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 a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies

Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x105cells/0.1 ml at 22degrees C and 1x106 cells/0.1 ml at 30degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 104cells/0.1 ml at 22degrees C and 30degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.