Serodiagnosis of canine Babesia gibsoni infection by enzyme-linked immunosorbent assay with recombinant P50 expressed in Escherichia coli

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.     

Immunodiagnosis of human dirofilariasis by enzyme-linked immunosorbent assay using recombinant DNA-derived fusion protein.

An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-Dirofilaria immitis recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human dirofilariasis. D. immitis-infected human sera reacted strongly with FP that was immobilized with anti-beta-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other filariasis. In detection of anti-D. immitis IgG antibody. ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

Description of an enzyme-linked immunosorbent assay for the detection of protein tyrosine kinase

A solid immunoassay for the detection of protein tyrosine kinases has been developed. It is based on the binding of the synthetic polypeptide poly(Glu.Na,Tyr) 4:1 to microELISA wells, where the phosphorylation reaction takes place in the presence of ATP and enzyme. The phosphorylated tyrosine residues produced in the reaction are finally detected, in the same well, by means of an ELISA using monoclonal antiphosphotyrosine antibody, peroxidase-labeled goat anti-mouse IgG antibody, and substrate. The amount of protein tyrosine kinase activity present in the sample is proportional to the color at 492 nm developed in each well.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B2 (TXB2). As a specific antigen, the bovine serum albumin conjugate of TXB2 was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB2 per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB2 per sample. The suitability of the assay was checked with TXB2-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r = 0.960 and r = 0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r = 0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

An enzyme-linked immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of rabbit serum antibody directed against the causative agent of Tyzzer's disease, Bacillus piliformis. Ninety-four percent agreement was found between the ELISA and an indirect fluorescent antibody test. The sensitivity of the ELISA was 95% and its specificity was 92% as compared to the indirect fluorescent antibody test (IFAT). The rabbit origin B. piliformis isolate used in this ELISA was found to be cross-reactive by ELISA and IFAT to B. piliformis isolates of rat, gerbil and horse origin. This suggests that a single B. piliformis isolate may be used as antigen for an ELISA utilizable for multiple species.     

Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate

A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 μl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n = 96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Development of an enzyme-linked immunosorbent assay system for the determination of asymmetric dimethylarginine using a specific monoclonal antibody

We produced a monoclonal antibody (mAb) against N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM-100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens

Background

Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection.

Methods

RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006–2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems.

Results

rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%.

Conclusions

Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

     

Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escherichia coli.

Several nonoverlapping epitopes were mapped on the primary sequence of the Sendai virus NP protein. After a complete cDNA clone of the Sendai virus NP gene was expressed in Escherichia coli, deletion constructs were used to generate a series of overlapping NP fragments deleted at their C termini. Immunoblot analyses with 11 monoclonal antibodies identified four antigenic sites. All of these sites resided in the C-terminal half of NP and were also the only sites detected with a polyclonal serum. These findings confirm and extend the evidence that the C terminus of the NP protein represents the domain exposed on the surface of the nucleocapsid. One of the monoclonal antibodies reacted with a site, comprising only 6 amino acids, lying with a hinge between an alpha-helix and a beta-strand in the predicted secondary structure of NP. Since this antibody is a potent inhibitor of in vitro viral RNA synthesis (K. L. Deshpande and A. Portner, Virology 139:32-42, 1984), the epitope may be critical to the flexibility of the NP molecule that makes the RNA template accessible during RNA synthesis.     

Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli

The surface antigen 1-related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli, and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum-positive and -negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum. Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.     

Detection of Escherichia coli O157:H7 in meat by an enzyme-linked immunosorbent assay, EHEC-Tek.

Investigation of the specificity of an enzyme-linked immunosorbent assay (ELISA) for detection of Escherichia coli O157:H7 in raw meats (EHEC-Tek; Organon Teknika Corp.) revealed that the target antigens of the detection reagent, monoclonal antibody 4E8C12, were present in numerous serotypes of E. coli and that their ELISA reactivity was influenced by bile salts, acriflavine, and heat. The specificity of the ELISA was improved by a modified test protocol incorporating immunocapture.     

Improvements in the microtitre GM1 ganglioside enzyme-linked immunosorbent assay for Escherichia coli heat-labile enterotoxin

A variant of the microtitre GM1-ELISA for Escherichia coli heat-labile enterotoxin was studied. The test was improved by both reducing the assay time from 2 1/2 d to 8 h and by determining the most appropriate GM1 coating concentration. Coating the plates with greater than or equal to 3 micrograms of GM1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure. Thus an optimal accuracy was obtained. This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay. The sensitivity of this ELISA and of the chinese hamster ovary cell monolayer assay were identical.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B₂ (TXB₂). As a specific antigen, the bovine serum albumin conjugate of TXB₂ was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB₂ per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB₂ per sample. The suitability of the assay was checked with TXB₂-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r=0.960 and r=0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r=0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli.

A colony enzyme-linked immunosorbent assay using the hydrophobic grid membrane filter format was developed for the isolation of verotoxigenic Escherichia coli from human and food samples. The method utilizes monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1- and/or 2-producing serotypes. E. coli that produced a minimum of 2 x 10(2) and 2 x 10(3) 50% cytotoxic doses per ml of verotoxins 1 and 2, respectively, were detectable. In a method comparison using human stool specimens, this procedure isolated 29% more E. coli O157 than did the standard sorbitol-MacConkey agar procedure, with no false-positive reactions. When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. coli of a variety of serotypes including O157:H7. Four false positives were noted. This procedure provides a sensitive means for the isolation of verotoxigenic E. coli and should facilitate recovery of those serotypes that are otherwise indistinguishable from nonpathogenic strains.     

Detection of serum antibodies to Bacillus piliformis in mice and rats using an enzyme-linked immunosorbent assay.

Rats and mice were infected with Bacillus piliformis organisms at a dosage which resulted in clinical signs of Tyzzer's disease in gerbils. Although rats and mice did not show clinical signs of disease, rising antibody titers to B. piliformis were detected by enzyme-linked immunosorbent assay (ELISA) 2 to 6 weeks post-inoculation and remained at positive levels 11 weeks post-inoculation. Western blot analyses of sera from experimentally infected animals revealed banding patterns nearly identical to those obtained using hyperimmune serum. Results indicated that elevated ELISA titers reflected production of specific antibodies directed against antigens of B. piliformis. ELISA and Western blot analyses of naturally infected animals yielded similar results. These findings suggest that immunoassays such as ELISA can be used to detect subclinically infected rats and mice in the absence of clinical signs or histopathologic evidence of Tyzzer's disease.     

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.