Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4478-4486.doi: 10.3864/j.issn.0578-1752.2021.20.020

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody

LI MinXue1(),LI JianNan1,ZHOU Hong1,XIAO Ning1,LIN HuiXing1,MA Zhe1,FAN HongJie1,2()   

  1. 1College of Veterinary Medicine, Nanjing Agricultural University, Nangjing 210095
    2Jiangsu Co-innovation Center for the Prevention and Control of Important Animal, Yangzhou University Yangzhou, Yangzhou 225009 Jiangsu
  • Received:2020-09-09 Accepted:2021-04-12 Online:2021-10-16 Published:2021-10-25
  • Contact: HongJie FAN E-mail:2018107038@njau.edu.cn;fhj@njau.edu.cn

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Abstract:

【Objective】 Lawsonia intracellularis (L. intracellularis) is an enteric pathogenic bacteria that causes porcine proliferative enteropathy (PPE), which mainly shows the decline of animal welfare and causes serious economic losses to the world swine industries. The objective of this study was to prepare monoclonal antibodies against SodC of L. intracellularis, and to establish an immunoperoxidase monolayer assay (IPMA) method for detecting L. intracellularis base the monoclonal antibody, while test its application in clinical practice, so as to provide a scientific and effective means for the diagnosis of L. intracellularis. 【Method】In this study, the commercial live attenuated L. intracellularis vaccine was selected as target strain. The sodc gene was amplified by PCR and cloned into the prokaryotic expression vector pGex-6p-1. The recombinant plasmid pGex-6p-1-sodc was confirmed to be constructed successfully and induced expression of recombinant SodC protein. The reactivity of the recombinant protein was analyzed by Western Blot. The primary antibody was a mouse anti-GST labeled antibody. BALB/c mice aged 4-6 weeks were immunized with purified SodC protein, and hybridoma cells were screened by conventional cell fusion, limited dilution and indirect ELISA, then ascites were prepared. It was confirmed that two monoclonal antibodies had good specificity through indirect immunofluorescence (IFA). Using the monoclonal antibody as the primary antibody, a method of IPMA for detecting L. intracellularis was developed, and the specificity, sensitivity and repeatability of the method were evaluated. The optimized IPMA method was used to detect ileal tissue samples from pig farms in Jiangsu Province and to evaluate the clinical value of the method. 【Result】After purification, the concentration of SodC protein was higher, and it specifically bound to the antibody against GST tag, indicating that the protein had good regenicity. After three times of subcloning, two strains positive hybrid tumor cells were screened, named 1D6 and 1F7, respectively. The titers of two monoclonal antibodies were both reached 1﹕204 000 by ELISA. The subclass identification results of antibodies showed the subclass of 1D6 was IgA,and subclass of 1F7 was IgG3. The result IFA showed that 1D6 and 1F7 had specific reaction with L. intracellularis, but did not cross-react with S. Cholerasuis, PEDV and TGEV. The ascites of the two monoclonal antibodies were both 1﹕1 024 000 by ELISA; IFA confirmed that the two monoclonal antibodies had good specificity. The optimized IPMA reaction conditions showed that when the dilution ratio of the primary antibody was 1:800 for 45 min, and the dilution ratio of the secondary antibody was 1﹕2 500 for 1h, the established IPMA exhibited the best performance. The specificity and sensitivity tests showed that S. Cholerasuis, PEDV, TGEV, PCV2 and PRV were all negative, and the minimum detection limit was 103L.intracellularis. The optimized IPMA method was used to detect the ileum tissue samples from pig farms in the surrounding areas of Jiangsu Province. A total of 92 positive samples were detected from 146 samples of ileal tissues. The positive rates of 3 different pig farms were 65.6%, 68.1% and 53.7%, respectively, and the overall positive rate was 63.0%. 82 positive samples were detected by PCR method. and the positive coincidence rate of the two methods was 94.6%. These results indicated that this method had clinical value. 【Conclusion】The monoclonal antibodies against the recombinant SodC protein were successfully prepared, and the IPMA method for L. intracellularis was established with good specificity and sensitivity, and the clinical samples were tested. In summary, these results further proved that the IPMA had certain clinical value, and provided an effective technical means for the isolation and identification of L. intracellularis in the laboratory, localization in infected cells, epidemiological investigation and quarantine.

Key words: Lawsonia intracellularis, SodC protein, monoclonal antibody, an immunoperoxidase monolayer assay (IPMA)

Fig. 1

SDS-PAGE analysis of recombinant protein SodC M: Molecular weight standard of protein; 1: pGex-6p-1 no-load particle; 2: Uninduced recombinant expression plasmid; 3: Whole bacterial liquid induced and expressed; 4: Supernatant after induced expression; 5: Inclusion body after induced expression; 6: Purified SodC protein"

Fig. 2

Western Blot analysis of SodC protein M: Molecular quality standard of pre-dyed protein; 1: Purified protein SodC; 2: Blank control"

Fig. 3

Specificity identification of monoclonal antibodies A: 1D6 monoclonal antibody reacts with bacterial infected cells; B: 1F7 monoclonal antibody reacts with healthy cells; C-E:monoclonal antibody reacts with S. Cholerasuis, PEDV and TGEV, respectively; F: Negative control"

Fig. 4

Determination of working concentration of primary antibody A: Monoclonal antibody reacts with LI-infected cells; B: Monoclonal antibody reacts with healthy cells"

Fig. 5

Determination of working concentration of secondary antibody A: monoclonal antibody reacts with LI-infected cells;B: monoclonal antibody reacts with healthy cells"

Fig. 6

The specificity test of IPMA A: Monoclonal antibody against SodC reacts with LI-infected cells; B: Mouse monoclonal antibodies against SodC react with S. Cholerasuis, PEDV, TGEV, PCV2,PRV infected cells"

Fig. 7

The sensitivity test of IPMA A-F: Monoclonal antibody against SodC reacts with 1×106, 1×105, 1×104、1×103, 1×102, 1×101 LI-infected cells"

Table 1

Detection of clinical samples by IPMA"

猪场
Pig farm		 样品数
sample		 IPMA PCR
阳性
Positive		 阴性
Negative		 检出率
Detection rate (%)		 阳性
Positive		 阴性
Negative		 检出率
Detection rate (%)
泰州Taizhou 61 40 21 65.6 34 27 44.3
镇江Zhenjiang 44 30 14 68.1 26 18 59.1
盐城Yancheng 41 22 19 53.7 22 19 53.7
总计Total 146 92 54 63.0 82 64 56.2
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