李敏雪^,1, 李剑男¹, 周红¹, 肖宁¹, 蔺辉星¹, 马喆¹, 范红结^,1,21南京农业大学动物医学院,南京 210095
²江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州大学,江苏扬州 225009

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

LI MinXue^,1, LI JianNan¹, ZHOU Hong¹, XIAO Ning¹, LIN HuiXing¹, MA Zhe¹, FAN HongJie^,1,21College 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

通讯作者: 范红结,Tel：025-84399592;E-mail： fhj@njau.edu.cn

责任编辑: 林鉴非
收稿日期:2020-09-9接受日期:2021-04-12

基金资助:

江苏省农业科技自主创新资金(CX192020) 江苏省重点研发计划(BE2017341)

Received:2020-09-9Accepted:2021-04-12
作者简介 About authors
李敏雪,Tel：13218030982;E-mail： 2018107038@njau.edu.cn。









摘要
【目的】胞内劳森菌（Lawsonia intracellularis,LI）是引起猪增生性肠炎（porcine proliferative enteritis, PPE）的肠道病原菌,主要表现为动物福利下降,给世界养猪业造成严重的经济损失。研究通过制备鼠抗LI SodC的单克隆抗体,建立一种针对LI的免疫过氧化物酶细胞单层试验（IPMA）抗原检测方法,检验其在临床上的应用性,从而为LI的病原诊断提供一种科学有效的手段。【方法】选取胞内劳森菌弱毒疫苗为研究对象,通过PCR扩增其sodc片段,将其克隆至原核表达载体pGex-6p-1上,成功构建出pGex-6p-1-sodc重组质粒,诱导表达重组蛋白SodC。Western Blot分析重组蛋白的反应原性,一抗使用鼠抗GST标签的抗体。以该重组蛋白为免疫原,免疫4—6周龄BALB/c小鼠,利用常规细胞融合技术、有限稀释法和间接ELISA技术筛选阳性杂交瘤细胞,并制备腹水。通过间接免疫荧光（IFA）鉴定该单抗的特异性。以该单抗为一抗,摸索并建立了LI IPMA抗原检测方法,并评价该方法的特异性、敏感性和重复性。用优化后的IPMA方法对来自江苏周边地区猪场回肠组织样品进行检测,评价该方法的临床价值。【结果】纯化后SodC蛋白浓度较高,与鼠抗GST标签的抗体发生特异性结合,表明该蛋白反应原性较好。经3次亚克隆后最终共筛选获得2株阳性杂交瘤细胞,分别命名为1D6和1F7。单抗亚型鉴定结果显示：1D6亚型为IgA,1F7亚型为IgG3;ELISA检测1D6单抗效价为1﹕1 024 000;1F7单抗效价为1﹕1 024 000;间接免疫荧光（IFA）结果表明2株单抗均与LI菌株发生特异性反应,与猪霍乱沙门氏菌（Salmonella Cholerasuis,S. Cholerasuis）、猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）等猪常见病原无交叉反应。优化后IPMA反应条件为：一抗稀释倍数为1﹕800,作用45min;二抗稀释倍数为1﹕2 500,作用1h,此时IPMA检测效果最佳。用该方法检测S. Cholerasuis、PEDV、TGEV、伪狂犬病毒（PRV）、猪圆环病毒2（PCV2）均为阴性;最低检测限为10³个/mL,说明该方法特异性强、敏感性高。临床样品检测结果显示146份病料中共检测出92份阳性样品,3个不同猪场的阳性样品检出率分别为65.6%、68.2%和53.7%,总体阳性率为63.0%。普通PCR方法检测出82份阳性样品,两种方法的阳性符合率为94.6%。【结论】成功制备了鼠抗LI SodC蛋白的单克隆抗体,建立了针对LI抗原的IPMA检测方法,并对临床样品进行了检测。该方法具有良好的特异性和敏感性,并具有一定的临床价值,为实验室LI的分离鉴定、在感染细胞中的定位以及流行病学调查和相关检疫提供一种有效的技术手段。
关键词： 胞内劳森菌;SodC蛋白;单克隆抗体;免疫过氧化物酶细胞单层试验

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.
Keywords：Lawsonia intracellularis;SodC protein;monoclonal antibody;an immunoperoxidase monolayer assay (IPMA)


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本文引用格式
李敏雪, 李剑男, 周红, 肖宁, 蔺辉星, 马喆, 范红结. 基于SodC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 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. Scientia Acricultura Sinica, 2021, 54(20): 4478-4486 doi:10.3864/j.issn.0578-1752.2021.20.020


