Validation of a newly generated oxytocin antibody for enzyme-linked immunosorbent assays

The biological and psychological significance of oxytocin is increasingly recognized; however, reliable assays of oxytocin in biological samples have not been developed. We raised a new oxytocin polyclonal rabbit antibody against synthetic oxytocin. The affinity of antibodies to oxytocin was examined by a radio-immunoassay and compared with that of a previously validated antibody. One antibody showed affinity for oxytocin in the radio-immunoassay. We developed a solid-phase ELISA for oxytocin using this antibody and compared it with existing methods. The newly developed ELISA showed comparable results using urine samples but not using serum samples. These results indicate that the new ELISA is useful for urinary oxytocin; further modifications, such as different extraction methods, are needed for its application to serum oxytocin.     

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.     

Characterization of Campylobacter jejuni in large-billed crows (Corvus macrorhynchos) in Tochigi prefecture,Japan

As free-living crows are a potential source of Campylobacter infections in broilers and cattle, we characterized Campylobacter spp. isolated from crows using multilocus sequence typing and antimicrobial susceptibility testing. We obtained 82 samples from 27 birds captured at seven different times using a trap set in Tochigi prefecture, Japan. Campylobacter jejuni was isolated from 55 (67.1%) of the 82 samples and classified into 29 sequence types, of which 21 were novel. Tetracycline and streptomycin resistance rates were 18.2% and 3.6%, respectively. These results show that most types of C. jejuni infecting crows differ from those isolated from humans, broilers, and cattle. Thus, the importance of free-living crows as reservoirs of Campylobacter infections in broilers and cattle may be limited.     

Detection of flagellar antigen ofCampylobacter jejuni andCampylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA) — New prospects for diagnosis

A new diagnostic procedure was developed to detect the flagellar antigen ofCampylobacter jejuni andCampylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected allC. jejuni orC. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative forC. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens ofC. jejuni andC. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.Abbreviations ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - OD optical density     

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.     

地方流行性牛白血病间接gp51-ELISA抗体检测试剂盒的研制

利用重组牛白血病病毒gp51蛋白作为包被抗原研制了gp51-ELISA诊断试剂盒.试剂盒主要成分包括抗原gp51包被96孔板、HRP标记的兔抗牛IgG和牛白血病阴性、阳性标准血清.对试剂盒进行了特异性、灵敏度、重复性及保存期试验.结果表明,试剂盒特异性好,灵敏度是AGlDT的4～8倍,批间和批内的变异系数分别小于10%和15%,于一20℃保存至少能保持6个月检测结果稳定.与法国Synbiotics公司同类试剂盒进行比对试验,诊断敏感性、特异性和符合率分别为75%、96.1%和94.6%.对采集于山东济南的164份奶牛血清和进口澳大利亚奶牛的398份血清进行检测,抗体阳性率分别6.10%和4.77%.     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的初步建立

为了研究玉米赤霉烯酮的间接竞争ELISA检测方法,试验采用牛血清白蛋白与玉米赤霉烯酮的耦联物(ZEN-BSA)做包被抗原,标准玉米赤霉烯酮(ZEN)做竞争抗原,以制备的可稳定分泌抗ZEN的单克隆抗体为基础,初步建立了ZEN间接竞争ELISA检测方法。结果表明:间接竞争ELISA检测方法线性范围为0.363 2～78.985 2μg/L,最低检测限为0.231 9μg/L;曲线回归方程为y=68.711-25.666x,其中R2=0.987 1,批内平均变异系数为3.10%,批间平均变异系数为6.26%,与相似毒素的交叉反应率均小于0.01%。说明建立的检测方法可以用于ZEN的检测。     

Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID₅₀)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.     

Development of an indirect enzyme-linked immunosorbent assay for detecting equine serum antibodies to the lipopolysaccharide of Salmonella abortusequi

An indirect enzyme-linked immunosorbent assay (IELISA) was developed for the detection of equine serum antibodies to lipopolysaccharide of Salmonella enterica subsp. enterica serovar Abortusequi (LPS), a causative organism of Equine Paratyphoid. The data presented demonstrates that horses immunized with S. abortusequi LPS developed antibodies detectable by the IELISA. By comparison, the tube agglutination test (TAT) did not detect antibody to S. abortusequi LPS as consistently as the IELISA. The data suggests that the IELISA may be a more suitable test for the detection of serum antibodies to S. abortusequi than the TAT.     

Detection of Corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbant assay was developed to measure naturally occurring Corynebacterium equi specific antibody in horse serum. Antibody against C equi was demonstrated in normal adults and was passively transferred to foals. Adult levels of specific antibody were reached by 5 to 6 months of age in healthy foals. Decreased early antibody levels were demonstrated in a limited number of foals with confirmed C equi infection.     

