Generation and characterization of a novel recombinant scFv antibody specific for Campylobacter jejuni

Ruramayi M Nzuma,Fuquan Liu,Irene R Grant

doi:10.1007/s00253-018-8949-x

Ruramayi M Nzuma, Fuquan Liu + Show 1 more

Open Access

https://doi.org/10.1007/s00253-018-8949-x

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Abstract

Campylobacter jejuni is a leading cause of foodborne illness worldwide, mainly due to consumption and handling of contaminated raw chicken. Rapid detection methods for C. jejuni are vital for monitoring contamination levels in chicken products and reducing human Campylobacteriosis cases. The ‘gold standard’ culture-based method of Campylobacter detection takes 3–5 days and is too slow to permit effective intervention. Immuno-based methods are faster, but usually necessitate use of animals or hybridoma technology to produce antibodies; making them difficult and expensive to produce. Here, we report the generation and characterization of recombinant single-chain variable fragment (scFv) antibodies specific for C. jejuni cells, and evaluation of one scFv antibody for an immunomagnetic separation-quantitative PCR (IMS-qPCR) method to rapidly, sensitively, and specifically detect low numbers of C. jejuni. An scFv antibody phage-display library was constructed using spleen mRNA derived from a rabbit immunized with gamma-irradiated C. jejuni cells. This library was screened by surface biopanning against C. jejuni whole cells. Enriched clones were analyzed by enzyme-linked immunosorbent assay (ELISA). Two scFv antibodies that strongly and specifically recognized C. jejuni cell were expressed in Escherichia coli. Western blot analysis showed that one antibody, scFv80, was expressed as a soluble protein and retained its specific and strong binding to C. jejuni cells. This recombinant monoclonal scFv antibody was purified and used to covalently coat paramagnetic beads to be used for IMS-qPCR. The IMS-qPCR method was able to specifically and sensitively detect C. jejuni in mixed cultures within 3 h.

Highlights

Campylobacter, especially C. jejuni, is one of the leading causes of bacterial foodborne illnesses worldwide (Silva et al 2011; Tram et al 2012)
After the immunization course was completed for two New Zealand White rabbits, a substantial increase in anti-C. jejuni IgG production was observed in one of the rabbits (Fig. S1; Fig. 1a) and spleen and bone marrow were harvested from this rabbit
Total RNA extracted from the spleen (Fig. 1b) was used to synthesize the first-strand complementary DNA from which individual variable light (VL)- and variable heavy (VH)-chain region genes of approximately 350 and 400 bp, respectively, were amplified by polymerase chain reaction (PCR) (Fig. S1, S2; Fig. 1c)

Summary

Introduction

Campylobacter, especially C. jejuni, is one of the leading causes of bacterial foodborne illnesses worldwide (Silva et al 2011; Tram et al 2012). Campylobacter spp. are the most common cause of foodborne illness in humans, ahead of Salmonella (Food Standards Agency 2009; Dominguez 2012). Campylobacter is a harmless commensal intestinal inhabitant of chicken (Doyle 1994), which makes it difficult to spot early infection of Campylobacter in a flock. It spreads quickly and 95% of a flock of 20,000 chickens can be infected by Campylobacter within 4 to 7 days after colonization of the first bird (Van Gerwe et al 2009). A significant correlation between the contamination of the broilers during rearing and the carcasses after processing has been observed (Hermani et al 2003)

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