A comparative evaluation of dengue diagnostic tests based on single acute serum samples for laboratory confi rmation of acute dengue

A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confi rmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient’s demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on singleacute serum samples, 190 of the 558 patients (34.1%) were laboratory-confi rmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confi rmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was signifi cantly more sensitive than the conventional RT-PCR.

Development of a Polyclonal Antibody-based AC-ELISA and Its Comparison with PCR for Diagnosis of Canine Parvovirus Infection / 中国病毒学·英文版

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCID50/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.

Detection of Platelet Specific Antibodies by Modified Antigen Capture ELISA Test / 대한진단검사의학회지

BACKGROUND: Autoimmune thrombocytopenia (AITP) is characterized by autoantibody-induced platelet destruction. Although several studies have shown that pathogenic autoantibodies are mainly IgG directed platelet glycoproteins (GP), a platelet GP specific test is not available in clinical laboratories. The aim of this study was to evaluate the clinical usefulness of a Modified Antigen Capture Enzyme-linked immunosorbent assay (MACE) test in the diagnosis of AITP. METHODS: We investigated fifty-seven patients who showed a platelet count lower than 100 x 10(9)/L and underwent a bone marrow examination. They were classified into primary AITP (P-AITP) (n=21), secondary AITP (S-AITP) (n=15), and non-immune thrombocytopenia (NITP) (n=21) by bone marrow findings and clinical diagnosis. Platelet GP (IIb/IIIa, Ia/IIa, Ib/IX, IV)-specific antibodies and anti-HLA class I antibody were detected by MACE test. RESULTS: Among 57 samples, platelet GP specific antibodies were detected in 8 (22.2%) of 36 patients with AITP and 1 (4.8%) of 21 patients with NITP. The specificities were as follows: GP IIb/IIIa (n=4), GP Ia/IIa (n=5), GP Ib/IX (n=3) and GPIV (n=2). Of the nine patients with platelet GP specific antibodies, four (44.4%) had more than two platelet GP specific antibodies. The sensitivity, specificity, positive predictive value and negative predictive values of the MACE test for AITP were 22.2%, 95.2%, 88.9%, 41.7%, respectively. A previous transfusion history was associated with a higher detection rate of anti-HLA class I antibodies (P<0.05). CONCLUSIONS: The MACE test is a convenient method to detect platelet GP specific antibody and is very specific to diagnose AITP. In clinical practice, even though it is not sensitive, the MACE test would be useful in differentiating AITP from NITP.

Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application

Interleukin-6 (IL-6) is introduced as a marker of disease. At present, a variety of method may be used to quantify expression of this protein. Antigen capture-ELISA is a sensitive and accurate quantification method previously used with ovine, rat, and human IL-6 proteins. However, it has never been reported to quantify porcine IL-6 protein using capture ELISA. In this study, we generated and characterized a set of IgY and mono-specific polyclonal antibodies to recombinant porcine IL-6 (rpIL-6), and combining these with a sensitive and specific capture-ELISA for a diagnostic purpose. cDNA encoding the mature protein coding region of porcine IL-6 was cloned and expressed with pQE-30UA expression vector. rpIL-6 was then expressed and purified by using Ni-NTA resin. Protein mass of 24 kDa was found with SDS-PAGE and the identity of the protein was confirmed by Western-blot. Production of polyclonal antibodies against rpIL-6 was performed using the purified rpIL-6 in mice and hens. An antigen capture-ELISA was developed with the antibodies after their extraction. To compare the IL-6 level in the different sanitary state of farms, pig sera were randomly collected and concentration of IL-6 in the sera was measured with the antigen capture-ELISA. The capture-ELISA with the optimal concentration of antibodies, in this study, was able to detect about 10 ng/ml of rpIL-6. IL-6 levels determined with the capture-ELISA in pig sera showed positive correlation with the sanitary states of the farms. These results suggested that the developed antigen capture-ELISA could be a good tool for the screening of microbial infection in pig farms.

Clinical Significance and Detection of Antibodies Against Platelet GP Ib/I X and Gp II b/IIIa in Childhood Chronic Idiopathic Thrombocytopenic Purpura / 대한소아혈액종양학회지

BACKGROUND: Chronic idiopathic thrombocytopenic purpura is an autoimmune disorder caused by sequestration of antibody-sensitized platelets in the reticuloendothelial system. However, uncertainty as to the specificity, frequency and clinical significance of such antibodies still remains. So, we tried to further clarify the above uncertainty in childhood chronic idiopathic thrombocytopenic purpura. METHODS: We analyzed sera from 29 patients. Twenty six patients were chronic ITP who were admitted or followed up to the Department of Pediatrics, Severance Hospital, Yonsei University Medical College from August 1996 to March 1997 by employing a modified antigen-capture ELISA(MACE), flow cytometry and electrophoresis(SDS-PAGE) and immuno-blotting(IB) assays. Three patients with ITP less than 6 months after onset of ITP were included to know the possibility to differrentiate between acute ITP and chronic ITP in this study. RESULTS: 1) Glycoprotein(GP)-specific antibodies were found in 28% (8/29) of patients, with 2 patients having antibodies directed solely to Gp II b/III a, no patients holding antibodies specific only for GPI b/I X and 6 possessing antibodies against both anti-GP I b/I X and Gp II b/III a antigen. 2) The detection rate of GP-specific antibodies of flow cytometry was about 10%. The positivity of anti-GPI b/I X antibodies by MACE and immunoblotting was 14% (4/29), respectively, the positivity of anti-Gp II b/III a antibodies by MACE and immunoblotting was, 21 % (6/29) respectively. The concordance rate between two assays(MACE and IB) was 79% (23/29). None of the three methods was good enough to stand alone. 3) Serum antibodies were not more frequently detected in active(p=1.0) or non-splenectomized(p=.54) chronic ITP patients. 4) No association was found between antibody specificity(anti-GPI b/I X, anti-Gp II b/ III a) and platelet counts(p : .87). CONCLUSION: We conclude that in korean childhood chronic ITP, antibodies against both anti-GPI b/I X and Gp II b/III a antigen were predominant antibody. But, the longterm follow-up in more cases is needed to further clarify the clinical significance of antral-platelet antibody in chronic ITP should be assessed.