Detection of postvaccination mumps virus antibody by neutralization test, enzyme-linked immunosorbent assay and sensitive hemagglutination inhibition test

A group of 251 children aged 2-3 years given live attenuated mumps virus vaccine PAVIVAC of Czechoslovak production were tested for antiparotitis antibody levels in pre- and postvaccination sera by neutralization test (NT), enzyme-linked immunosorbent assay (ELISA) and sensitive hemagglutination inhibition test, enhanced by heterologous antibody to human immunoglobulin G (E-HIT). The prevaccination findings were as follows: positive ELISA IgG titres, neutralization antibodies and hemagglutination inhibition antibodies were present in, respectively, 35%, 25.9% and 27.9% of the sera. Postvaccination seroconversions were evaluated in 159 susceptible vaccinees whose prevaccination sera had been negative by all three tests. The lowest seroconversion was detected by NT (74.2%), seroconversions by ELISA and E-HIT were appreciably higher (82.4% and 86.8%, respectively). The seven children showing a seroconversion by E-HIT but not by ELISA had a 4 fold increase of anti-mumps ELISA IgG antibodies as well, but the rise of antibody titres was at a level falling in the range below the positivity criterion for ELISA. The statistically evaluated detection rate for antibodies was significantly higher (significance test "t") by ELISA as compared with neutralization test. However, antibody levels (geometric mean titres) were 8-10 times lower in postvaccination sera than in convalescent sera of 30 children with mumps in all three tests.     

Hybridomas specific for carbohydrates; synthetic human blood group antigens for the production, selection, and characterization of monoclonal typing reagents

A general method for the production of carbohydrate-specific hybridoma antibodies is illustrated by generation of monoclonal antibody to the antigenic determinant of human blood group B. This trisaccharide determinant was chemically synthesized and covalently coupled to bovine serum albumin and human blood group O red cells. Soluble protein antigen and the 'artificial' B red cells were used to immunize BALB/c mice before fusion of spleen cells with the Sp2/0 plasmacytoma cell line. ELISA screening of putative hybrids for B-specific binding activity was facilitated by the availability of a second synthetic conjugate, B-horse hemoglobin. IgM-producing clones were identified by class-specific ELISA reagents and by hemagglutination assay. In this way, clones suitable for blood typing were rapidly identified. The precise antigenic specificity and Ig class of such monoclonal antibodies were defined by inhibition of precipitation and by gel filtration. Hybridoma antibodies were obtained from two separate fusion experiments. One of these, clone 3E-4, was of the IgM class and possessed a binding site that was completely satisfied (100% inhibition) by the trisaccharide determinant of the B blood group. This antibody is shown to be suitable for use in blood typing.     

Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial.

The human immune response to monoclonal antibody-enzyme conjugates has been studied in patients included in the pilot clinical trial of ADEPT. Each patient received murine monoclonal anti-CEA antibody fragments (A5B7-F(ab')2, conjugated to bacterial enzyme, carboxypeptidase G2 (CPG2) followed by a galactosylated monoclonal anti-CPG2 antibody (SB43), 36-48 h after the conjugate. Some patients were also given a dose of 131I-labeled conjugate (4-8 mg, 7-15 mCi) for blood clearance and gamma camera image studies. All patients studied developed human antimouse antibodies (HAMA) and anti-CPG2 antibodies within 10 d after a single course of treatment with the conjugate. In most cases, IgM response was detected at 7 d after the conjugate followed by the IgG response 14 d later. In one patient, HAMA and anti-CPG2 antibodies of the IgG type could still be detected at 10 mo after treatment. Anti-CPG2 antibodies in serum of one patient were found to inhibit CPG2 activity in vitro. Generation of neutralizing antibodies limits the use of repeat cycles of ADEPT in patients. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying the appearance of HAMA and anti-CPG2 antibodies. Patients given cyclosporin A before and during ADEPT are currently being studied for HAMA and anti-CPG2 response.     

Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial

The human immune response to monoclonal antibody-enzyme conjugates has been studied in patients included in the pilot clinical trial of ADEPT. Each patient received murine monoclonal anti-CEA antibody fragments (A5B7-F(ab')2, conjugated to bacterial enzyme, carboxypeptidase G2 (CPG2) followed by a galactosylated monoclonal anti-CPG2 antibody (SB43), 36–48 h after the conjugate. Some patients were also given a dose of¹³¹I-labeled conjugate (4–8 mg, 7–15 mCi) for blood clearance and gamma camera image studies. All patients studied developed human antimouse antibodies (HAMA) and anti-CPG2 antibodies within 10 d after a single course of treatment with the conjugate. In most cases, IgM response was detected at 7 d after the conjugate followed by the IgG response 14 d later. In one patient, HAMA and anti-CPG2 antibodies of the IgG type could still be detected at 10 mo after treatment. Anti-CPG2 antibodies in serum of one patient were found to inhibit CPG2 activity in vitro. Generation of neutralizing antibodies limits the use of repeat cycles of ADEPT in patients. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying the appearance of HAMA and anti-CPG2 antibodies. Patients given cyclosporin A before and during ADEPT are currently being studied for HAMA and anti-CPG2 response.     

Use of monoclonal antibodies in the assay of hepatitis B core antigen and antibody

Hybridoma cells secreting antibody against hepatitis B core antigen (HBc Ag) were prepared. BALB/c mice were immunized with 0.2 ml of purified HBc Ag, and their spleen cells were fused with mouse myeloma (P3U1) cells by means of polyethylene glycol 1000. Activities of antibodies against HBc Ag (anti-HBc) were tested by the immune adherence hemagglutination (IAHA) and reverse passive hemagglutination inhibition (RPHI) techniques. Hybridoma cells found to contain antibodies accounted for 26.5% by IAHA and 52.1% by RPHI, respectively. Among 32 monoclonal anti-HBc antibodies, 18 were found to be positive by both IAHA and RPHI, and the remaining 14 positive by RPHI only. After cloning, they were injected intraperitoneally into ascitic mice. The highest anti-HBc activity with an IAHA titer of 1:4 X 10(6) and with an RPHI titer of 1:1 X 10(5) was detected in this ascitic fluid. Enzyme immunoassay (EIA) and RPHI with monoclonal antibody containing the highest anti-HBc activity were developed. All the sera in which anti-HBc was detected by IAHA and RPHI with polyclonal antibody were positive in EIA. RPHI titers obtained with monoclonal antibody were in good agreement with usual IAHA and RPHI titers obtained with polyclonal antibody. These results indicate that monoclonal antibody can be used in the HBc Ag and anti-HBc assay system.     

A shortened dengue IgM capture ELISA using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody

A shortened IgM capture ELISA for the detection of dengue IgM antibodies using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody was described. The shortened two-step assay was compared with the four-step IgM capture ELISA on sera from dengue patients confirmed by the hemagglutination inhibition (HI) test. When paired acute and convalescent sera were tested, the shortened ELISA showed 100% agreement with HI results. It detected dengue IgM antibodies in the acute sera of 66% of patients with a primary dengue infection, 60% of patients with a secondary infection, and 98% of patients with a presumptive secondary infection. When the results of 151 dengue patients were combined, 75% of the acute sera were positive by the shortened IgM capture ELISA.     

Analysis of triiodothyronine and thyroxine-binding autoantibodies in chickens susceptible to autoimmune thyroiditis

This report reveals a surprisingly high incidence of thyroid hormone (T3 and T4) autoantibodies (THA) and thyroglobulin autoantibodies in a closed flock of untreated Cornell strain (CS) White Leghorn chickens. This flock is closely related to the Obese strain chicken, which develops a severe spontaneous autoimmune thyroiditis. A sensitive electrophoretic autoradiographic assay for THA was developed. This assay was applied to the study of autoantibodies to T3 and T4 in the sera of adult female CS chickens. Of 109 females, 29.4% had antibodies to T3 and 18.4% had antibodies to T4. The incidence of thyroglobulin antibodies, determined by passive hemagglutination, was 15.6%. The presence of THA affected RIA measurements because serum T3 and T4 hormone concentrations appeared elevated in those birds with moderate to high antibody levels. There was major variance in the electrophoretic heterogeneity of the THA from individual chickens; i.e., some of the sera contained antibodies to T3 or T4 that appeared to be monoclonal, whereas other sera exhibited polyclonal multi-banded patterns. To determine if antibodies reactive with T3 and T4 (which are haptens) were generated by antibody responses to the T3/T4 sites on the thyroglobulin molecule, competitive binding assays were performed to determine the relative binding affinities of the antibodies for the haptens (T3/T4) and the "hapten-conjugate" (thyroglobulin). In these assays, thyroglobulin competed with the haptens, thus supporting the above hypothesis.     

