Immunoblot diagnosis of infection with Neospora caninum in cattle based on recombinant NcSAG4 Antigen

The Neospora caninum (N. caninum) NcSAG4 gene was subcloned into a pET-28a (+) vector and successfully expressed in Escherichia coli as inclusion body, and confirmed by SDS-PAGE and Western blot using anti-His monoclonal antibody. The purified protein was then purified using Ni-NTA affinity chromatography column and recognized by positive serum from N. caninum-infected cattle. Immunoblot (IB) method based on purified recombinant NcSAG4 (rNcSAG4) antigen to detect antibodies against N. caninum in cattle was developed. Subsequently, both IB and ELISA kit were used to test sera (52) from naturally infected/uninfected cattle. Results showed that 50 and 48 out of 52 was positive for IB and ELISA kit, respectively, revealing that IB is more or at least as sensitive as ELISA when used for serodiagnosis of infected cattle     

Development of Rapid Immunochromatographic Test with Recombinant NcSAG1 for Detection of Antibodies to Neospora caninum in Cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

Development of an immunochromatographic test with recombinant EMA-2 for the rapid detection of antibodies against Babesia equi in horses

An immunochromatographic test (BeICT) for the rapid detection of antibodies against Babesia equi was developed. It clearly differentiated B. equi-infected horses from B. caballi-infected and uninfected horses. The agreement with enzyme-linked immunosorbent assay results was 96.7% in the detection of field sera. The results suggest that BeICT is rapid, simple, reliable, and suitable for use to detect B. equi infection in the field.     

Sensitive and specific identification of Neospora caninum infection of cattle based on detection of serum antibodies to recombinant Ncp29

Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Comparative study for the detection of antibodies to Neospora caninum in milk and sera in dairy cattle in southern Romania

The study aimed to assess the within-herd Neospora caninum exposure in dairy cattle in southern Romania, based on the detection of specific antibodies in milk and serum. A total of 104 paired samples of milk and serum were collected from four dairy farms. Individual samples were analyzed for N. caninum antibodies by ELISA: IDEXX Neospora Ab (Idx) (three farms: A, B, C; n = 60) and ID-VET Lab (Idv) (farm D; n = 44). Additionally, four pooled milk samples, one per each farm (A, B, C) and a composed one (A+B+C), were analyzed with Idx ELISA. Optimized cut-off values for milk samples were determined by receiver operating characteristic (ROC) analysis, with serum results considered as true status. The agreement was expressed by K values. The overall seroprevalence of N. caninum infection was 45% in the farms tested by Idx ELISA and 56.8% in the farm tested by Idv ELISA. A good agreement between serum and milk was obtained for both ELISA kits (K = 0.72 and 0.77, respectively). The specificity and sensitivity at optimized cut-off of S/P>0.704 for Idx and S/P%>7.966% for Idv were 100% and 70.37% for Idx and 89.47% and 88% for Idv. Testing pooled milk samples, there were identified as N. caninum positive the dairy farms with a 15% or higher within-herd seroprevalence at the cut-off value of S/P>0.51. This is the first study in Romania in which milk samples were tested to determine the N. caninum infection status in dairy farms, providing a base for further researches.     

Performance characteristics of a rapid new immunochromatographic test for detection of antibodies to human immunodeficiency virus

A new immunochromatographic rapid test (Rapid Check HIV 1 and 2; Núcleo de Doen?as Infecciosas) for the detection of antibodies to human immunodeficiency virus type 1 and type 2 in human samples (whole blood, serum, and plasma) was evaluated and compared to the commercially available Determine (Abbott Laboratories). When whole-blood samples were evaluated, the specificity and sensitivity of both tests were 100%. However, when plasma samples were used, sensitivity for the Rapid Check HIV 1&2 and the Determine tests were 100 and 98.58%, respectively. The observed specificity for plasma samples was 98.94% for the Rapid Check HIV 1&2 and 96.97% for the Determine test. The results presented here are encouraging and support the adoption of both tests as an alternative to enzyme-lined immunosorbent assay and/or Western blots in regions where laboratorial infrastructure is not available or for use in the management of occupational accidents for healthcare workers.     

Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats

An immunochromatographic test using recombinant truncated surface antigen 2 for detection of antibodies against Toxoplasma gondii was developed. Evaluation of detection of the antibody in mice and cats suggests that this test is rapid, simple, accurate, relatively inexpensive, and suitable for use under field conditions.     

Serological response over time to recombinant Neospora caninum antigens in cattle after a neosporosis-induced abortion.

Recombinant Neospora caninum tachyzoite antigens were evaluated in an enzyme-linked immunosorbent assay (ELISA) for recognition by serum antibodies (Ab) from Neospora-infected cattle. Serum samples were obtained every 2 to 3 weeks for 8 to 15 months from 10 cows with histories of Neospora-associated abortion. Serum samples were also obtained from offspring of these animals and from a large number of cows that had aborted a fetus, due to infection by Neospora or other organisms, at various times during gestation. All 10 cows had positive ELISA Ab titers to both recombinant N. caninum tachyzoite antigens after abortion, during subsequent gestation, and after parturition. In three cows, there was a noticeable peak in Ab titers early in gestation. Calves born to Neospora-infected cows also had positive titers of Ab to the recombinant tachyzoite antigens, and these titers remained elevated for at least 4 months after birth. A portion of the serum immunoglobulin in calves may have been derived from colostrum of infected cows. A calf born from a seronegative mother had a positive ELISA titer only after being fed colostrum from a seropositive cow. However, precolostral titers in calves born from Neospora-infected cows were high at birth, suggesting that the parasite was transmitted to the fetus via the placenta and induced a humoral immune response therein. The recombinant tachyzoite antigens were also useful for corroborating clinical diagnoses of Neospora-induced abortion. A significant difference (P < 0.05) between anti-recombinant antigen Ab titers in cows that aborted due to Neospora and those in cows that aborted from other causes was found.     

Development of an immunochromatographic test to detect antibodies against recombinant Em18 for diagnosis of alveolar echinococcosis

An immunochromatographic test (ICT) for the rapid detection of antibodies to Echinococcus multilocularis was developed. The ICT showed a sensitivity of 94% and a specificity of 95.4%. High degrees of agreement were observed between the ICT and an enzyme-linked immunosorbent assay (κ = 0.93) and between the ICT and immunoblot analysis (κ = 0.97). It is expected that the ICT developed in this study will be useful for the serodiagnosis of alveolar echinococcosis.     

Development of a lateral-flow immunochromatographic test device for the rapid detection of difloxacin residues

In this study, a rapid competitive immunochromatographic test strip has been developed for specifically testing residues of difloxacin (DIF). 1H₁₀-2B₂, one of the three hybridoma lines that were tested and selected by enzyme linked immunosorbent assay, was used in the test strip. The monoclonal antibody has a good sensitivity with an IC₅₀ of 2.2 ng/mL to DIF, 0.24% cross-reactivity towards danofloxacin and no cross-reactivity towards other related compounds and other drugs. The calibration curve of DIF test strip presented a typical sigmoidal curve. In practice, the lower limit of detection using a strip reader was 0.5 ng/ml while 2 ng/ml by unaided visual assessment. Samples of milk were spiked at 2–8 ng/mL, presenting recoveries between 94.5 and 107%, and the coefficient of variation (CV,%) (1.6–9%). The data above suggests that the strip meets the requirements of high sensitivity, good specificity, simplicity and speed, as well as the characteristics of reproducibility and accuracy.     

Development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus

The genes encoding the Erns and E2 antigen epitopes of classical swine fever virus (CSFV) were expressed as a chimeric protein in Escherichia coli BL21 by pET expression system. The antigenicity of the expressed protein CnC2 was identified by indirect enzyme-linked immunoabsorbant assay (ELISA) and immunoblot with anti-CSFV antibodies. Based on the CnC2 protein, an immunochromatographic strip was developed to evaluate the antibody titer of serum samples from swine vaccinated with CSFV vaccine rapidly. The chimeric protein used as a detector was labeled with colloidal gold. Staphylococcal protein A (SPA) and anti-CnC2 monoclonal antibodies (mAbs) were blotted onto the nitrocellulose membrane as the test and control lines, respectively. The strip assay could be performed within 5min, which did not require any special equipment or skills. Through testing sera against various strains of CSFV, the sensitivity of the strip was determined to be 97.0% (65/67) and the specificity was 100% (98/98). The strip results were consistent with those of the existing commercial ELISA kit, and their correlation coefficient was 0.935. In conclusion, the immunochromatographic strip was an acceptable method for surveying CSFV-antibody titers in pigs.     

