ABSTRACT
Objective To study the sensitivity and specificity of a double monoclonal antibody sandwich enzyme-linked immunosorbent assay (DMcAbS-ELISA)for the detection of F1 antigen of Yersinia pestis (Y.pestis).Methods Viscera (viz.liver and spleen)specimens of infected mice with virulent Y.pestis and negative control mice were detected by bacteriological test,DMcAbS-ELISA and reverse indirect hemagglutination assay (RIHA) for the F1 antigen.Results The 225 control specimens were all negative tested by plague bacteriology testing,DMcAbS-ELISA and RIHA.A total of 308 plague-infected mouse organ specimens were tested,and the positive detection rate was 92.21％ (284/308),90.91％(280/308) and 89.61％ (276/308),respectively,with germiculture,DMcAbS-ELISA and RIHA,and the difference was not statistically significant(x2=5.65,P＞0.05).The coincidence rate of DMcAbS-ELISA and bacterial culture was 97.00％[(274+243)/533],Kappa =0.940;RIHA in line with the rate was 99.25％[(276+253)/533],Kappa =0.985.Authenticity comparison of F1 antigen detection in viscera specimens:sensitivity,specificity,positive predictive value,negative predictive value,adjusted agreement and Youden's index was 96.48％(274/284),97.59％(243/249),97.86％ (274/280),96.05 ％(243/253),96.99％[1/4×(274/280+274/284+243/253+243/249)]and 0.9407,respectively,for DMcAbS-ELISA and 96.13％(273/284),98.80％(246/249),98.91％(273/276),95.72％(246/257),97.39％[1/4×(273/276+273/284+246/257±246/249)]and 0.9492,respectively,for RIHA.The detection sensitivity of DMcAbS-ELISA and RIHA was 2.7×104 cfu/ml and 2.2×105 cfu/ml,for Y.pestis,respectively,and was 10 μg/L for F1 antigen.Conclusions DMcAbS-ELISA assay is a sensitive,specific,simple and fast method for detection of the F1 antigen,and it has a potential application value in rapid diagnosis of plague.
ABSTRACT
Objective To study the sensitivity and specificity of gold-immunochromatography assay (GICA) for detection of Yersiniapestis(Y. pestis ) F1 antigen. Methods Viscera organ(liver and spleen) specimens of 308 mice with virulent Y. pestis infection and 225 control specimens of rats(217 Spermophilus dauricus, 5 mice,3 guinea pigs) were detected by GICA dipstick with monoclonal antibody against plague F1 antigen (F1MAb).Meanwhile, micro-method of reverse indirect hemagglutination assay(RIHA) and bacteria culture were carried out for parallel testing. Results Bacteriological examination of 225 control specimens, and F1 antigen detected with GICA and RIHA were all negative. No cross-reaction with related Yersinia pseudotuberculosis at 1 x 108 cfu/ml level was found in GICA and RIHA. Detection sensitivity of Y. pestis by GICA and RIHA were 2.5 × 105 cfu/ml and 2.0 × 105 cfu/ml, respectively, and of F1 antigen were 1μg/L and 10 μg/L, respectively. Coincidence was 97.94% (522/533) between GICA and bacteriological test, Kappa = 0.959, and the difference was statistically insignificant(x2 = 0.36, P ＞ 0.05); and 97.94%(522/533) between GICA and RIHA, Kappa = 0.959, with statistically significant difference in the positive detection rates(x2 = 9.09, P ＜ 0.05). Out of the 308 infected mice, 284 were positive of plague bacterial cultured, In 284 samples with positive bacterial culture, there were 280 of positive detected by GICA for F1 antigen, positive rate of F1 antigen was 98.59%, higher than that by RIHA[the positive rate of 96.13%(522/533)], with statistically significant difference(x2 = 5.14, P ＜ 0.05). Sensitivity of GICA was 98.59% (280/284), specificity was 97.19% (242/249), positive predictive value (PPV) was 97.56% (280/287),negative predictive value ( NPV ) was 98.37% (242/246), and Youden index was 0.9578. Conclusions GICA is sensitive and specific, fast and simple in detection of F1 antigen of the plague. It's a valuable detection technique for early and rapid diagnosis of plague.
ABSTRACT
Objective To study the doses and methods of F1 antigen(F1Ag) to immune Balb/c mice during the establishment of hybridoma cell strains. Secreting McAbs against F1Ag of Yersinia pestis. Methods Balb/c mice of seven to nine weeks old were randomly divided into six groups. The first four groups were 150, 100, 50 and 25 μg F1Ag inoculated group, having multipoint hypodermic inoculation of F1Ag of 150, 100, 50 and 25 μg followed by multipoint hypodermic inoculation of F1Ag of 100 μg for a second time and then intraperitoneal injection of 100 μg. Next, hypodermically inoculated group received F1Ag of 100 μg for three times in multiple points. Finally, the intraperitoneal injection group was intraperitoneally inoculated with F1Ag of 100 μg for three times. Emulsification liquid of F1Ag + Complete Frednd's adjuvant(CFA) of equivalence was used in the first inoculation, emulsification liquid of F1Ag + Incomplete Frednd's adjuvant(IFA) balanced mix in the second, F1Ag liquid in the third. One week afterwards, tail blood of the mice was collected to test antibody titers of anti-F1Ag by double antigens sandwich enzyme linked immunosorbent assay (DAgS-ELISA) and trace indirect hemagglutination assay(IHA). Results The levels of antibody of anti-F1Ag in 150,100,50 and 25 μg groups had statistics difference (DAgS-ELISA method: G = 12 173.87,13 440.37,15 024.19 and 4466.72, F= 3.11, P< 0.05;IHA: G = 19 972.32,18 089.40,23 170.47 and 4871.08, F = 4.11, P < 0.05). Immune effect of the 3 groups of 150, 100 and 50 μg was almost the same (P> 0.05), and excelled as compared with that in 25 μg group with statistics difference(DAgS-ELISA method: t = 2.18,2.39,2.73, P < 0.05;IHA: t = 2.54,2.73,3.13, P< 0.05). The titer of F1 antibody had an increasing trend from the 100 μg group to hypodermic group and intraperitoneal injection groups, but without statistics difference (DAgS-ELISA method: G = 8933.44, 9986.16, 13 440.37;IHA: G = 13 777.25,16 384.00, 18 089.40, F = 0.66,0.25, all P > 0.05). Conclusions Hyodermical inoculation of F1Ag with the first dose of 50 μg in multiple points for mouse is appropriate, and a strengthening dose of 100 μg in an intraperitoneal injection may shorten the immune period.