河豚毒素中和性单抗的制备、鉴定及TTX检测方法的建立

目的研制河豚毒素中和性单抗,建立基于河豚毒素单抗的河豚毒素检测方法。方法用TTX-KLH免疫Balb/c小鼠,用TTX-BSA间接ELISA筛选,建立杂交瘤细胞系,腹腔接种Balb/c小鼠诱生腹水,Protein A Sepharose CL4B亲和柱纯化,SDS-PAGE、间接ELISA鉴定;用常规法确定TTX对昆明小鼠的LD50;将单抗和TTX混合物注入小鼠腹腔,检测单抗对TTX的中和能力;建立检测TTX的竞争ELISA法。结果获得了2株TTX中和性单抗,腹水用Protein A Sepharose CL 4B纯化后抗体纯度大于95%;常规间接ELISA检测,显示单抗5E7的结合能力高于5E4。单抗对2 LD50 TTX攻击昆明小鼠的保护率为50%,建立了基于中和性单抗的TTX检测方法,TTX的最小检出浓度为1.56μg/mL。结论获得了TTX中和性单抗,对致死剂量TTX攻击昆明小鼠的保护率为50%,建立了基于中和性单抗的TTX检测方法,TTX的最小检出浓度为1.56μg/mL。     

Evaluation of iodovinyl antibody conjugates: comparison with a p-iodobenzoyl conjugate and direct radioiodination

The preparations and conjugations of 2,3,5,6-tetrafluorophenyl 5-[125I/131I]iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluorophenyl 3,3-dimethyl-5-[125I/131I]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[131I]iodobenzoate (PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-T method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was approximately a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate.     

Separation of histones from Physarum polycephalum by ion-paired, reverse-phase high-performance liquid chromatography

A rapid, one-step method for the efficient purification of murine monoclonal antibodies from tissue culture supernatants is described. This process is based on affinity chromatography on protein A-Sepharose columns. It was found that murine monoclonal antibodies raised against tick-borne encephalitis virus frequently eluted at more than one pH value and these pH values did not always correspond to those of antibodies of the same subclass from polyclonal mouse sera. The two populations of antibody molecule eluting at different pH values showed no variation in molecular weight, isoelectric profiles, specific enzyme-linked immunosorbent assay titer, or antibody subclass.     

Seroepidemiology of herpes simplex virus in Yogyakarta, Indonesia

Sera from 487 individuals in Yogyakarta, Indonesia, were tested for antibodies to herpes simplex virus (HSV) by a passive hemagglutination method. Age-specific incidence rates for antibodies to HSV were calculated. For sera from persons other than prostitutes, in the age group from 10 to 19, the positive rate was 48% but in the age group higher than 20, it was more than 87%. Fifty of 59 pregnant women (85%) were positive. The positive rate and the distribution of antibody levels in prostitutes were higher than in the general population.     

Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

Molecular cloning of ribosomal P protein in Toxoplasma gondii and the availability to detect antibody against recombinant protein in toxoplasmosis patients

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy. The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5'-RACE as 1,592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.     

Characterization of anti-Citrobacter 036 specific polysaccharide monoclonal antibodies

Monoclonal mouse antibodies specific for the 0 antigen of Citrobacter 036, a homopolymer of beta (1----2)-linked 4-deoxy-D-arabinohexose, were generated by the hybridoma technique. Balb/c mice were immunized with killed whole-cell vaccine and initial selection of active clones was based on enzyme-linked immunosorbent assay (ELISA) employing purified lipopolysaccharide (LPS). Concentrated culture supernatants from selected hybrid cultures were used to identify 10 0-antigen specific monoclonal antibodies using the multiple criteria of immunoprecipitation of 0 chains and LPS, inhibition by acid hydrolyzed 0 chains in the screening ELISA, and antibody class analysis. Four monoclonal antibodies were chosen for further study using dose-dependent 0-chain inhibition of ELISA and passive hemagglutination, passive hemolysis, and bacterial agglutination titres. When screened with Citrobacter serotypes known to contain the sugar 4-deoxy-D-arabinose, passive hemagglutination tests showed that the two monoclonal antibodies examined possessed titres which could be correlated with the reported 4-deoxy-D-arabinohexose content of the respective LPS's. This sugar is an antigenically important unit of several Citrobacter serotypes as defined by these well-characterized monoclonal antibodies.     