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0 引言

【研究意义】胞内劳森菌（Lawsonia intracellularis, LI）是引起猪增生性回肠炎（porcine proliferative enteritis, PPE）的一种专性细胞内寄生菌^[1,2,3]。该菌主要感染保育猪且寄生在回肠末端,临床上以细胞增生和黏膜增厚为主要特征^[4]。感染胞内劳森菌的猪表现为生长迟缓、饲料转化率低,严重影响了猪的经济价值和动物福利,给世界养猪业造成重大经济损失^[3,5-10]。在猪群中,PPE主要呈现增生性出血性肠病(PHE)和猪肠道腺瘤病(PIA)两种形式。PHE常以散发形式出现,其临床症状和严重程度与动物年龄有关^[11];而PIA是PPE临床上最常见的一种形式,以生长缓慢为主要特征^[12,13,14]。该病于1931年首次报道,广泛见于欧洲、北美及亚洲的一些猪场,呈世界范围内流行^[15,16]。近年来,我国集约化猪场中该病的发生率呈不断升高趋势,普遍存在亚临床型感染。郭建超等^[17]采集广东不同地区、不同日龄的577份猪粪便样品进行流行病学调查,结果显示总体阳性率为16.29%;曲向阳等^[18]调查华东地区三省一市规模化猪场的PPE感染情况,发现所检测粪便样品的总体阳性率达59.81%;黄忠等^[19]采用ELISA方法对华南5省区的22个猪场的1 100份猪血清进行检测,发现22个猪场抗体检测结果均为阳性。SodC蛋白是抗氧化系统的主要成员。研究显示SodC是布鲁氏菌重要的毒力因子,影响该菌的胞内存活,能够保护细菌免受过氧化氢的损伤,亦为重要的保护性抗原^[20]。研究表明,感染LI的肠上皮细胞对Cu提取转运蛋白的激活与胞内劳森菌内Zn/Cu-SOD的高表达有关,表明LI通过吸收胞浆内的Cu,表达SodC,逃避宿主细胞中超氧基团的损伤^[21]。胞内劳森菌是严格胞内寄生菌,目前无法在培养基中进行培养,需要依赖细胞进行细菌培养^[22],检测出细胞内LI是该菌成功分离与培养的前提。【前人研究进展】目前,已报道建立了PCR、qPCR、LAMP等方法检测该菌。孙娜等^[23]根据16SrRNA建立PCR检测方法,检测限为32.8μg·L^-1;彭忠等^[24]建立了巢式PCR方法,从粪便中检测该菌的最低限为2.2×10²拷贝/μL;PEDERSEN等^[25]建立了qPCR方法,能对LI进行定量分析;LI等^[26]建立了LAMP方法,可以用于猪粪便中的LI检测。但PCR仅用于感染排菌期猪粪便样品检测,检测阴性并不能说明未感染。此外,粪便中存在PCR抑制因子,可造成假阴性,且不适合大批样品的检测^{[27,28,29,30]}。【本研究切入点】LI可感染特定细胞系,如IPEC-J2、PK-15、McCoy等。临床上从病料中分离该菌常用小鼠肠上皮细胞McCoy,因此可用IPMA对McCoy中分离的细菌进行检测。与PCR、IFA等检测方法相比,IPMA特异性好,敏感性高,操作简便,判断直观,结果可以长期保存^{[31,32,33,34,35,36]}。同时适合大量样品筛查,特别是临床上大规模分离、鉴定该菌 。目前国内外建立的IPMA方法均用于PPE的血清流行病学调查,未有针对LI抗原IPMA方法的报道。【拟解决的关键问题】LI特殊的培养特性给该菌的分离、培养带来一定的困难。目前国内对其临床感染情况的相关研究报道还很少,仍处于初级阶段,研究LI的病原检测方法对于防治PPE显得尤为重要。本试验基于制备的SodC特异性单克隆抗体,建立了一种针对LI的IPMA检测方法,可用于LI的实验室和临床检测。

1 材料与方法

1.1 试验材料

胞内劳森菌疫苗株（B3903）,购于Boehringer Ingelheim公司;SPF级BALB/c小鼠购自青龙山动物繁殖场;McCoy细胞购自ATCC;HRP标记的羊抗鼠IgG购自北京博奥森生物科技有限公司;小鼠单克隆抗体亚型鉴定试剂盒购自南京迅贝生物科技有限公司;3-氨基-9-乙基咪唑（AEC）显色剂试剂盒购自上海翊圣生物科技有限公司;二氨基联苯胺（DAB）显色液购自凯基生物技术股份有限公司。

