Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3283-3293.doi: 10.3864/j.issn.0578-1752.2024.16.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Development and Application of Indirect ELISA Kits for Antibody Detection of Porcine Proliferative Enteropathy

ZHANG CongYue1(), ZHOU Hong1, LIN HuiXing1, FAN HongJie1,2()   

  1. 1 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095
    2 Jiangsu Collaborative Innovation Center for the Prevention and Control of Important Animal Diseases, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2024-03-02 Accepted:2024-05-10 Online:2024-08-16 Published:2024-08-27
  • Contact: FAN HongJie

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Abstract:

【Objective】 The objective of this study was to develop an indirect ELISA kits to detect Lawsonia intracellularis (LI) antibodies in pig, so as to provide a tool for the monitoring of LI infection and vaccine evaluation.【Method】 The outer membrane protein (Omp2) of LI was expressed and purified, and an indirect ELISA method based on it to detect LI antibodies was established. The reaction conditions of the indirect ELISA and assemble the test kit was further optimized. Based on this, the prototype kit was used to test the sensitivity of the kit with serially diluted LI-positive sera, and other pathogen-positive sera were tested to study the specificity of the kit. The practicality of the kit was evaluated by testing 1 000 clinical pig serum samples collected at different times from different farms. From these clinical serum samples, the positive or negative effect of 50 sampleswere tested for by imported commercial kits, and the conformity of the kit were compared and verified.【Result】 The Omp2 protein was successfully expressed and purified, with a purity of 90.31%, and was identified to react with LI-positive serum via Western blot. The optimal ELISA conditions were: coating with Omp2 at 200 ng/well at 4 °C for 12-16 hours; serum diluted 1﹕50 and incubated at 37 °C for 30 minutes; goat anti-pig IgG-HRP diluted 1﹕20 000, and incubated at 37°C for 30 minutes; TMB substrate developed at 37 °C for 15 minutes, and the reaction was stopped with 0.5 mol·L-1 sulfuric acid. The highest detection dilution of LI-positive sera was 8-fold, indicating that the established ELISA method had the good sensitivity. The test kit tested negative for E. coli, App, Mhp, SS2, PRRSV, and PRV, demonstrating the good specificity of the ELISA method. Sensitivity and specificity testing standards for the kit have been established. The kit remains stable in terms of appearance, sensitivity, and specificity for 15 months when stored at 4 °C. The criteria for the kit were set as follows: OD450nm ≥1.25 for positive standard sera and OD450nm <0.3 for negative standard sera. The intra-batch and inter-batch variability coefficients of the prototype kit were all less than 10%. The concordance rate with foreign commercial ELISA test kits reached 86%. The developed ELISA test kit detected LI-positive samples at a rate of 59.90% among 1 000 clinical pig serum samples from the eastern part of China, indicating the widespread presence of LI in the region. 【Conclusion】 The LI indirect ELISA antibody test kit in pig developed in this study had high specificity and sensitivity, and a high concordance rate with commercial test kits, making it suitable for clinical detection of LI antibodies.

Key words: Lawsonia intracellularis, outer membrane protein 2, indirect enzyme-linked immunosorbent assay, specificity, sensitivity, coincidence rate

Table 1

Kit component specifications and loading volume"

试剂盒组分Kit component 数量 Quantity
LI抗原包被板 LI antigen-coated plates 96孔/板×2块96 wells/plate×2 plates
LI阳性对照血清 LI positive control serum 1 mL/管×1管 1 mL/vial×1vial
LI阴性对照血清 LI negative control serum 1 mL/管×1管 1 mL/vial× 1 vial
酶标记物 Enzyme conjugate 20 mL/瓶×1瓶 20 mL/bottle×1 bottle
样品稀释液 Sample diluent 20 mL/瓶×1瓶 20 mL/bottle×1 bottle
底物溶液A Substrate solution A 12 mL/瓶×1瓶 12 mL/bottle×1 bottle
底物溶液B Substrate solution B 12 mL/瓶×1瓶 12 mL/bottle×1 bottle
终止液 Stop solution 12 mL/瓶×1瓶 12 mL/bottle×1 bottle
20×洗涤液 20× Wash buffer 30 mL/瓶×2瓶 30 mL/bottle×2 bottles

Fig. 1

Identification of purified recombinant protein effect by SDS-PAGE and Western blot A: Identification of recombinant protein purification effect by SDS-PAGE (M: Protein marker; 1: bacterial lysate; 2: recombinant protein Omp2); B: Western blot of recombinant protein (M: Protein marker; 1: Recombinantprotein Omp2, 2: Uninduced strains)"

Fig. 2

Determination of cut-off value A: The distribution of 120 positive and negative pig serum with the ELISA; B: Receiver operating characteristic curve"

