Measurement of antibody to Jo-1 by ELISA and comparison to enzyme inhibitory activity

Antibody to the Jo-1 antigen (histidyl-tRNA synthetase) is found almost exclusively in myositis patients, usually those with adult PM, but has been found in only 30% of that group by immunodiffusion or other techniques thus far reported. We have reexamined the prevalence of antibody to Jo-1 in sera from 130 patients and 82 controls by using the sensitive ELISA technique. The ELISA used affinity-purified, enzymatically active bovine Jo-1 antigen. A wide range of antibody level by ELISA was found among 24 immunodiffusion positive sera. Six myositis and two control sera had apparent specific antibody detectable only by ELISA. Overall, however, the antibody continued to show high myositis specificity with predominance in adult PM (35.8% in that group). Because the antibody inhibits enzymatic activity of the synthetase antigen, we also studied the quantitative inhibitory activity of these sera to compare with the antibody activity as determined by ELISA. Twenty-four immunodiffusion-positive sera, 29 immunodiffusion-negative sera, and 15 normal sera were tested at 1/50 dilution in the reaction mixture. There was background inhibition by all normal sera tested that averaged 30.5%. All but one immunodiffusion negative myositis sera (a high binder by ELISA) inhibited less than 50% of the average with normal serum. Twenty-three of 24 immunodiffusion positive sera inhibited greater than 80% of this normal average; the other inhibited 66%. The serum dilution giving 50% inhibition was highly correlated (R = 0.83) with the ELISA activity. Thus, inhibition of histidyl-tRNA synthetase activity is a relatively accurate measure of Jo-1 antibody. This method should be applicable to measuring antibody to other aminoacyl-tRNA synthetases.     

ELISA for the detection of specific IgM and IgG in human leptospirosis

ELISA was used to detect specific IgM and IgG in sera from humans with current or past leptospirosis. A serological pattern of a high IgM titre (greater than or equal to 1280), or moderately increased IgM (160-640) in conjunction with a low IgG titre (less than or equal to 20), with serovar copenhageni antigen was characteristic for approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in the first two months of the disease. The antigen was the supernatant of a heated and centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni, grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa and hardjo infections showed cross-reactivity when different antigens were used. In past infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM and IgG allows the rapid diagnosis of acute leptospirosis.     

ELISA for the routine determination of antitoxic immunity to tetanus

Serum samples from 727 persons with different vaccination histories were assessed for tetanus antitoxin content in an enzyme linked immunosorbent assay (ELISA) and tested for tetanus toxin neutralization activity in mice in order to compare the results obtained by the two methods. Neutralizing antibody activities in sera from individuals previously completely vaccinated correlated well with results obtained by ELISA and the accuracy increased with increasing antitoxin concentration in serum. This correlation was observed in sera from persons vaccinated recently as well as in sera from persons vaccinated many years ago. In sera from persons with an incomplete vaccination history ELISA was found to be an unreliable tool for the prediction of in vivo results. Many of these sera had antitoxin levels by ELISA far above the in vivo values, probably due to the presence of non specific or low avidity antitoxin which is detected in ELISA. The lowest ELISA value reliably predictive of protective antibody activity in serum irrespective of vaccination history was found to be 0.16 IU/ml. It was concluded that ELISA is useful for larger population studies as an initial test, but sera with an antitoxin content below 0.16 IU/ml should also be assessed in a neutralization system.     

Schistosoma mansoni: immunodiagnosis is improved by sodium metaperiodate which reduces cross-reactivity due to glycosylated epitopes of soluble egg antigen

ELISA with soluble egg antigen (SEA) from Schistosoma mansoni is widely used in the diagnosis of schistosomiasis, but cross-reactivity with other intestinal helminths, overestimating the true prevalence, represents a great limitation. The role of glycoproteins of SEA in cross-reactions was investigated. SEA was oxidized with sodium metaperiodate (SMP) in ELISA and immunoblot. One hundred schistosomiasis-negative individuals sera were submitted to SMP-ELISA improving the specificity from 73% without SMP treatment to 97% with SMP. On the other hand, 94 S. mansoni-positive sera were evaluated showing that 99% were positive in ELISA either with or without SMP treatment, indicating the maintenance of high sensitivity under SMP treatment. By immunoblot, 24 sera from persons with schistosomiasis and 10 sera from schistosomiasis-free persons were assayed under reducing and nonreducing conditions with or without SMP, looking for specific infection markers and cross-reactivity markers. Reactivity from positive sera showed that specific molecules were mainly low-molecular-mass antigens and seem to have predominant proteic epitopes. The unspecific molecules reacting with some schistosomiasis-negative individuals harboring other intestinal parasites (false-positive sera) were mostly larger than 60 kDa and seemed to be basically glycosylated. Glycosylated epitopes have an important role in cross-reaction and SMP can successfully be used to reduce the false reactivity of SEA with no decrease in sensitivity, especially in ELISA as an immunodiagnostic screening surveillance method, which is useful in areas of low schistosomiasis transmission.     

Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

Determination of antibodies to Streptococcus group A polysaccharide in human sera by an immunoenzyme method

Use was made of the ELISA to develop a highly sensitive quantitative method for detection of antibodies against Streptococcus group A polysaccharide (polysaccharide A) in human sera. The main advantage is that one can use only one optimal dilution of the sera together with the reference serum. Sera of 53 healthy volunteers and 77 patients with a history of Streptococcus group A infections were screened for the presence of polysaccharide A antibodies. Highly reproducible results were obtained in 97% of cases. The specificity of the method was shown with the polysaccharide A-induced inhibition of the reaction. Positive reactions obtained with the tested sera in gel immunodiffusion correlated with the data derived by the ELISA. Using the latter high level of specific antibodies was found in some of the sera that yielded negative reactions when tested by gel immunodiffusion. This may be associated with the presence of non-precipitating antibodies.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.     

Expression of the porcine circovirus type 2 capsid protein subunits and application to an indirect ELISA

Porcine circovirus type 2 (PCV2) is considered to be associated with post-weaning multisystemic wasting syndrome (PMWS), which is a newly emerged economically important swine disease. The entire coding region of open reading frame 2 (ORF2), encoding the viral capsid protein (Cap), of PCV2 was cloned and sequenced from the clinical specimen obtained from PMWS-affected piglets. Six recombinant subunits, A-F, spanning the defined regions of Cap were produced by E. coli expression system and used as antigens for testing their reactivities with swine sera in the indirect ELISA. The recombinant Cap subunit-based ELISA was evaluated by examining a panel of 12 PCV2-negative and 26 PCV2-positive sera. When the positive/negative cut-off value was set at the mean value of negative sera plus 3 standard deviations, all subunits-based ELISA demonstrated 100% specificities. The N-terminal subunits, A and B, revealed poor reactivity with positive swine sera, whereas, greater immunoreactivity was observed for the C-terminal subunits of which subunits C and D demonstrated good sensitivities of 96.2% and 84.6%, respectively. The recombinant Cap subunits possessing defined antigenicity are easy to produce and the subunit-based ELISA was developed with a high specificity and sensitivity that may provide a useful method for routine serodiagnosis of PCV2 infection.     

Yolk-sac transmission and post-hatching ontogeny of serum immunoglobulins in the duck (Anas platyrhynchos)

1. To investigate the immunoglobulin (Ig) class which is active in yolk-sac transmission of maternal antibodies in ducks, sera from laying ducks, yolks from their eggs, and sera from ducklings 0-87 days of age were examined by immunoelectrophoresis (IE), and Na2SO4-precipitated Igs from these sera and yolks were run in polyacrylamide gel electrophoresis (PAGE) under reducing conditions. 2. In yolk, 7.8S IgG was greatly enriched compared to 5.7S IgG and IgM, and was the only Ig to reach the sera of newly hatched ducklings. 3. The levels of maternally derived 7.8S IgG in duckling sera decreased after 5 days of age, reaching minimum levels at about 14 days of age. 4. Increases in the serum levels of 7.8S IgG, 5.7S IgG and IgM occurred after 20 days of age, reflecting de novo synthesis by the duckling, and the adult serum profile was achieved by 71 days of age. 5. The involvement of 7.8S rather than 5.7S IgG in yolk-sac transmission was probably determined by the additional heavy chain components of this molecule.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Sendai virus antibody in mouse and guinea pig sera by an enzyme-linked immunosorbent assay with protein A

An enzyme-linked immunosorbent assay using horseradish peroxidase (HRPO)-labeled protein A (P-ELISA) was established for detection of Sendai virus (SV) antibody in mouse and guinea pig sera. Sensitivity and specificity of P-ELISA were compared with those of ordinary ELISA using HRPO-labeled immunoglobulin G (IgG-ELISA) and the hemagglutination inhibition (HI) test. P-ELISA was 100 to 1,000 times more sensitive than the HI test for detection of the antibody in SV-naturally infected mice. P-ELISA and IgG-ELISA showed similar sensitivities for detection of the antibody in naturally infected mouse and guinea pig sera. A high specificity was demonstrated in P-ELISA with a cut-off optical density value of 0.2 (492 nm), while a non-specific reaction was observed when IgG-ELISA was used to both mouse and guinea pig sera at a low dilution (1:10-20). The antibody in rat sera was not detected by P-ELISA although it was realized by IgG-ELISA.     

A new capture test using conjugated peptides for the detection of HIV antibodies.

A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591-611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314-329) IRIQRGPGRAFVTIGK and the p27 sequence (182-198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N-hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen

Aims

A new liquid-phase piezoelectric immunosensor (LP-PEIS), which can detect Schistosoma japonicum (Sj) circulating antigens (SjCAg) quantificationally, was developed.

Methods

The IgG antibodies were purified from the sera of rabbits which had been infected or immunized by Sj and were immobilized on the surface of piezoelectric quartz crystal in LP-PEIS by staphylococcal protein A (SPA). It was used to detect SjCAg in sera of rabbits which had been infected by Sj in order to acquire some optimum conditions for detecting SjCAg. Finally, the LP-PEIS with optimum conditions was used to detect SjCAg in sera of patients who had been infected by Sj, and was compared with sandwich ELISA.

