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

RichHTML

9

PDF

68

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
[1]
PAGE A E, SLOVIS N M, HOROHOV D W. Lawsonia intracellularis and equine proliferative enteropathy. Veterinary Clinics of North America: Equine Practice, 2014, 30(3): 641-658.
[2]
LAWSON G H K, ROWLAND A C. Intestinal adenomatosis in the pig: a bacteriological study. Research in Veterinary Science, 1974, 17(3): 331-336.

pmid: 4614347
[3]
KROLL J J, ROOF M B, HOFFMAN L J, DICKSON J S, HANK HARRIS D L. Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis. Animal Health Research Reviews, 2005, 6(2): 173-197.
[4]
BOUTRUP T S, BOESEN H T, BOYE M, AGERHOLM J S, JENSEN T K. Early Pathogenesis in Porcine Proliferative Enteropathy caused by Lawsonia intracellularis. Journal of Comparative Pathology, 2010, 143(2/3): 101-109.
[5]
MØLBAK L, JOHNSEN K, BOYE M, JENSEN T K, JOHANSEN M, MØLLER K, LESER T D. The microbiota of pigs influenced by diet texture and severity of Lawsonia intracellularis infection. Veterinary Microbiology, 2008, 128(1/2): 96-107.
[6]
LAWSON G, MCORIST S, ROWLAND A, MCCARTNEY E, ROBERTS L. Serological diagnosis of the porcine proliferative enteropathies: implications for aetiology and epidemiology. Veterinary Record, 1988, 122(23): 554-557.

pmid: 3046108
[7]
XIAO N, LI J N, LI M X, HU Y T, LIN H X, FAN H J. Preparation and characterization of a new monoclonal antibody specific against Lawsonia intracellularis and its application in indirect immunofluorescence and immunocytochemistry assay. Frontiers in Veterinary Science, 2021, 8: 753610.
[8]
HOLYOAKE P K, EMERY D, GONSALVES J, DONAHOO M, COLLINS A. Prevalence of antibodies to Lawsonia intracellularisin pig herds in Australia. Australian Veterinary Journal, 2010, 88(5): 186-188.
[9]
WATARAI M, YAMATO Y, HORIUCHI N, KIM S, OMATA Y, SHIRAHATA T, FURUOKA H. Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with proliferative enteropathy. Journal of Veterinary Medical Science, 2004, 66(6): 735-737.
[10]
LI M X, XIAO N, LI J N, LIN H X, FAN H J. Evaluation of immune efficacy of Omp2 protein against Lawsonia intracellularis in mice. Veterinary Microbiology, 2021, 263: 109274.
[11]
范红结, 肖宁, 周红, 蔺辉星, 李剑男, 胡雨婷, 李敏雪. 一种胞内劳森菌的IPMA抗体检测方法: CN113376375A[P]. 2021-09-10
FAN H J, XIAO N, ZHOU H, LIN H X, LI J N, HU Y T, LI M X. A type of IPMA detection method for Lawsonia intracellularis antibody: CN113376375A[P]. 2021-09-10. (in Chinese)
[12]
李敏雪, 李剑男, 周红, 肖宁, 蔺辉星, 马喆, 范红结. 基于Sod C单克隆抗体的胞内劳森菌IPMA抗原检测方法的建立及应用. 中国农业科学, 2021, 54(20): 4478-4486.

doi: 10.3864/j.issn.0578-1752.2021.20.020
LI M X, LI J N, ZHOU H, XIAO N, LIN H X, MA Z, FAN H J. Establishment and preliminary application of Lawsonia intracellularis IPMA antigen detection method based on sod C monoclonal antibody. Scientia Agricultura Sinica, 2021, 54(20): 4478-4486. (in Chinese)
[13]
JACOBSON M, GERTH LÖFSTEDT M, HOLMGREN N, LUNDEHEIM N, FELLSTRÖM C. The prevalences of Brachyspira spp. and Lawsonia intracellularisin Swedish piglet producing herds and wild boar population. Journal of Veterinary Medicine, Series B, 2005, 52(9): 386-391.
[14]
STEGE H, JENSEN T K, MØLLER K, BÆKBO P, JORSAL S E. Prevalence of intestinal pathogens in Danish finishing pig herds. Preventive Veterinary Medicine, 2000, 46(4): 279-292.