1.2 试验时间及地点

试验于2019年3—6月在南京农业大学实验动物中心进行。

1.3 SodC蛋白的原核表达及纯化

以猪胞内劳森菌弱毒疫苗为模板,常规PCR扩增sodc片段,鉴定为阳性后切胶回收sodc片段。以pGex-6p-1为载体,酶切并连接pGex-6p-1和上述PCR扩增产物,构建pGex-6p-1-sodc重组质粒。连接产物热激转化至感受态DH5ɑ,PCR鉴定为阳性的菌液提质粒转化BL21感受态,含有重组质粒的BL21命名为pGex-6p-1-sodc/BL21。

将重组菌pGex-6p-1-sodc/BL21接种到含氨苄青霉素和氯霉素的LB液体培养基中,置于37℃、180 r/min震荡培养至OD_600nm为0.6—0.8时,加入终浓度0.5 mmol·L^-1 IPTG,于16℃、90 r/min诱导18 h,离心收集菌体,超声破碎后进行SDS - PAGE电泳鉴定。

1.4 重组SodC蛋白的Western Blot 分析

纯化后的SodC蛋白经SDS-PAGE电泳后,Western Blot分析该单抗与蛋白的反应原性。一抗使用鼠抗GST标签的抗体,显色液使用二氨基联苯胺（DAB）显色液。

1.5 鼠抗SodC蛋白单抗的制备

使用GST亲和层析柱对重组SodC进行纯化。纯化后SodC蛋白免疫4—6周龄BALB/c小鼠,采用皮下多点注射方式,免疫剂量为50 μg/只。一免后第14和21天各加强免疫一次,免疫剂量与免疫途径同首免。三免后7 d选取效价最高的小鼠进行冲击免疫,采用腹腔注射的方式,免疫剂量为100μg/只,冲击免疫后3 d取小鼠脾细胞与SP2/0细胞进行融合。以间接ELISA筛选阳性杂交瘤细胞,经3—5次亚克隆后获得稳定分泌抗体的杂交瘤细胞株。以1×10⁶个/只的数量注射小鼠腹腔,制备腹水单抗,检测效价。

1.6 SodC蛋白单克隆抗体的鉴定

1.6.1 单克隆抗体亚型鉴定 用小鼠单抗亚型鉴定试剂盒进行亚型鉴定,操作按说明书进行。

1.6.2 单克隆抗体特异性鉴定 采用常规间接免疫荧光（IFA）鉴定单抗与LI反应的特异性,阴性对照孔用S. Cholerasuis,PEDV,TGEV感染细胞。

1.7 IPMA检测方法的建立

1.7.1 IPMA检测反应板的制备

（1）将McCoy细胞从液氮中取出复苏,加入含10%胎牛血清的DMEM培养基中,置于37℃,5% CO₂温箱中培养,传代5次后即可使用。将生长状态良好的McCoy细胞接种于96孔细胞培养板内,消化后调整细胞密度为1×10⁴—1×10⁵个/mL,置于37℃、5% CO₂培养箱培养24 h;

（2）形成单层细胞后,加入稀释后的猪LI弱毒疫苗,菌量为10^3.9 TCID₅₀/mL,阴性对照孔加入等量的DMEM培养液,细胞培养板室温2 000×g离心10—15 min,置于含8.0%O₂、8.8%CO₂和83.2%N₂的环境三气培养箱中培养3 h;

（3）更换培养液为含万古霉素（100 μg·mL^-1）、新霉素（50 μg·mL^-1）和两性霉素B（2 µg·mL^-1）的培养液,置于三气培养箱中培养至24 h;

（4）加入0.1% PBS稀释的Triton X-100,室温透化10 min;

（5）加入4%多聚甲醛在室温固定15 min,干燥后于4℃保存备用。

1.7.2 IPMA检测步骤 取出IPMA反应板于室温预热,按1﹕800比例加入SodC腹水单抗,37℃孵育1 h;按1﹕2 500比例加入HRP标记羊抗鼠IgG,37℃孵育1 h;加入AEC底物,室温显色20 min,甩去底物,加入PBS终止反应,加入苏木素染液染色30 s,彻底干燥后用光学显微镜观察结果。每个步骤之间需用PBS清洗3次。阳性判定标准：SodC单抗与感染细胞的细菌反应,胞核呈蓝色,胞浆呈棕红色;阴性判定标准：SodC单抗与健康细胞不反应,胞核呈蓝色,胞浆不着色。