Results

A lot of optimum conditions of LP-PEIS for detecting SjCAg had been acquired. In the detection of patients' sera with acute Schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%). However, there were no significant difference between LP-PEIS and sandwich ELISA.

Conclusions

LP-PEIS can quantificationally detect SjCAg in patients' sera as well as sandwich ELISA.     

Prealbumin locus in chickens

Three different phenotypes of a weakly staining prealbumin migrating near the borate line during starch gel electrophoresis were found in chicken sera and egg yolks. It is shown that the synthesis of these phenotypes is determined by one autosomal locus with two alleles, Pa ^A and Pa ^B.
The paper also presents the results of partial isolation of polymorphic prealbumins from cock sera. Rivanol®– ammonium sulphate precipitation and chromatography on Sephadex G-200® were used for this purpose.     

Toxoplasma gondii infection: analysis of serological response by 2-DE immunoblotting

The potency testing of Clostridium perfringens mono- and multicomponent veterinary vaccines is currently performed with the mouse neutralisation test (MNT) to estimate levels of C. perfringens beta- and epsilon-antitoxin levels in the sera of rabbits immunised with the vaccine. Two in vitro methods based on monoclonal antibodies (mAb) have been developed for the determination of specific antibodies against C. perfringens beta-toxin (capture ELISA) and epsilon-toxin (competitive ELISA) in these sera. Both test systems show high specificity and good reproducibility. These ELISA procedures were used in addition to the routine batch potency test in mice (MNT) to determine beta- and epsilon-antitoxin levels in 523 samples of rabbit serum. There was good agreement between the rank order of sera determined in vivo and the rank order determined in vitro. Linear regression analysis gave correlation coefficients of 0.88 for the capture ELISA and 0.41 for the competitive ELISA, with a significance level of P < 0.01 in both cases. Furthermore, a prevalidation study was carried out in four laboratories to evaluate the transferability of the ELISA procedures and the interlaboratory reproducibility of the results. Three coded serum samples were tested several times. The results indicated that both ELISA systems are suitable candidates to replace the MNT used for the potency testing of beta- and epsilon-toxoid in mono- and multicomponent veterinary vaccines. However, these assays still need to be validated in an international collaborative study.     

A new capture test using conjugated peptides for the detection of HIV antibodies

Abstract A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591–611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314–329) IRIQRGPGRAFVTIGK and the p27 sequence (182–198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N -hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

口蹄疫病毒非结构蛋白3A、3B和2C基因的表达及产物纯化与活性检测

[目的]口蹄疫病毒(FMDV)非结构蛋白(NSP)3A、3B和2C基因的表达及产物纯化与活性检测.[方法]利用原核表达系统表达了FMDV NSP 3A、3B和富含B细胞抗原位点序列的2C蛋白.利用高浓度尿素裂解包涵体,采用稀释法和氧化型、还原型谷胱甘肽系统相结合方法对2C蛋白进行复性.用金属鳌合亲合层析的方法对表达的FMDV NSP 3A、3B和2C进行纯化.采用ELISA方法对比检测了3种纯化蛋白在检测羊血清NSP抗体的效果.[结果]检测得知3A和3B为可溶性表达蛋白,2C以包涵体形式表达.通过Western-blot分析,表明纯化后蛋白能与FMDV感染动物血清发生特异性反应.纯化的3A、3B和复性后的2C融合蛋白与3ABC抗原的检测结果具有很高的符合性.[结论]该研究为建立鉴别FMDV自然感染动物和灭活疫苗免疫动物的酶联免疫电转移印迹技术(EITB)提供了所需的材料.     

Modification of the ELISA for the estimation of tetanus antitoxin in human sera

The use of indirect ELISA for the quantitation of tetanus toxin neutralizing antibodies in human sera is limited by marked overestimations in low titered sera. The reasons for the discrepancy between the results obtained by ELISA and by in vivo assay and modifications of the ELISA to overcome the problem were investigated. Catching ELISA and indirect ELISA using trays coated with the contaminant proteins in toxoid preparations indicated that antibodies to contaminants were only partly responsible for the discrepancy and the introduction of these modifications did not solve the problem. In ELISA competition experiments with toxin neutralizing monoclonal antibodies, the human immunoglobulins irrelevant in toxin neutralization, but detectable in indirect ELISA, were found to be difficult to inhibit in their binding to the solid antigen phase. These might represent antitoxins bound bivalently to the solid phase but with affinities in monovalent binding insufficient for toxin neutralization or other coupled antibodies due to conformational changes of the antigen. A competition ELISA with toxin in solution was therefore developed to assess selectively the antitoxin capable of binding the antigen in solution and by this approach the in vivo activities of even low titered sera were accurately predicted. This antigen competition ELISA may be easily introduced into routine tetanus serology and the principle may also be of value for the in vitro detection of functional antibodies to other antigens.     

Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.