pmid: 10960714
[15]
SUH D K, SONG J C. Prevalence of Lawsonia intracellularis, Brachyspira hyodysenteriae and Salmonella in swine herds. Journal of Veterinary Science, 2005, 6(4): 289-293.
[16]
LEE S W, KIM T J, PARK S Y, SONG C S, CHANG H K, YEH J K, PARK H I, LEE J B. Prevalence of porcine proliferative enteropathy and its control with tylosin in Korea. Journal of Veterinary Science, 2001, 2(3): 209.
[17]
XIAO N, LÜ Y Y, LI J N, CHEN C F, LIN H X, FAN H J. Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis. Journal of Integrative of Agriculture, 2023, 22(9): 2824-2833.
[18]
支康发, 李复坤, 钟梦龙, 王红彩, 李波, 欧兰欣, 黄福强, 余新刚, 齐冬梅, 张浩吉. 饲料禁抗背景下规模化猪场胞内劳森菌的流行病学调查. 广东畜牧兽医科技, 2023, 48(2): 65-69.

doi: 10.19978/j.cnki.xmsy.2023.02.12
ZHI K F, LI F K, ZHONG M L, WANG H C, LI B, OU L X, HUANG F Q, YU X G, QI D M, ZHANG H J. Epidemiological investigation of Lawsonia intracellularis in large scale pig farm under the scenarios of antibiotic-free feed policy. Guangdong Journal of Animal and Veterinary Science, 2023, 48(2): 65-69. (in Chinese)
[19]
GUEDES R M C, GEBHART C J. Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis. Veterinary Microbiology, 2003, 91(2/3): 135-145.
[20]
NOGUEIRA M G, COLLINS A M, DONAHOO M, EMERY D. Immunological responses to vaccination following experimental Lawsonia intracellularis virulent challenge in pigs. Veterinary Microbiology, 2013, 164(1/2): 131-138.
[21]
GUEDES R M C, GEBHART C J. Preparation and characterization of polyclonal and monoclonal antibodies against Lawsonia intracellularis. Journal of Veterinary Diagnostic Investigation, 2003, 15(5): 438-446.
[22]
XIAO N, LI J N, LI M X, ZHOU H, LIN H X, FAN H J. Isolation and in Vitro cultivation of Lawsonia intracellularis from China. Veterinary Microbiology, 2022, 270: 109457.
[23]
BALDASSO D Z, GUIZZO J A, DAZZI C C, PARABONI FRANDOLOSO G C, FERONATO C, VON BERG S, CARVALHO GUEDES R M, WILSON H L, KREUTZ L C, FRANDOLOSO R. Development and validation of a flow cytometry antibody test for Lawsonia intracellularis. Frontiers in Immunology, 2023, 14: 1145072.
[24]
CORZO C A, FRIENDSHIP R, DEWEY C, BLACKWELL T. Comparison of 2 serologic tests for the diagnosis of porcine proliferative enteropathy. La Revue Veterinaire Canadienne, 2005, 46(5): 433-435.
[25]
KROLL J J, EICHMEYER M A, SCHAEFFER M L, MCORIST S, HARRIS D L, ROOF M B. Lipopolysaccharide-Based Enzyme- Linked immunosorbent assay for experimental use in detection of antibodies to Lawsonia intracellularisin pigs. Clinical and Vaccine Immunology, 2005, 12(6): 693-699.
[26]
JACOBSON M, WALLGREN P, NORDENGRAHN A, MERZA M, EMANUELSON U. Evaluation of a blocking ELISA for the detection of antibodies against Lawsonia intracellularis in pig sera. Acta Veterinaria Scandinavica, 2011, 53(1): 23.
[27]
吴艳阳, 杨东东, 高冬生, 李永涛, 常洪涛, 王川庆, 赵军. 胞内劳森菌LsaA蛋白的原核表达及间接ELISA抗体检测方法的建立. 畜牧兽医学报, 2018, 49(4): 786-793.
WU Y Y, YANG D D, GAO D S, LI Y T, CHANG H T, WANG C Q, ZHAO J. Prokaryotic expression of LsaA protein of Lawsonia intracellularis and development of a LsaA-based indirect ELISA for antibody detection. Chinese Journal of Animal and Veterinary Sciences, 2018, 49(4): 786-793. (in Chinese)
[28]
YANG L J, LAI F J, HE L, LU Y J, ZHONG Q W, LAI C D, DAI Y M. LI1035, a putative effector secreted by Lawsonia intracellularis, targets the MAPK pathway and regulates actin organizationin yeast and mammalian cells. Veterinary Microbiology, 2019, 235: 127-135.
[29]
FOURIE K R, CHOUDHARY P, NG S H, OBRADOVIC M, BROWNLIE R, ANAND S K, WILSON H L. Evaluation of immunogenicity and protection mediated by Lawsonia intracellularis subunit vaccines. Veterinary Immunology and Immunopathology, 2021, 237: 110256.
[30]
张鹏云, 陈敏, 刘明星, 周红, 蔺辉星, 范红结. 副猪嗜血杆菌间接ELISA抗体检测试剂盒的研制与应用. 中国农业科学, 2023, 56(8): 1606-1614.