Development of an immunochromatographic test kit for rapid detection of bovine viral diarrhea virus antigen

An immunochromatographic test was developed for rapid diagnosis of bovine viral diarrhea virus (BVDV) infections using monoclonal antibodies against the nonstructural protein, NS3, of the virus. The kit detected specifically the NS3 of various BVDV strains. Using the kit, leukocyte extracts of cattle infected persistently with BVDV were found positive while those of healthy cattle were negative. The sensitivity and specificity of this kit in compared with virus isolation were 100% and 97.2%, respectively. Furthermore, the test also gave positive results for calves infected acutely with BVDV in experimental infection. The BVDV antigen was detected in 1 ml of blood using a relatively simple procedure. This test kit should be useful for rapid diagnosis of BVD.     

Direct agglutination test for serologic diagnosis of Neospora caninum infection

A direct agglutination test was evaluated for the detection and quantitation of IgG antibodies to Neospora caninum in both experimental and natural infections in various animal species. As compared with results obtained by the indirect fluorescent antibody test, the direct agglutination test appeared reliable for the serologic diagnosis of neosporosis in a variety of animal species. The direct agglutination test should provide easily available and inexpensive tools for serologic testing for antibodies to N. caninum in many host species. Received: 26 June 1997 / Accepted: 5 August 1997     

Evaluation of an indigenously manufactured rapid immunochromatographic test for detection of HBsAg

A 'rapid' one step immunochromatographic, visually read, antigen capture assay--the "HEPACARD" (J Mitra & Co. Ltd., New Delhi, India) used for rapid screening of HBsAg was evaluated. Thousand consecutive sera sent to our laboratory for the purpose of HBsAg screening were tested by this device and by a third generation enzyme immunoassay (EIA) (Auszyme Monoclonal, Abbott Laboratories, Chicago, Illinois) or an automated Axsym microparticle enzyme immunoassay (MEIA) (Axsym HBsAg V2, Abbott Laboratories, Abbott Park, Chicago, Illinois, ISA). Hepacard showed a sensitivity of 79% (CU: 57.3-92%) and specificity of 98.9% (CI: 97.9-99.4%) when compared to the findings by the third generation EIA assays. This study suggested that this particular rapid HBsAg test results have to be confirmed by either an EIA or MEIA where the facility exists. The test may be used only in a small hospital setting where the facilities for enzyme immuno assays do not exist.     

Comparison of a new immunochromatographic rapid test with a commercial EIA for the detection of Puumala virus specific IgM antibodies.

BACKGROUND: Hantaviruses are associated with two human diseases: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV), which is one of the Hantaviruses, is a causative agent of nephropathia epidemica (NE), a mild form of HFRS. OBJECTIVE: a new 5 min rapid test, POC PUUMALA (Erilab Ltd, Finland), for detecting IgM antibodies to PUUV was evaluated and compared with the commercially available Hantavirus (Puumala) IgM ELISA test (Progen, Germany). Discrepant test results between the two tests were confirmed by a mu-capture reference EIA. STUDY DESIGN: Two hundred and thirty five serum samples, which had earlier been analyzed with the Progen IgM ELISA, were assayed with the POC PUUMALA rapid test. Five persons, without knowing the Progen IgM ELISA test results, interpreted independently the rapid test results. In addition, a panel of 48 serum samples was analyzed in parallel with the rapid test and the Progen IgM ELISA by one technician in daily routine diagnostics in a clinical microbiology laboratory. RESULTS: the agreement between the results of the five interpreters was 95%, and the congruence of the results between individual readers and commercial ELISA test varied from 93 to 96%. Diagnostic efficacy of the rapid test varied between 98 and 99% compared with 96% of the Progen IgM ELISA. The POC PUUMALA rapid test showed higher or similar sensitivity compared with the Progen IgM ELISA, whereas both the tests had similar levels of specificity. CONCLUSIONS: the analytical performance of the POC PUUMALA rapid test was found to be as good or even slightly better than the analytical performance of the Progen IgM ELISA. In addition, the rapid and straightforward procedure makes the POC PUUMALA a feasible tool for the diagnosis of the acute PUUV infection.     

