Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay

Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus.     

Production of monoclonal antibodies against human growth hormone releasing hormone and their use in an enzyme-linked immunosorbent assay (ELISA)

Two murine monoclonal antibodies (mAbs) specific for human growth hormone releasing hormone (GHRH-44-NH2) were produced from a fusion of spleen cells from a BALB/c mouse immunized with GHRH-conjugated BSA with SP 2/0 myeloma cells. The antibodies were of the IgG1 kappa, and IgG2b-kappa isotypes. The binding of both antibodies to GHRH-coated plates was inhibited by a 30-44 amino acid fragment but not by a 1-26 fragment. Thus, both antibodies are directed against the carboxy terminus of the peptide. Furthermore, both antibodies bind to the same epitope on the 30-44 amino acid portion since they cross-inhibit each other's binding to intact GHRH. Using these mAbs, a direct binding GHRH enzyme-linked immunosorbent assay (ELISA) was developed which had a least detectable dose of 30 pg. The availability of these antibodies and their use in ELISA methodology permits consistent and specific detection of GHRH in a non-isotope assay. They should prove of value in screening acromegalic patients for ectopic sources of GHRH secretion and in studies of ontogenic analysis of GHRH production.     

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.     

Production of subgroup-specific monoclonal antibodies against human rotaviruses and their application to an enzyme-linked immunosorbent assay for subgroup determination

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid. Using the three monoclonal antibodies (S2-37, YO-5, and YO-156), an enzyme-linked immunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.     

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.     

Detection of human group C rotaviruses by an enzyme-linked immunosorbent assay using monoclonal antibodies.

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical samples which were found to contain group C rotaviruses were all strongly positive, whereas 9 samples containing group A rotaviruses and 51 samples lacking rotaviruses were all negative with this test.     

An enzyme-linked immunosorbent assay for the quantitation of human IgG subclasses using monoclonal antibodies

An enzyme-linked immunosorbent assay was established for the quantitation of human IgG1, IgG2, IgG3 and IgG4 using IgG subclass-specific monoclonal antibodies. The method could detect 1-10 ng/ml of the Ig subclasses. The technique is suitable for measuring IgG subclass concentration in sera of healthy adults and in supernatants from human lymphocytes cultured in the presence of pokeweed mitogen.     

Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies.     

Development of an enzyme-linked immunosorbent assay for serotyping ureaplasma urealyticum strains using monoclonal antibodies

Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of U. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.     

Typing of herpes simplex virus by an enzyme-linked immunosorbent assay with monoclonal antibodies.

We developed a method that uses monoclonal antibodies for typing herpes simplex virus type 1 and type 2 strains. Clinical isolates from GMK cells were seeded directly into a monolayer of GMK cells. After incubation overnight, monoclonal antibodies were added to the infected monolayer, and antibody binding was indicated by a peroxidase enzyme-linked immunosorbent assay. Using prototype strains and previously typed patient strains, we verified the specificity of this technique. This method is now used routinely for typing herpes simplex strains in our laboratory. We have also used this technique for specific staining of type 1 plaques in a mixture of type 1 and type 2 plaques. With this method it is possible to find a single type 1 plaque among several hundred type 2 plaques on a single petri dish. Infectious virus can also be recovered from stained, unfixed type 1 plaques.     

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the ³H-labeled natural isomers of nicotine or cotinine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10⁸ M⁻¹ were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly- -lysine werecoated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5–10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and ³H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.     

Production of novel anti-recombinant human erythropoietin monoclonal antibodies and development of a sensitive enzyme-linked immunosorbent assay for detection of bioactive human erythropoietin

Erythropoietin (EPO) is a growth factor, regulating the proliferation and differentiation of erythroid progenitor cells. In this study, we generated five monoclonal antibodies (mAbs) that reacted specifically with recombinant human EPO (rhEPO). Epitope exclusion and other experiments showed that the mAbs obtained were divided into two groups, differing in recognition sites for rhEPO: group 1 mAbs recognize the N-terminal region of rhEPO, whereas group 2 mAbs seem to recognize a conformation-dependent epitope. Although most of the previously reported anti-EPO antibodies that recognized the N-terminal region of EPO lacked the EPO-neutralizing activity, the group 1 mAbs obtained here had the rhEPO-neutralizing activity. Therefore, the group 1 mAbs may be useful for future study on structure-function relationship of EPO. One of the group 2 mAbs, 5D11A, showed the highest affinity for rhEPO with K(D) value 0.52 nM and had the highest rhEPO-neutralizing activity. Using this mAb, we developed a reproducible and sensitive enzyme-linked immunosorbent assay for the quantification of bioactive rhEPO.     