Protective efficacy of stockpiled vaccine against H5N8 highly pathogenic avian influenza virus isolated from a chicken in Kumamoto prefecture,Japan, in 2014

H5 highly pathogenic avian influenza (HPAI) viruses have spread worldwide, andantigenic variants of different clades have been selected. In this study, the nationalstockpiled vaccine prepared from A/duck/Hokkaido/Vac-1/2004 (H5N1) strain was evaluatedfor the protective efficacy against H5N8 HPAI virus isolated in Kumamoto prefecture,Japan, in April 2014. In the challenge test, all of the vaccinated chickens survivedwithout showing any clinical signs and reduced virus shedding. It was concluded that thepresent stockpiled vaccine was effective against the H5N8 HPAI virus.     

Detection of antibodies against Akabane virus in bovine sera by enzyme-linked immunosorbent assay

An enzyme-linked immunonosorbent assay was established for detection of antibodies to Akabane virus in bovine sera. The assay was shown to be a useful serological tool for studies on Akabane virus infection.     

An enzyme-linked immunosorbent assay using canine coronavirus-infected CRFK cells as antigen for detection of anti-coronavirus antibody in cat

From the reasons that canine coronavirus (CCV) grows more efficiently than feline coronavirus in a cell culture and they are mutually related in their antigenicities, an enzyme-linked immunosorbent assay (ELISA) using CCV-infected feline kidney (CRFK) cells as substrate antigens was developed for detection of anti-coronavirus antibodies in cats. It was indispensable for generating coronavirus-specific ELISA antibody activities that the sample was applied to the mock-infected, normal CRFK cells in parallel with the CCV-infected cells and then the optical density values given by the mock-infected cell antigen were subtracted from those given by the virus-infected cell antigen. On the basis of ELISA antibody titers obtained in sera from the cats experimentally infected with CCV and from the spontaneous feline infectious peritonitis (FIP) cases, the ELISA described in the present study was found to be applicable as a simple and easy serologic test which was able to detect anti-coronavirus antibodies as efficiently as the indirect immunofluorescence assay with homologous FIP virus.     

Prevalence of trichinosis in Canadian swine determined serologically by enzyme-linked immunosorbent assay.

Fifteen thousand three hundred and eighteen porcine sera from all regions of Canada were examined for the presence of anti-Trichinella antibodies using the enzyme-linked immunosorbent assay with an excretory-secretory antigen. Four sera (0.026%) revealed the presence of anti-Trichinella antibodies, with titers (optical density readings) that fell in the low positive or high negative range on repeated examinations. One animal originated in British Columbia and three in Ontario. Serological examination of swine in the herds at time of traceback did not reveal further animals with anti-Trichinella antibodies.     

Demonstration of antibodies to Eimeria species in lambs by an enzyme-linked immunosorbent assay

Soluble extracts were prepared from sporulated oocysts, unsporulated oocysts and merozoites of Eimeria crandallis, E faurei and E ovinoidalis. They were assessed for antigenicity and specificity by ELISA using rabbit antisera to sporulated oocysts or merozoites. Antibody levels were examined in sera from colostrum-deprived coccidia-free lambs, conventionally reared lambs and lambs which had received experimental infections. Maternal antibody was demonstrated in colostrum and in serum taken at 24 hours from all conventionally reared animals but not colostrum-deprived animals. Antibody levels in conventional animals dropped over the first five weeks of life and rose again during the next five weeks. Antibody was not detected in coccidia-free animals. Monospecific infections of E faurei or E ovinoidalis demonstrated antibody responses to primary and secondary infections. Some specificity of response was suggested with E faurei infection. The antigen preparations showed considerable cross-reactions between species. These serum antibody responses, although appearing too late for individual diagnosis, may assist diagnosis on a flock basis.     

Prevalence of interfering antibodies in dogs and cats evaluated using a species‐independent assay

Background

Interfering antibodies in human serum and plasma are known to react with mammalian antibodies in immunoassays and cause false‐positive test results. Although this phenomenon was recently shown in companion animals, knowledge regarding immunoassay interference in veterinary medicine is very limited.

Objectives

The aims of this study were to set up a species‐independent immunoassay procedure to detect interference in serum samples, to screen for interference in a cross‐section of canine and feline patient samples from an animal hospital, and to determine if the detected interference could be neutralized using an immunoassay based on nonmammalian reagents.

Methods

A 2‐site sandwich‐type interference assay was set up using commercially available mouse reagents. A total of 369 serum samples from 320 dogs and 263 samples from 218 cats were analyzed using the interference assay. Multiple samples were submitted from 36 dogs and 39 cats. Nineteen samples identified as interference‐positive were analyzed in an assay using chicken antibodies.

Results

Interference was detected in samples from 28 dogs (9%) and 10 cats (5%) screened with the interference assay. Except for 1 cat, consistent results were obtained for all 75 dogs and cats that submitted more than 1 sample. The interference was eliminated when analyzed in the chicken‐based assay (P < .001).

Conclusions

Substances with reactivity toward mouse IgG can be detected in serum samples from dog and cat patients using a 2‐site interference assay. The detected substances are most likely interfering antibodies, possibly originating from immunization with other mammalian species.