously been judged to be seropositive for BDV by an immunofluorescence or immunoblot test. These findings suggested that false-positive cases of infection due to nonspecific reactions may be included in previous seroepidemiological information with regard to BDV.     

Specificity of a sandwich enzyme-linked immunosorbent assay for detecting Aspergillus galactomannan.

The specificity of a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was tested with exoantigens of 29 fungi cultured from clinical specimens. Cross-reactivity was observed with Penicillium chrysogenum, Penicillium digitatum, and Paecilomyces variotii. Furthermore, 40 serum samples obtained from bacteremic patients with hematologic malignancies were retrospectively tested by sandwich ELISA. False-positive reactions with the serum were reproducible but did not correspond with the results of culture of specific microorganisms. Moreover, the microorganisms cultured from the blood showed no reactivity by the sandwich ELISA.     

An enzyme-linked immunosorbent assay for detection of antibodies against halothane-altered hepatocyte antigens

Patients with massive liver cell necrosis that may follow halothane anaesthesia have a high incidence of circulating antibodies against halothane-induced hepatocyte antigens. In order to provide an objective and quantitative method for the detection of these antibodies, an enzyme-linked immunosorbent assay has been developed. Sera, after absorption with normal rabbit liver microsomal fraction, are tested for binding to microsomal fractions from control and halothane-pretreated rabbits. Those containing antibodies against halothane-induced determinants give significantly enhanced binding to halothane-altered fractions; this specificity was verified by absorption experiments. Using this method, halothane-related antibodies were detected in sera from 16/24 patients with halothane-associated liver failure, at titres ranging from 1:100 to 1:25600. Such antibodies were not detectable in sera from 26 normal blood donors, 5 healthy anaesthetists, 12 patients who had received multiple halothane anaesthetics but had normal liver function tests and 32 patients with a variety of other liver diseases. This rapid and reproducible assay should be of value for the detection of antibodies and for detailed investigation of patient antibody responses, and also for characterization of the route of production and metabolism of the antigen.     

Outer membrane protein antigens in an enzyme-linked immunosorbent assay for Salmonella enteric fever and meningococcal meningitis.

Outer membrane protein preparations were obtained from strains of Salmonella and Neisseria meningitidis. Solubilized cell envelope (CE) fractions from S. typhi and Salmonella groups A, B, C, and E had very similar electrophoretic mobilities on polyacrylamide gel, and common antigens were demonstrated by immunodiffusion. CE appeared to be a more satisfactory antigen than the more purified preparation (T/TEI) in the enzyme-linked immunosorbent assay (ELISA) with sera from typhoid and paratyphoid patients. With either antigen, however, the presence of antibodies was demonstrated in acute- and vonvalescent-phase sera. In the case of N. meningitidis infections, the crude (STA) and the more purified antigens (T/TEI) were equally satisfactory, and a rise in antibody titer could easily be demonstrated with paired acute- and convalescent-phase sera. The ELISA appears to be a simple but highly sensitive test for the detection of antibodies by using outer membrane protein antigens.     

A quantitative enzyme-linked immunosorbent assay for rat insulin

A simple, quantitative micro-ELISA (Enzyme-Linked Immunosorbent Assay) has been developed for rat insulin. The micro-ELISA is a solid phase, indirect, competitive immunoassay. The useful range of the micro-ELISA was superior to that of a commercial RIA for rat insulin (e.g. 0.4 to 46.0 ng/ml for the ELISA; 0.2-8.6 ng/ml for the RIA). The ELISA's sensitivity (the lower limit of detection) was 1.0 ng/ml +/- 0.13 ng/ml, (mean, +/- SEm; 9 assays) or 20 +/- 2.6 pg/determination, and compared favorably with the sensitivity of a radioimmunoassay (RIA) for rat insulin (0.38 +/- 0.10 ng/ml; 4 assays). The ELISA measured pure rat insulin standards accurately. Correlation experiments showed that the results of the ELISA agreed with those of the RIA (r = 0.91), when rat insulin was assayed in crude extracts of isolated pancreatic Islets of Langerhans. When the standard curve was plotted as a log of the dose response curve, a sigmoidally shaped curve was obtained which could be transformed into a straight line relationship with a logit-log program. The goodness of fit of the transformed standard curve to a straight line relationship was excellent (r = 0.97 to 0.99: n = 4 ELISAs). The transform facilitated dose interpolation, tests of parallelism, and quality control. Tests of parallelism showed that the ELISA was specific for rat insulin. The precision of the ELISA was superior to the precision of the rat insulin RIA tested. The intraassay precision of the ELISA was always less than 10% (CV%), and its interassay precision was always less than or equal to 15% (CV%). The micro-ELISA is versatile, since it can be used to measure human, porcine, rat, and probably mouse insulin.     

Correlation between enzyme-linked immunosorbent assay and immunofluorescence assay with lytic antigens for detection of antibodies to human herpesvirus 8

The purpose of this study was to evaluate, in Kaposi's sarcoma patients, the correlation between antibody titers to the lytic antigens of human herpesvirus 8, as assessed by immunofluorescence assay, and values obtained by an enzyme immunoassay. The methods showed a stringent correlation, r = 0.625 (P<0.001).     

A direct enzyme-linked immunosorbent assay for hexoestrol residues

A simple enzyme-linked immunosorbent assay for the detection of hexoestrol (HES) residue has been developed. The HES derivatives, hexoestrol-mono-ether-butyrate-ethyl (HES-MEBE) and hexoestrol-mono-caroxyl-propyl-ethyl (HES-MCPE) were synthesised to enable the coupling of HES and proteins together. The immunogen HES-MCPE-BSA and enzyme-linked antigen HES-MCPE-HRP were prepared by mixed anhydride methods. After the antibody was obtained by immunisation of New Zealand rabbits, a direct enzyme-linked immunosorbent assay was developed for the determination of HES residue. Several parameters were optimised. Excellent linearity (R ²=0.9905) was demonstrated from 0.01 to 8.1 ng/ml. The 50% inhibition value (IC₅₀) and the limit of detection (LOD) were 0.23 and 0.01 ng/ml, respectively. The specificity of the immunoassay was evaluated by determining cross-reactivity of six hormones (diethylstilbestrol, dienestrol, 19-nortestosterone, medroxyprogesterone, testosterone and estrone), and all cross-reactivity rates were <0.5%. The recoveries from beef and fish ranged from 61 to 102%. The stabilisation of the coated plates was also studied. The ELISA developed was used to detect the residues in chicken muscle tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). This proposed method could be applied to routine residue analysis.     

Development of enzyme-linked immunosorbent assay for detecting antibodies to replication-competent murine leukemia virus

A method for detecting the antibodies to replication-competent retrovirus (RCR) was developed. Specific fragments of murine leukemia virus (MLV) Gag or Env protein were cloned and expressed in Escherichia coli, and used subsequently to develop the ELISA system. It was found that CA of Gag and SU of Env, but not the transmembrane portion of Env, could be used in ELISA. ELISA conditions such as coating buffer and blocking solution were optimized using sera obtained from mice immunized with amphotropic MLV particles. In an optimized ELISA system, serum samples from normal healthy individuals provided very low absorbance values. ELISA was performed using serum samples from patients who had received skin fibroblasts engineered with MLV-based retroviral vector. Experimental samples presented absorbance values comparable to those found with control serum samples from normal, healthy individuals, showing no evidence of RCR infection.     

Optimization of a human papillomavirus-specific enzyme-linked immunosorbent assay

A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.