羊副结核分枝杆菌间接ELISA抗体检测方法的建立与初步应用

doi:10.3864/j.issn.0578-1752.2026.03.013

Abstract

Abstract:

【Objective】This study aimed to develop an indirect ELISA detection method for antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in sheep, for providing an efficient and reliable technique for the epidemiological surveillance and serological testing of Johne's disease (JD) in sheep. 【Method】In this study, the culture supernatant of the strain (MAP-XJB13) was isolated in the laboratory during earlier stage, which was selected as the MAP-coated antigen. The optimal reaction conditions and critical values for indirect ELISA were determined through the systematic screening and optimization of various parameters, including the coating solution and conditions, blocking solution and conditions, antigen coating concentration, serum dilution ratio, antibody incubation and color development time, brand of color development solution, sample dilution solution and enzyme-labeled secondary antibody protective solution. The efficacy of the developed indirect ELISA antibody detection method for sheep MAP was assessed in terms of sensitivity, specificity, repeatability, preservation period, and coincidence rate. Finally, the initially assembled reagent kits were utilized for the clinical detection of samples from in Heilongjiang and Inner Mongolia. 【Result】The optimal conditions for the ELISA were determined as follows: the coating solution utilized as CBS buffer, with the coating condition process conducted at 37 ℃ for 4 hours. The blocking solution comprised 5% fish gelatin, 5% trehalose, and 12% PEG4000, with the blocking procedure also performed at 37 ℃ for 2 hours. The antigen coating concentration was set at 80 µg·mL^-1, the serum dilution ratio was 1:40, and the dilution ratio for the enzyme-labeled secondary antibody was 1:30 000. The incubation parameters included primary antibody incubation at 25 ℃ for 30 minutes, followed by a 30-minute incubation of the enzyme-labeled secondary antibody, and a 15-minute color development phase. The color development solution employed was Biodragon, while the sample dilution solution consisted of 1% ovalbumin and 0.5% trehalose. Additionally, the protective solution for the enzyme-labeled secondary antibody contained 0.1% ovalbumin. The critical threshold for the developed indirect ELISA method for detecting antibodies against sheep MAP was determined to be 0.460, with a sensitivity of 95.89% and a specificity of 96.12%. The cross-reactivity analysis demonstrated that, based on the premise that the positive and negative results were valid, there was no cross-reactivity with the following: positive serum for Brucella in sheep, positive serum for Mycoplasma mycoides in sheep, positive serum for Corynebacterium pseudomycosis in goats, positive serum for tuberculosis in sheep, positive serum for peste des petits ruminants virus in goats, positive serum for peste des petits ruminants in sheep, and positive serum for poxvirus in sheep. The intra-batch and inter-batch coefficients of variation ranged from 0.754% to 7.812% and 1.252% to 7.277%, respectively, and the stability of the results was maintained for up to 8 months. The sheep MAP indirect ELISA antibody detection kit exhibited a positive concordance rate of 95.89% and a negative concordance rate of 95.55% when compared to the ID.vet MAP ELISA antibody detection kit, resulting in an overall concordance rate of 98.56%. The prevalence of MAP antibodies in sheep from Heilongjiang and Inner Mongolia was found to be 10.81%. 【Conclusion】This study successfully developed an indirect ELISA method for the detection of antibodies MAP in sheep. The method exhibited exceptional specificity, high sensitivity and a long shelf life, thereby offering robust technical support for the prevention and management of JD.

Key words: Mycobacterium avium subsp. paratuberculosis, sheep paratuberculosis, indirect ELISA, antibody detection, MAP- XJB13 stain

GAO YunJie, GUO RuoNan, CHEN ChunWen, DUAN YiFan, ZHANG ZhenJun, NIE ChanChan, LI MengYu, WANG Hui, FENG TingTing, CUI YingYing, DANG GuangHui, LIU SiGuo. Establishment and Preliminary Application of Indirect ELISA Antibody Detection Method for Mycobacterium avium subsp. paratuberculosis in Sheep[J].Scientia Agricultura Sinica, 2026, 59(3): 655-667.

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Figures/Tables 12

Table 1

Fig. 1

Table 2

Test validity determination"

	阳性血清 Positive serum	阴性血清 Negative serum
标准差 SD	0.010	0.100
平均值 $x ¯$	0.072	1.662
$x ¯$ -3SD	1.362	0.042
$x ¯$ +3SD	1.962	0.102

Table 2

Fig. 2

Table 3

Table 4

Table 5

Table 6

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Table 8

Table 9

Table 10

References 33

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阳性血清Positive serum			阴性血清 Negative serum
编号Number	来源Source	数量Quantity	编号Number	来源Source	数量Quantity
1-53	黑龙江Heilongjiang	53	1-77	黑龙江Heilongjiang	77
54-67	内蒙古Inner Mongolia	14	78-126	内蒙古Inner Mongolia	49
68-73	新疆Xinjiang	6	127-129	新疆Xinjiang	3
合计Total		73	合计Total		129

