Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7x10(4) CFU/mL and 2.0x10(6) CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Evaluation of SD BIOLINE Chagas Ab Rapid Kit / 대한진단검사의학회지

BACKGROUND: Chagas' disease is caused by Trypanosoma cruzi, a protozoan parasite, which is transmitted by blood-sucking bugs or through blood transfusion or organ transplantation. It is endemic in Central and South America. The objective of this study was to compare the performance of immunochromatographic SD Bioline Chagas Ab Rapid (Standard Diagnostics, Korea) with three immunochromatographic kits for the detection of antibodies to T. cruzi. METHODS: A total of 320 serum specimens (140 positive and 180 negative) from National Reference Laboratory for Chagas and Leishmaniasis (NRLCL, Honduras) were used for the evaluation of four different test kits: SD Bioline Chagas Ab Rapid, Chagas Stat-Pak Assay (Chembio Diagnositc Systems, USA), OnSite Chagas Ab Rapid test-Cassette (CTK Biotech, USA), and Trypanosoma Detect Rapid Test (InBios International, USA). The results of four kits were compared with those of NRLCL. Cross-reactivity with other parasites was also evaluated. RESULTS: Compared with the results of NRLCL, sensitivity and specificity were 99.3% and 100% for both of SD and Chembio kits, 97.2% and 100% for InBios kit, and 97.9% and 98.8% for CTK kit. None of other parasites showed cross-reactivity. CONCLUSIONS: SD Bioline Chagas Ab Rapid kit showed test results highly correlating with those of National Reference Laboratory for Chagas and Leishmaniasis. It can be used for a rapid detection of Chagas' disease in endemic region and monitoring the disease among overseas travelers in Korea.

Development of Test System for Detection of Antibody to Human Immunodeficiency Virus Type 1 Subtype O

In Korea, all domestic made test systems for detecting antibodies in HIV-1 contain the antigens from human immunodeficiency type 1 (HIV-1) subtype B. However, because HIV-1 subtype O is significantly different in amino acid sequences from all other subtypes of HIV-1, there has been a need for developing a test for detecting antibodies in subtype O. For this purpose, the entire nucleotide sequence corresponding to the extracellular domain of the transmembrane glycoprotein of HIV-1 subtype O was synthesized with consideration of Escherichia coli cordon usage. Various regions of the extracellular domain were cloned into E. coli expression vectors and tested for levels of protein production. The nucleotide sequence, named ECTM, that can encode a 129 amino acid-long peptide, was found to be expressed at a high level in E. coli. The protein of approximately 17 kDa specifically reacted with sera from individuals infected with HIV-1 subtype O. The ECTM protein was purified to near homogeneity by the CM-T gel chromatography, using concentrated, denatured inclusion bodies. In Western blot analysis, the purified viral antigen reacted with sera from individuals infected with subtype O more efficiently than subtype B. The enzyme linked immunoabsorbent assay (ELISA) system was developed using the subtype O viral protein and compared with the commercially available kit lacking the antigens from subtype O. The ELISA kit containing the subtype O antigen ECTM alone efficiently reacted with sera from individuals infected with subtype O. The subtype O antigen-containing kit produced a positive absorbence even when sera were diluted 512-fold, suggesting a high sensitivity. The commercially available kit also reacted with subtype O sera, but produced a negative result at a dilution of 8-fold. Our results suggest that the currently available kit may not be able to efficiently detect subtype O sera and that the viral protein developed in this study may be added to the current system to maximize the detection of sera from individuals infected with subtype O.

An Evaluation of Commercial Reagent Kits for Detecting HCV Antibodies: GenediaTM HCV ELISA 3.0, GenediaTM HCV Rapid and GenediaTM HCV Confirm 4.0 / 대한임상병리학회지

BACKGROUND: This study was conducted to evaluate accuracy of newly developed HCV Ab test kits by Korea Green Cross Co.(Yongin, Kyunggi), namely GenediaTM HCV ELISA 3.0 (ELISA) for routine test, GenediaTM HCV Rapid (RAPID) for quick screening. and GenediaTM HCV Confirm 4.0 (CONFIRM) for confirmation. METHODS: Performance of ELISA was compared with that of Ortho HCV 3.0 ELISA (Neckargemund, Germany; ORTHO ELISA) using 990 patients' sera. Accuracy of RAPID was evaluated by testing on 114 HCV Ab negative and 86 positive specimens by ELISA. Discrepant results obtained by RAPID were confirmed by Chiron RIBA HCV 3.0 Strip Immunoblot Assay (Ca, USA; RIBA) and HCV Blot 3.0 (Genelabs Diagnostics, Singapore; BLOT). Accuracy of CONFIRM test was compared between RIBA and BLOT using 78 ELISA positive sera. To elucidate prevalence of viremia, RT-PCR was performed on 165 serum samples and results were compared with that of ELISA and RAPID. RESULTS: Agreement of test results between ELISA and ORTHO ELISA was 99.6% (986/990). On HCV Ab negative specimens 99.1% (113/114) agreed among RAPID, ELISA and ORTHO ELISA. However, on seropositive specimens 91.7% (79/86) agreed between RAPID and ELISA. Agreement between CONFIRM and RIBA was 83.3% (65/78). Core antigen showed the highest reactivity and NS5 antigen showed the lowest reactivity with CONFIRM. HCV RNA was detected in 58.3% (28/48) of ELISA positive specimens, however, it was not detected in ELISA negative specimens. There was no correlation between prevalence of HCV RNA and 5 antigens used in ELISA test. CONCLUSIONS: Newly developed Korea Green Cross GenediaTM HCV ELISA 3.0, Rapid and HCV Confirm were considered to be clinically accurate routine, quick screening and confirmatory test for detecting HCV Ab in serum samples.