1.8 IPMA反应条件的确定

1.8.1 一抗及二抗工作浓度的测定 制备IPMA反应板,将一抗分别进行1﹕50、1﹕100、1﹕200、1﹕400、1﹕600、1﹕800、1﹕1 000、1﹕2 000稀释,按照IPMA操作程序,分别加入1﹕500、1﹕1 000、1﹕2 500、1﹕3 000、1﹕4 000、1﹕5 000、1﹕6 000稀释的二抗,进行IPMA检测,确定一抗及二抗的最佳稀释度。

1.8.2 一抗最佳孵育时间的确定 制备IPMA反应板,加入最佳工作浓度一抗,于37℃作用30、45、60、90、120 min,进行IPMA操作,确定一抗最佳孵育时间。

1.8.3 二抗最佳孵育时间的确定 制备IPMA反应板5块,将二抗稀释到最佳工作浓度,分别于37℃作用30、45、60、90、120 min,按照IPMA操作程序,

确定二抗最佳孵育时间。

1.8.4 IPMA方法的特异性、敏感性和重复性试验 用S. Cholerasuis,PEDV,TGEV,PCV2分别进行IPMA检测,确定其特异性;将胞内劳森菌培养物稀释为1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹个菌,进行IPMA检测,确定其最低检测限;取不同批次和相同批次制备的IPMA 反应板进行3次重复试验,比较检测结果的重复性。

1.8.5 IPMA对临床样品的检测 分别从江苏3个不同地区猪场采集146份猪回肠组织样品,用优化后的IPMA对临床样品进行检测。

2 结果

2.1 SodC蛋白表达与纯化

SDS - PAGE电泳结果（图1）显示,在40 kD左右可见一条蛋白条带,与目的蛋白大小一致,表达量高,主要以上清形式表达。纯化后的蛋白浓度达1.02 mg·mL^-1,纯度较高,可作为免疫原免疫动物。

图1

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图1重组蛋白SodC的SDS-PAGE分析

M：蛋白分子量标准;1：pGex-6p-1空载体质粒;2：未诱导的重组表达质粒;3：诱导表达后的全菌液;4：诱导表达后的上清;5：诱导表达后的包涵体;6：纯化后的SodC蛋白
Fig. 1SDS-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

2.2 单克隆抗体的Western Blot分析

采用Western Blot方法检测SodC蛋白的反应原性。结果显示,SodC蛋白与一抗抗发生了特异性结合,在40 kD处出现一条明显的单一蛋白条带（图2）,结果与预期大小相符,进一步证明该蛋白在原核载体中成功表达,并具有良好的抗原性。

图2

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图2重组SodC蛋白的Western Blot分析

M：预染蛋白质分子质量标准;1：SodC纯化蛋白;2：空白对照
Fig. 2Western Blot analysis of SodC protein

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

2.3 单克隆抗体效价测定

经筛选获得1D6和1F7这2株阳性杂交瘤细胞。免疫小鼠制备腹水,间接ELISA检测结果显示,1D6单抗效价为1﹕1 024 000,1F7单抗效价为1﹕1 024 000。

2.4 单克隆抗体亚型鉴定

经过亚型鉴定,1D6、1F7单抗重链亚型分别为IgA链、IgG3链,2株单抗轻链均为κ链。

2.5 单克隆抗体特异性鉴定

IFA结果显示,2株单抗均能与LI发生反应,胞核呈蓝色,胞浆内产生绿色荧光,而与S. Cholerasuis、PEDV、TGEV无特异性反应（图3）。

图3

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图3单克隆抗体特异性鉴定

A：1D6与LI感染细胞反应;B：1F7与LI感染细反应;C-E：单抗分别与S.Cholerasuis、PEDV、TGEV反应;F：阴性对照
Fig. 3Specificity 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

2.6 IPMA反应条件的确定

经优化后最终确定IPMA反应条件为：一抗稀释浓度1﹕800,作用45min;二抗稀释浓度为1﹕2 500,作用1h时,此时IPMA显色效果最佳（图4、5）。

图4

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图4一抗工作浓度的确定

A：单抗与LI感染细胞反应;B：单抗与健康细胞反应
Fig. 4Determination of working concentration of primary antibody

A: Monoclonal antibody reacts with LI-infected cells; B: Monoclonal antibody reacts with healthy cells

图5

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图5二抗工作浓度的确定

A：单抗与LI感染细胞反应;B：单抗与健康细胞反应
Fig. 5Determination of working concentration of secondary antibody

A: monoclonal antibody reacts with LI-infected cells;B: monoclonal antibody reacts with healthy cells

2.7 IPMA的特异性

IPMA方法除了与LI发生特异性反应外,与S. Cholerasuis、PEDV、TGEV、PCV2、PRV均无交叉反应,表明建立的IPMA方法具有良好的特异性（图6）。

图6

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图6IPMA的特异性试验

A：单抗与LI感染细胞反应;B-F：单抗分别S. Cholerasuis、PEDV、TGEV、PCV2 、PRV感染细胞反应
Fig. 6The 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

2.8 IPMA的敏感性

将LI以1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹个/mL共6个梯度胞内劳森菌感染细胞,经IPMA检测,发现当细菌浓度稀释至为10³个/mL时,镜下可见较少的棕红色反应,而当细菌浓度稀释至为10²个/mL时,未见棕红色反应,说明该方法最低检测限为10³个/mL（图7）。

图7

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图7IPMA敏感性试验
A-F：单抗分别与1×10⁶ 、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹个/mL LI感染细胞反应
Fig. 7The 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

2.9 IPMA的重复性

通过检测IPMA 96孔检测板的批间和批内重复性,3次检测结果显示,IPMA反应板批间与批内检测结果一致,重复性良好。

2.10 IPMA对临床样品的检测

用优化后的IPMA方法对猪回肠组织样品进行检测,146份病料中共检测出92份阳性样品,3个不同地区屠宰场的阳性样品检出率分别为65.6%、68.2%和53.7%,总体阳性率为63.0%。普通PCR方法检测出82份阳性样品,两种方法的阳性符合率为94.6%,阴性符合率为91.5%。（表1）

Table 1
表1
表1IPMA对临床样品的检测
Table 1Detection 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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3 讨论

LI主要感染动物回肠末端,严重时可扩展至整个肠道^[3]。该菌主要定植于未成熟的肠隐窝上皮细胞中,造成腺瘤样增生、黏膜增厚、出血性炎症等临床症状^[37,38]。近年来,随着我国生猪养殖规模化程度越来越高,大部分猪场存在LI感染。目前国内对LI的流行现状和致病机理等方面的研究仍处于初级阶段,鉴于该病给我国养殖业造成的巨大经济损失,LI病原诊断与检测受到广泛关注。LI是严格细胞内寄生菌,但体外可感染IEC-18、IPEC-J2、PK-15、McCoy等细胞,并在这些细胞系上生长增殖^{[39,40,41,42,43]}。迄今为止,只有McCoy细胞被系统地评估了LI诱导的增殖,因此可用IPMA方法对LI进行检测。

近年来,国内外相继建立了LI IPMA抗体检测方法,拟用于增生性肠炎的血清流行病学调查,但敏感性存在不足^[34,44]。本研究基于SodC单克隆抗体,首次建立了检测LI的IPMA方法。通过优化,确定一抗最佳稀释度为1﹕800,作用45 min;二抗最佳稀释度为1﹕2 500,作用1 h,检测效果最佳。该单抗仅与LI发生特异性反应,与表达sodc的Salmonella Cholerasuis以及其他常见肠道病原,如PEDV、TGEV、PRV、PCV2均无交叉反应,进一步说明该方法特异性强。此外,该方法具有较高的敏感性,其最低检测限为10³个/mL。常规PCR虽可以对细胞内LI进行鉴定,但是细胞中基因组提取的纯度会直接影响后续酶解和PCR反应,导致假阴性。IFA虽然也可以检测细胞内的LI,但是对仪器设备要求较高,检测结果不能长时间保存。且IFA二抗为荧光抗体,假阳性结果的可能性增大;而IPMA检测仅需要光学显微镜可以进行亚细胞定位,肉眼即可判定阳性结果,显色板可保存几个月。

在LI IPMA抗原检测方法成功建立的基础上,本试验对3个不同地区猪场的146份回肠组织病料进行检测,共检测出92份阳性样品,总体阳性率为63.0%;而常规PCR阳性检出率为56.2%,敏感性低于IPMA抗原检测方法,可能的原因是模板基因组裂解过程中多种因素影响了PCR反应,从而降低了检测的敏感性。本文建立的IPMA检测方法,当样品中细菌含量低于10³个/mL时,亦不能发生特异性的显色反应。但是,实验室用McCoy分离临床LI样品时,初传阳性样本细胞LI的量可达到1×10³至1×10⁴个/mL,所以本研究建立的IPMA检测方法可用于实验室LI的检测。

4 结论

本研究基于SodC单克隆抗体成功建立了胞内劳森菌IPMA抗原检测方法,该方法具有较高的特异性、敏感性和重复性,适用于实验室胞内劳森菌的分离鉴定以及临床样品的快速检测。

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