血清 Serum	血清稀释度 Serum dilution
血清 Serum	1:40	1:80	1:160	1:320	1:640	1:1 280	1:2 560	1:5,120
N1	2.659	2.514	2.21	1.95	1.278	0.86	0.472	0.273
N2	2.107	1.697	1.27	1.112	0.45	0.248	0.164	0.117
N3	0.982	0.802	0.426	0.28	0.127	0.085	0.071	0.074
N4	2.091	2.342	1.509	1.152	0.598	0.355	0.229	0.147
N5	2.474	2.392	2.15	1.921	1.438	0.958	0.627	0.396
N6	2.519	2.446	1.647	1.208	0.529	0.286	0.186	0.127
N7	2.542	2.383	1.706	1.225	0.627	0.337	0.215	0.142
N8	1.716	1.272	0.676	0.454	0.239	0.148	0.096	0.094
N9	0.173	0.153	0.119	0.106	0.071	0.066	0.062	0.07
N10	0.419	0.332	0.182	0.137	0.083	0.068	0.065	0.072
N11	0.301	0.204	0.136	0.114	0.07	0.065	0.063	0.066
N12	0.338	0.218	0.152	0.122	0.097	0.068	0.062	0.072
N13	0.711	0.459	0.279	0.198	0.128	0.085	0.073	0.073
阳性对照Positive control	1.550		1.749		1.535		1.447
阴性对照Negative control	0.094		0.101		0.064		0.087

血清 Serum	血清稀释度 Serum dilution
血清 Serum	1:40	1:80	1:160	1:320	1:640	1:1280	1:2560	1:5120
N1	1.650	1.557	1.361	1.194	0.761	0.491	0.241	0.113
N2	1.295	1.031	0.755	0.654	0.227	0.097	0.043	0.013
N3	0.570	0.454	0.212	0.118	0.019	-0.008	-0.017	-0.015
N4	1.284	1.446	0.909	0.679	0.322	0.166	0.085	0.032
N5	1.531	1.478	1.322	1.175	0.864	0.554	0.341	0.192
N6	1.560	1.513	0.998	0.716	0.278	0.121	0.057	0.019
N7	1.575	1.473	1.036	0.726	0.341	0.154	0.076	0.029
N8	1.043	0.757	0.373	0.230	0.091	0.033	-0.001	-0.002
N9	0.049	0.036	0.014	0.005	-0.017	-0.020	-0.023	-0.018
N10	0.207	0.151	0.054	0.025	-0.009	-0.019	-0.021	-0.016
N11	0.131	0.069	0.025	0.011	-0.018	-0.021	-0.022	-0.020
N12	0.185	0.101	0.054	0.033	0.015	-0.005	-0.010	-0.002
N13	0.449	0.271	0.144	0.087	0.037	0.007	-0.002	-0.002

血清Serum	20240601	20240602	20240603
N1	0.305	0.12	0.111
N2	0.683	0.269	0.183
N3	0.120	0.117	0.096
N4	0.364	0.336	0.269
N5	0.390	0.261	0.186
N6	0.183	0.181	0.131
N7	0.47	0.495	0.324
阳性对照 Positive control	1.529	1.592	1.587
阳性对照 Positive control	1.536	1.721	1.593
阴性对照 Negative control	0.097	0.075	0.085
阴性对照 Negative control	0.099	0.074	0.081

血清Serum	20240601	20240602	20240603
N1	0.144	0.029	0.019
N2	0.408	0.123	0.066
N3	0.015	0.027	0.009
N4	0.185	0.165	0.123
N5	0.204	0.118	0.068
N6	0.059	0.067	0.032
N7	0.259	0.266	0.160

Establishment and Preliminary Application of Indirect ELISA Antibody Detection Method for Mycobacterium avium subsp. paratuberculosis in Sheep

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血清稀释比例 Serum dilution ratio	1个月 1 month	2个月 2 months	4个月 4 months	6个月 6 months	8个月 8 months
1:40	1.311	1.097	1.186	1.190	1.037
1:80	0.968	0.918	0.871	0.888	0.764
1:160	0.606	0.633	0.533	0.633	0.521
1:320	0.291	0.279	0.231	0.293	0.209
阳性对照 Positive control	1.591	1.316	1.282	1.53	1.44
阳性对照 Positive control	1.575	1.659	1.462	1.315	1.414
阳性对照 Positive control	0.063	0.076	0.069	0.077	0.096
阳性对照 Positive control	0.06	0.077	0.081	0.08	0.088

血清稀释比例 Serum dilution ratio	1个月 1 month	2个月 2 months	4个月 4 months	6个月 6 months	8个月 8 months
1:40	1.245	1.154	1.194	1.158	1.114
1:80	0.852	0.823	0.907	0.922	0.743
1:160	0.516	0.639	0.569	0.634	0.486
1:320	0.290	0.314	0.224	0.319	0.247

实验室自制试剂盒 Laboratory made kit	IDV.et试剂盒IDV.et kit
实验室自制试剂盒 Laboratory made kit	阳性Positive	阴性Negative	合计Total
阳性Positive	70	11	81
阴性Negative	3	231	234
合计Total	73	242	315
符合率 Rate of conformity	95.89%	95.55%	95.56%

实验室自制试剂盒 Laboratory made kit	省份Provinces
实验室自制试剂盒 Laboratory made kit	黑龙江 Heilongjiang	内蒙古 Inner Mongolia	合计 Total
阳Positive	82	60	142
阴Negative	597	569	1166
合计Total	679	629	1308
阳性率Positive rate	12.08%	9.54%	10.87%

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