Production of polyclonal antibodies against spores ofClostridium tyrobutyricum, a contaminant affecting the quality of cheese: characterisation of the immunodominant protein

Abstract

The effect of different treatments in the production of polyclonal antibodies against Clostridium tyrobutyricum spores and its immunodetection was studied. Moreover, the main protein that causes the immunological response has been characterised. Antisera from rabbits immunised with non-treated, heated or sonicated spores were tested by ELISA. Heated and sonicated spores gave a faster increase in antibody titre than non-treated spores a long time after immunisation. Heat treatment improved reactivity when it was applied to the spores prior to immunodetection. However, when the highest titre was reached, the treatment effect was not observed either in the immune response or in the immunodetection. The immunodominant protein was identified in a spore extract by transferblotting, analysed by on-line liquid chromatography tandem mass spectrometry, and characterised by comparison of its peptide fragmentation spectrum with the NCBI database. Five proteins from the family of chaperonins were shown related to the immunodominant protein of C. tyrobutyricum spore extract.

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores

“…However, in our case, obtaining functionalized paramagnetic beads had to be implemented from preparation of appropriate ligands. Previously obtained antibodies and peptides with affinity to C. tyrobutyricum spores [9,29] have been used. Their application together with magnetic beads and biomagnetic separation of this microorganism was studied in the present work, since promising results using this technique had been observed in separation and detection of microorganisms in milk [35,36] and even in the detection of other substances, for example, antibiotics [37].…”

“…Specific polyclonal antibodies against spores of C. tyrobutyricum were obtained by rabbit immunization as previously described [29]. Briefly, animals were injected in multiple subcutaneous injections with 0.5 mL of 10 9 spores/mL suspension in 0.15 M NaCl, 10 mM K 2 HPO 4 ⁄KH 2 PO 4 buffer, pH 7.4, mixed with the same volume of Freund's complete adjuvant (Sigma-Aldrich) as primary dose, and followed by monthly boost of the same dose in Freund's incomplete adjuvant (Sigma).…”

Detection ofClostridium tyrobutyricumspores using polyclonal antibodies and flow cytometry

“…tyrobutyricum were shown to react with spores and vegetative cells and showed some cross‐reactivity with other microbial species, especially with Cl. sporogenes when they were analysed by an ELISA (Lavilla et al. 2008).…”

“…Rabbit polyclonal antisera raised against spores of Cl. tyrobutyricum in our laboratory was performed as previously described (Lavilla et al. 2008).…”

“…Presence of specific antibodies against Cl. tyrobutyricum was checked by immunodotting, and antibody titres were determined by ELISA as previously described (Lavilla et al. 2008).…”