doi: 10.3864/j.issn.0578-1752.2023.08.015
ZHANG P Y, CHEN M, LIU M X, ZHOU H, LIN H X, FAN H J. Development and application of indirect ELISA kits for antibody detection of Haemophilus parasuis. Scientia Agricultura Sinica, 2023, 56(8): 1606-1614. (in Chinese)
[31]
LEBRET A, AUVIGNE V, MOREL SAIVES A. Longitudinal evaluation of three commercial diagnostic assays for Lawsonia intracellularis infection in pigs. Veterinary Record, 2013, 172(13): 341.
[32]
COLLINS A, GONSALVES J, FELL S, BARCHIA I. Comparison of a commercial ELISA with an indirect fluorescent antibody test to detect antibodies toLawsonia intracellularisin experimentally challenged pigs. Australian Veterinary Journal, 2012, 90(3): 97-99.
[33]
PAGE A E, STILLS H F, HOROHOV D W. Sub-isotypic differences in the immunoglobulin G response to Lawsonia intracellularis in vaccinated, seropositive, and equine proliferative enteropathy-affected horses. Veterinary Immunology and Immunopathology, 2014, 162(3/4): 162-167.
[1] YU Ru, SONG JiaShen, ZHANG HongYuan, CHANG FangDi, WANG YongQing, WANG XiQuan, WANG Jing, WANG WeiNi, LI YuYi. Effects of Straw Interlayer Combined with Spring Irrigation on Saline- Alkali Soil Respiration and Its Temperature Sensitivity in Hetao Irrigation District [J]. Scientia Agricultura Sinica, 2023, 56(12): 2341-2353.
[2] LI GuiXiang,LI XiuHuan,HAO XinChang,LI ZhiWen,LIU Feng,LIU XiLi. Sensitivity of Corynespora cassiicola to Three Common Fungicides and Its Resistance to Fluopyram from Shandong Province [J]. Scientia Agricultura Sinica, 2022, 55(7): 1359-1370.
[3] CAO HanBing,XIE JunYu,LIU Fei,GAO JianYong,WANG ChuHan,WANG RenJie,XIE YingHe,LI TingLiang. Mineralization Characteristics of Soil Organic Carbon and Its Temperature Sensitivity in Wheat Field Under Film Mulching [J]. Scientia Agricultura Sinica, 2021, 54(21): 4611-4622.
[4] LI MinXue,LI JianNan,ZHOU Hong,XIAO Ning,LIN HuiXing,MA Zhe,FAN HongJie. Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody [J]. Scientia Agricultura Sinica, 2021, 54(20): 4478-4486.
[5] DAI YuLi,GAN Lin,TENG ZhenYong,YANG JingMin,QI YueYue,SHI NiuNiu,CHEN FuRu,YANG XiuJuan. Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis [J]. Scientia Agricultura Sinica, 2020, 53(3): 527-538.
