Chung Chungwon, Wilson Carey, Bandaranayaka-Mudiyanselage Chandima-Bandara, Kang Eunah, Adams D Scott, Kappmeyer Lowell S, Knowles Donald P, McElwain Terry F, Evermann James F, Ueti Massaro W, Scoles Glen A, Lee Stephen S, McGuire Travis C
1Chungwon Chung, VMRD Inc., 425 NW Albion Drive, Pullman, WA 99163.
J Vet Diagn Invest. 2014 Jan;26(1):61-71. doi: 10.1177/1040638713511813. Epub 2013 Dec 6.
The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the commercial rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the commercial cELISA, a new recombinant antigen designated rMSP5-glutathione S-transferase (GST) was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption.
从用于包被平板的重组抗原中去除麦芽糖结合蛋白（MBP），将提高商业化无形体抗体竞争酶联免疫吸附测定（cELISA）的特异性。在使用未吸附MBP的重组主要表面蛋白5（rMSP5）-MBP cELISA进行检测时，无形体阴性牛群中358份具有显著MBP抗体结合（≥30%I）的血清中有139份（38.8%）。使用吸附了MBP的商业化rMSP5-MBP cELISA检测时，除8份外，所有MBP结合阳性血清均变为阴性（<30%I），特异性达97.8%。该特异性高于一些先前的报道，因此为提高商业化cELISA的特异性，开发了一种新的重组抗原，即rMSP5-谷胱甘肽S-转移酶（GST），从抗原中去除了MBP，无需进行MBP吸附。使用rMSP5-GST cELISA检测时，358份无形体阴性血清中只有1份呈阳性，这139份血清在未吸附MBP的rMSP5-MBP cELISA中具有显著（≥30%I）的MBP结合。这使得诊断特异性提高到99.7%。未吸附MBP的rMSP5-GST cELISA与吸附了MBP的rMSP5-MBP cELISA具有相当的分析灵敏度，在用巢式聚合酶链反应定义的135份阳性血清进行检测时，诊断灵敏度为100%。此外，rMSP5-GST cELISA解决了使用吸附了MBP的rMSP5-MBP cELISA从选定血清中获得的103例假阳性反应，并提高了其他31份血清中29份血清的分辨能力。总之，与吸附了MBP的rMSP5-MBP cELISA相比，rMSP5-GST cELISA检测速度更快、操作更简便，具有更高的特异性、相当的灵敏度和更好的分辨能力。