一种快速检测人腺病毒的免疫层析分析方法的建立

目的:建立一种基于免疫层析法(Immunochromatographic assay, ICA)的快速简便的人腺病毒(Human adenoviruses,HAdVs)检测方法。方法:利用抗原结合和病毒斑点实验检测发现单克隆抗体3C11和7E6能特异性结合所有测试的人腺病毒,以这两个抗体建立ICA方法。使用培养病毒及1...     

Comparison of a new immunochromatographic test to enzyme-linked immunosorbent assay for rapid detection of immunoglobulin m antibodies to hepatitis e virus in human sera

An immunochromatographic test for rapid detection of IgM antibodies in patients with acute hepatitis E infection was developed utilizing the well-characterized recombinant protein EP2.1 and monoclonal antibody 4B2. The new rapid test based on a novel reverse-flow technology was able to generate a positive result within 2 to 3 min. Our study showed that this test was able to detect anti-HEV IgM antibodies in 96.7% of the patient samples tested (n = 151) while maintaining an excellent specificity of 98.6% with samples from various patient or healthy control groups (total n = 208). Furthermore, this rapid test gave a good specificity of 90.9% when tested with rheumatoid factor (RF)-positive sera (RF value of < or =850 IU/ml; n = 11) although a higher concentration of RF in samples might cause cross-reactivity. The new test has a good agreement of 97.2% with a kappa value of 0.943 when compared with a reference enzyme-linked immunosorbent assay. The positive predictive value and the negative predictive value for the rapid test thus reached 98.0 and 97.6%, respectively. This is the first rapid, point-of-care test for hepatitis E and will be especially useful for the diagnosis of acute hepatitis E virus infection in field and emergency settings and in resource-poor countries.     

胶体金免疫层析试纸条快速检测乙型流感病毒

目的建立有效、简便的胶体金免疫层析试纸条快速检测乙型流感病毒感染的方法。方法通过对乙型流感病毒核蛋白单克隆抗体进行胶体金标记,成功研制了乙型流感病毒免疫层析检测试纸条。结果该试纸条操作简单,肉眼于10～15 min内判定结果,对乙型流感病毒具有高度特异性,与甲型H1N1、H3N2亚型流感病毒等其他重要呼吸道病毒无交叉反应。试纸条在室温保存12个月、2～8℃保存18个月,其特异性和灵敏度无明显变化。对从内蒙自治区医院收集的流感样症状病人的702份鼻咽部分泌物进行检测,与美国Quidel公司同类产品的符合率为95%。结论建立的乙型流感病毒免疫层析检测方法具有简便、快速、特异、敏感和稳定等特点,对乙型流感病毒感染疾病的临床检测与早期诊断具有重要意义。     

Rapid immunochromatographic test for hantavirus andes contrasted with capture-IgM ELISA for detection of Andes-specific IgM antibodies

Hantavirus is associated with hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The clinical diagnosis of hantavirus infections has been confirmed routinely by the immunofluorescence antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA). A rapid and easy diagnostic test for hantavirus infection is required. A new immunochromatographic assay for hantavirus, POC-PUU, useful for the diagnosis of epidemic nephrophaty associated with hantavirus Puumala in Europe, was evaluated in Chile. This test is based on recombinant N-protein of hantavirus Puumala, and cross-reacts with other hantaviruses. Eighty human sera were selected at random from patients from Southern Chile who were suspected with HPS. The hantavirus capture-IgM ELISA was compared with a commercially available POC-PUU test (POC PUUMALA, Reagena Ltd., Toivala, Finland). The test sensitivity and specificity of the POC-PUU test were 97 and 90%, respectively. It is important to note that although the test is not specific for Andes virus the sensitivity and specificity were above 90%, which indicates good reactivity to the Puumala nucleoprotein antigen. As this test is cost-effective, with a high negative value, rapid and easy to carry out, specialized personnel are not necessary, nor does it require specialized equipment. Its usefulness for diagnosis is important in hospitals far from reference centers and areas with a high incidence of HPS cases.