Comparison of Methods for Removal of Nonspecific Inhibitors of Arbovirus Hemagglutination

Human sera were treated with kaolin, acetone, and dextran sulfate to determine the best method for removing nonspecific hemagglutination inhibitors. Results indicated that on surveys for group A, group B, and some group C arbovirus HI antibodies, dextran sulfate treatment of sera could be used effectively. This method, however, has limited usefulness for detecting HI antibody for a number of arboviruses, particularly some members of the Bunyamwera supergroup since nonspecific inhibitors for these antigens were not completely removed. HI antibodies in sera drawn early after dengue and Venezuelan equine encephalitis infection were detected more readily after dextran sulfate treatment than after kaolin treatment. Kaolin, but not dextran sulfate, was shown to remove antibody from IgM fractions of sera.     

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay

A novel simultaneous measurement method for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.     

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.     

The association of enhancing antibodies with seroconversion in humans receiving a dengue-2 live-virus vaccine

A group of human subjects, some with yellow fever (YF) antibodies, volunteered for testing of a live-attenuated dengue-2 (DEN-2) vaccine. Serum samples taken before DEN-2 vaccination were tested for their ability to enhance infection of human monocytes by DEN-2 virus. A significantly greater proportion of enhancing antibodies (Eab) were found in YF-immune (YFI) individuals (50%) as compared to those with no evidence of flavivirus infection (9.5%). Geometric mean titers of neutralizing and hemagglutination inhibiting antibodies to DEN-2 virus in YFI subjects with Eab were fourfold to seven-fold higher than in the YFI subjects without Eab in prevaccine sera and 10- to 35-fold higher than in non-immune volunteers. Additionally, levels of Eab in prevaccine sera were directly related to antibody titers found in postvaccine sera. The presence of Eab in the serum of a human subject before DEN-2 vaccination was a good predictor of the immune response after vaccination, and may in part be responsible for the higher seroconversion rate in YF immunes (90%) as compared to nonimmunes (61%) receiving this vaccine. This is the first human study to demonstrate that circulating Eab in non-DEN-immune persons is associated with an augmented immune response to DEN virus infection. This finding supports the hypothesis that cross-reactive antibodies against one flavivirus enhance an infection with another closely related flavivirus.     

Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum

A specific and sensitive immunoassay based on magnetic microbead separation for schistosomiasis japonica screening is presented in this article. So far as we know, this is the first time that magnetic microbead-based enzyme-linked immunoassay (MEIA) has been used for the determination of Schistosoma japonicum (Sj) antibody in human serum. Fluorescein isothiocyanate (FITC)-labeled soluble egg antigen (SEA) and polymer-coated magnetic beads, to which anti-FITC monoclonal antibodies were immobilized, were used as separation support in MEIA. Immunoassay parameters were optimized based on a direct immunoreaction of SEA on the magnetic microbead and Sj antibody in serum samples. The laboratory experimental results showed that the MEIA method was more sensitive and more precise than traditional SEA-ELISA (enzyme-linked immunosorbent assay). In the field test, human sera collected from 513 infected humans and 2260 uninfected humans were tested with indirect hemagglutination assay (IHA), dipstick dye immunoassay (DDIA), and MEIA. IHA and DDIA were then compared with MEIA, and a lower false negative rate (0.97%) was obtained.     

Enhanced neutralization of flavivirus by monoclonal antibodies against Negishi virus

Ten monoclonal antibodies against Negishi virus were classified into 3 groups and 7 types according to the reaction patterns to Negishi virus by the hemagglutination inhibition (HI) test and by several kinds of neutralization tests. When kinetic neutralization was applied to these monoclonal antibodies at the same HI titer of 1:64, the initial slope and the persistent fraction of the kinetic curve was varied in each antibody. In the double-kinetic neutralization test, neutralization did not proceed further when the second antibody was the same type as the first one. When the second antibody was a different type from that of the first one, neutralization proceeded further. The mixtures of 4 monoclonal antibodies classified as different groups and types remarkably enhanced neutralization in the kinetic assay. These results suggested that an assortment of antibodies is needed for effective neutralization of Negishi virus and that a multi-hit model is likely operating in the neutralization of Negishi virus.