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.     

Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay

The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin. Enterohemorrhagic E. coli produces elevated levels of SLT-I, SLT-II, or both cytotoxins (also called Verotoxins). SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I. In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933. Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA). Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-II but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E. coli that causes edema disease of swine. All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit. Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass. All five lines had kappa light chains. These neutralizing monoclonal antibodies have been used as probes in a colony ELISA to detect SLT-II-positive bacterial colonies. The colony ELISA with these monoclonal antibodies is a specific, sensitive test with potential diagnostic value.     

Anti-endometrial antibodies in women measured by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed tomeasure anti-endometrial antibody concentrations in the serumof women with endometriosis. Pooled cytosolic protein extractsfrom the endometrial gland cells of 10 women were used as anantigen source. Serum samples were obtained from women withendometriosis before (n = 51) and after 6 months treatment withdanazol or nafarelin (n = 30). Control sera came from womenwith a normal pelvis at laparoscopy, performed for sterilization(n = 23) or the investigation of pain and/or infertility (n= 22), 13 women with Rokitansky syndrome, and 10 umbilical cordbloods and adult males. There were no significant differencesin serum anti-endometrial antibody concentrations before andafter treatment, or between women with endometriosis and withoutendometriosis. Concentrations were lower in male and cord bloodserum than in female's serum (P < 0.0001). We conclude thatthe ELISA is not a useful diagnostic tool for endometriosisunless more specific antigens can be isolated.     

Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and application of the antibodies for enzyme-linked immunosorbent assay

A total nine hybridoma cell lines that produced monoclonal antibodies against thermostable direct hemolysin (Vp-TDH), a possible pathogenic toxin, of Kanagawa phenomenon-positive Vibrio parahaemolyticus was isolated and characterized. These monoclonal antibodies (mAbs) were divided into a minimum of five different specificity groups, including mAbs specific to Vp-TDH and common to Vp-TDH and Vp-TRH, a Vp-TDH-related hemolysin produced by Kanagawa phenomenon-negative V. parahaemolyticus. An enzyme-linked immunosorbent assay (ELISA) using mAb-1-D, a mAb specific for Vp-TDH, was developed for specific detection of Vp-TDH. On the other hand, the ELISA using mAb-9-D, and mAb common to both Vp-TDH and Vp-TRH, could be used for detection of both Vp-TDH and Vp-TRH. Thus, by combining these two ELISAs differential detection of Vp-TDH and Vp-TRH can be performed. Hence, the two ELISAs were applied for various strains of V. parahaemolyticus and it was found that most Kanagawa phenomenon-positive and -negative clinical isolates produced Vp-TDH and Vp-TRH, respectively, but all environmental strains, that were Kanagawa phenomenon-negative, produced neither toxin.Supported by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Developmental Scientific Research from the Ministry of Education, Science, and Culture of Japan     

Evaluation of an enzyme-linked immunosorbent assay for quantitation of antibodies to Junin virus in human sera

An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.     

Detection of antibodies to Mycoplasma pulmonis by an enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay, which entails the use of antigen-coated tubes and enzyme-labeled anti-immunoglobulins, was applied for the detection of antibodies against Mycoplasma pulmonis in mice. A lysate of M. pulmonis was used as the antigen, and anti-mycoplasmal antibodies of the different immunoglobulin classes were detected by class-specific anti-immunoglobulin labeled with alkaline phosphatase. The optimal conditions for the test were determined, the specificity was evaluated, and the assay was compared with other procedures for detection of mycoplasmal infection. The enzyme-linked immunosorbent assay was found to be a specific, highly sensitive, and reliable procedure for detecting anti-mycoplasmal antibodies in mice.     

An improved enzyme-linked immunosorbent assay of anti-collagen antibodies in human serum

An improved enzyme-linked immunosorbent assay (ELISA) for the determination of anti-collagen antibodies in human serum has been developed. The method is based on the use of serum samples diluted to 1/50 with heat-inactivated normal rabbit serum adjusted to pH 8.0 with solid Tris (0.05 M), NaCl (0.15 M) and 2 M HCl. The use of normal rabbit serum minimizes non-specific adsorption of immunoglobulin G onto the plastic surface of microtiter plate. The applicability of the method for the quantitation of anti-collagen antibodies in human serum is demonstrated with 290 specimens of sera from normal controls (194) and patients with rheumatoid arthritis (96).