Development of Listeria monocytogenes specific antibodies using a proteomics/genomics approach and expression of antibody-specific antigens InlB and ActA under different environments

Amanda A Lathrop, Purdue University

Abstract

Foodborne pathogen Listeria monocytogenes (LM) is a significant public health risk. Detecting and eliminating LM from the food supply is difficult for the food industry because current rapid detection methods lack specificity to LM. Here a proteomiclgenomic approach to identify LM specific target immunogens was used for the development of polyclonal antibodies. When the LM genome was compared to the nonpathogenic L. innocua genome, 22 surface proteins were unique to LM. Nine surface proteins were chosen and segments of these proteins were synthesized and used to develop antibodies in rabbits. All nine antibodies were screened against a panel of Listeria and non-Listeria bacteria. Two of the nine antibodies, PAb Lm404 and LmC639, showed a specific reaction to LM and the corresponding surface proteins were In1B and ActA, respectively. Both antibodies are currently being considered for use in ELISA and in a surface plasmon resonance sensor to detect LM from test samples. Environmental factors are known to affect ActA and InlB expression. Thus, expression of these antigens when present in food or in the selective or non-selective media was done. InlB and ActA expression in 13 LM strains representing one from each serotype was determined by Western blotting. In BHI (Brain Heart Infusion), 7 strains expressed ActA and 11 strains expressed InlB. When grown in LB (Luria-Bertani) only one strain showed ActA expression while all but one strain showed InlB expression. In Listeria-enrichment broths, expression of InlB was observed in only one strain, while ActA expression was seen in 12 strains in BLEB (Buffered Listeria Enrichment Broth) and 11 strains in UVM (University of Vermont Medium). These data were compared with immunoelectron microscopy and RT-PCR. Overall, InlB expression was poor in selective media; while, ActA expression was poor in non-selective media. Only a few strains showed expression of both InlB and ActA under the same conditions. Therefore, PAb Lm404 and PAb LmC639 could be used for specific detection of LM with appropriate selective media to reduce cross-reaction from other non-Listeria species and to insure expression of InlB or ActA antigens.

Degree

Ph.D.

Advisors

Bhunia, Purdue University.

Subject Area

Food science

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