[6] WANG JunJie,WANG HaiGang,CAO XiaoNing,CHEN Ling,LIU SiChen,TIAN Xiang,QIN HuiBin,QIAO ZhiJun. Comprehensive Evaluation of Photoperiod Sensitivity Based on Different Traits of Broomcorn Millet [J]. Scientia Agricultura Sinica, 2020, 53(3): 474-485.
[7] HE LiFei, CHEN LeLe, XIAO Bin, ZHAO ShiFeng, LI XiuHuan, MU Wei, LIU Feng. Establishment of Sensitivity Baseline and Evaluation of Field Control Efficacy of Fludioxonil Against Fulvia fulva [J]. Scientia Agricultura Sinica, 2018, 51(8): 1475-1483.
[8] ZHU Yang,LIU YongFeng,WEI YanChao,SHEN Qian,WANG YiFan. Qualitative and Quantitative Detection Methods of Pork in Beef and Its Chinese Processing Products [J]. Scientia Agricultura Sinica, 2018, 51(22): 4352-4363.
[9] HUANG XuePing, SONG YuFei, LUO Jian, ZHAO ShiFeng, MU Wei, LIU Feng. Sensitivity of Sclerotinia sclerotiorum to Fluopyram and Evaluation of Its Application Potential in Controlling Sclerotinia Stem Rot [J]. Scientia Agricultura Sinica, 2018, 51(14): 2711-2718.
[10] JIA XiaoPing, YUAN XiLei, LI JianFeng, ZHANG Bo, ZHANG XiaoMei, GUO XiuPu, CHEN ChunYan. Comprehensive Evaluation of Main Agronomic Traits of Millet Resources Under Different Light and Temperature Conditions [J]. Scientia Agricultura Sinica, 2018, 51(13): 2429-2441.
[11] KONG YouBin, LI XiHuan, ZHANG CaiYing. Construction and Activity Analysis of the Promoter of Purple Acid Phosphatase Gene GmPAP4 in Soybean [J]. Scientia Agricultura Sinica, 2017, 50(3): 582-590.
[12] CUI KaiDi, HUANG XuePing, HE LeiMing, ZHAI YongBiao, MU Wei, LIU Feng. The Inhibition Effect of Microbial Volatile Compound Benzothiazole on Botrytis cinerea [J]. Scientia Agricultura Sinica, 2017, 50(19): 3714-3722.
[13] ZHANG YanJun. Effect of Long-Term Fertilization on Temperature Sensitivity of Soil Respiration During Fallow Season [J]. Scientia Agricultura Sinica, 2017, 50(16): 3164-3174.
[14] XING HuiMin, XIANG ShiYao, XU XinGang, FENG HaiKuan, YANG GuiJun, CHEN ZhaoXia. Global Sensitivity Analysis of AquaCrop Crop Model Parameters Based on EFAST Method [J]. Scientia Agricultura Sinica, 2017, 50(1): 64-76.
[15] WANG Fang, FENG Yu, ZHANG Ge, JIANG Hui, ZHU Liang-quan, DING Jia-bo. Development of Indirect ELISA for Antibody of Brucella abortus [J]. Scientia Agricultura Sinica, 2016, 49(9): 1818-1825.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd