用于检测食品和水样中伤寒沙门氏菌的富集酶联免疫吸附测定法。

Enrichment-ELISA for detection of Salmonella typhi from food and water samples.

作者信息

Kumar S, Balakrishna K, Batra H V

机构信息

Division of Microbiology, Defence R & D Establishment, Jhansi Road, Gwalior 474002, India.

出版信息

Biomed Environ Sci. 2008 Apr;21(2):137-43. doi: 10.1016/S0895-3988(08)60019-7.

DOI:10.1016/S0895-3988(08)60019-7

PMID:18548853

Abstract

OBJECTIVE

Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay (sELISA) for rapid detection of Salmonella enterica serovar typhi (S. typhi) from food and water samples and optimization of enrichment procedures for use with the developed sELISA to increase the detection sensitivity of the assay.

METHODS

Spleen cells from BALB/c mice immunized with flagellin (H=d) antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An sELISA was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the sELISA, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water (BPW) and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed sELISA with preceding enrichment step in BPW (Enrichment-ELISA) was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food (30) and water (35) samples collected from field were also tested by Enrichment-ELISA and culture method.

RESULTS

Out of four specific clones to H=d antigen, one clone (# 2/56, IgG2a isotype) was used in sELISA. The sELISA had the detection limit of 10(4)-10(5) cfu of S. typhi. Of the various broths used with sELISA, BPW was found to yield maximum ELISA values. Enrichment-ELISA, when tested in artificially inoculated food samples, generally, could detect 10(2) S. typhi cfu/mL within 10 h from various food rinses (meat, vegetable) and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples (water) gave false positive result by Enrichment-ELISA.

CONCLUSION

In comparison to culture, the Enrichment-ELISA is a rapid, sensitive, and specific method for detection of S. typhi from food or water samples. This method may be used as rapid screening procedure for environmental monitoring during outbreak situation.

摘要

目的

开发基于单克隆抗体的夹心酶联免疫吸附测定法（sELISA），用于快速检测食品和水样中的伤寒沙门氏菌（伤寒杆菌），并优化富集程序，以配合所开发的sELISA提高检测灵敏度。

方法

用伤寒杆菌鞭毛蛋白（H=d）抗原免疫的BALB/c小鼠的脾细胞与Sp2/0骨髓瘤细胞融合。建立了对H=d抗原特异的杂交瘤细胞系，进行了表征，并针对其中一个克隆制备了腹水。在新西兰白兔中制备了针对鞭毛蛋白抗原的超免疫血清。使用多克隆抗体作为捕获抗体、单克隆抗体作为检测抗体开发了一种sELISA。为设计与sELISA配合使用的高效培养策略，对不同的预富集和富集肉汤进行了评估。这些培养基包括用于预富集的缓冲蛋白胨水（BPW）和脑心浸液肉汤，以及作为富集肉汤的亚硒酸盐F肉汤和Rappaport-Vassiliadis肉汤。在人工接种伤寒杆菌的各种食品样品中评估了在BPW中进行预富集步骤后的所开发的sELISA（富集-ELISA）。还通过富集-ELISA和培养方法对从现场采集的各种食品（30份）和水（35份）样品进行了检测。

结果

在针对H=d抗原的四个特异性克隆中，一个克隆（#2/56，IgG2a亚型）用于sELISA。该sELISA对伤寒杆菌的检测限为10⁴-10⁵ cfu。在所使用的与sELISA配合的各种肉汤中，发现BPW产生的ELISA值最高。当在人工接种的食品样品中进行检测时，富集-ELISA通常能够在10小时内从各种食品冲洗液（肉类、蔬菜）和牛奶样品中检测到10² cfu/mL的伤寒杆菌。在BPW中过夜富集后，每10 mL牛奶、肉类冲洗液和鸡肉冲洗液中低至2个细菌都能被检测到。通过富集-ELISA检测，现场样品中只有一份（水）给出了假阳性结果。