Table 2

The result of specificity test（OD450 nm）"

阳性血清
Positive serum		 平均值
AM		 标准差
SD		 判定结果
Result
N2 0.333 0.038 阴性Negative
N3 0.344 0.026 阴性 Negative
N4 0.341 0.037 阴性 Negative
N5 0.253 0.016 阴性Negative
N6 0.317 0.016 阴性Negative
N7 0.357 0.014 阴性Negative
N8 0.381 0.009 阴性Negative
N9 0.313 0.017 阴性Negative
N10 0.316 0.006 阴性Negative

Table 3

The result of sensetivity test (OD450 nm)"

血清稀释倍数
Serum dilution		 平均值
AM		 标准差
SD		 判定结果
Result
P1 1.756 0.024 阳性Positive
P1（1:2） 1.309 0.068 阳性Positive
P1（1:4） 0.833 0.073 阳性Positive
P1（1:8） 0.584 0.038 阳性Positive
P1（1:16） 0.297 0.039 阴性Negative

Table 4

Sensitivity test mean and standard deviation calculation (S/N)"

P P不同稀释度 Different dilutions of P
1﹕2 1﹕4 1﹕8 1﹕16
平均值AM 10.729 6.586 4.015 2.441 0.905
标准差SD 0.909 0.676 0.500 0.289 0.154
AM±3SD 8.002-13.456 4.558-8.614 2.515-5.515 1.574-3.308 0.443-1.367

Table 5

Sensitivity test standards"

血清 Serum S/N
P 8.002-13.456
P（1:2） 4.558-8.614
P（1:4） 2.515-5.515
P（1:8） 1.574-3.308
P（1:16） <2.1

Table 6

Specificity test mean and standard deviation calculation (S/N)"

血清
Serum		 平均值
AM		 标准差
SD		 AM±3SD
N1 0.982 0.075 0.758-1.206
N2 1.693 0.126 1.315-2.071
N3 1.400 0.117 1.050-1.750
N4 1.402 0.115 1.057-1.748
N5 1.596 0.117 1.246-1.946
N6 1.304 0.105 0.988-1.620
N7 1.595 0.116 1.246-1.944
N8 1.600 0.129 1.212-1.988
N9 1.249 0.085 0.994-1.505
N10 1.616 0.119 1.259-1.973

Table 7

Sensitivity test results (S/N value)"

时间（月）
Time (month)		 P P不同稀释度 Different dilutions of P 阳性对照OD450nm
Positive control OD450nm		 阴性对照OD450nm
Negative control OD450nm
1﹕2 1﹕4 1﹕8 1﹕16
0 11.841 6.886 3.641 2.343 0.799 1.761 0.154
3 10.305 7.149 4.020 2.427 0.990 1.713 0.151
6 9.190 6.381 3.946 2.544 0.859 1.703 0.153
9 11.151 6.987 4.796 2.296 0.908 1.745 0.152
12 11.617 6.717 4.387 2.755 0.942 1.710 0.150
15 10.061 6.306 3.767 2.277 0.924 1.741 0.151

Table 8

Specificity test results (S/N value)"

时间（月）
Time (month)		 N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 阳性对照
OD450nm Positive control OD450nm		 阴性对照
OD450nm Negative control OD450nm
0 0.834 1.618 1.241 1.279 1.466 1.152 1.520 1.464 1.234 1.643 1.761 0.152
3 0.891 1.603 1.295 1.281 1.524 1.207 1.502 1.522 1.276 1.638 1.738 0.148
6 0.990 1.727 1.382 1.369 1.583 1.271 1.611 1.660 1.257 1.626 1.727 0.153
9 1.007 1.679 1.420 1.402 1.659 1.287 1.589 1.584 1.284 1.613 1.724 0.152
12 0.979 1.732 1.447 1.414 1.625 1.331 1.571 1.706 1.211 1.643 1.715 0.152
15 0.848 1.645 1.262 1.300 1.490 1.172 1.545 1.488 1.255 1.670 1.744 0.150

Table 9

Test results of clinical serum samples"

样品
Sample		 地区 District
江苏 Jiangsu 安徽 Anhui 浙江 Zhejiang 总数 Total
阳性 Positive 185 346 68 599
阴性 Negative 172 177 52 401
总数 Total 357 523 120 1000
阳性率 Positive rate (%) 51.82 66.16 56.67 59.90

Table 10

Compliance test"

商品化ELISA检测试剂盒 Commerciallized ELISA kits Omp2-ELISA检测试剂盒 Omp2-ELISA kits 符合率
Conincidence rate (%)
血清 Serum 数量 Quantity 阳性 Positive 阴性 Negative
阳性 Positive 50 46 4 92.0
阴性 Negative 50 8 42 84.0
总数 Total 100 54 46 88.0
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