Research On Immunochromatography Assay For Simultaneous Detection Of Five Kinds Of Common Salmonella Serotype Based On Colloidal Gold

Salmonella spp. is a kind of food-borne pathogen, which can infect humans andanimals through contaminated food, feed, animal manure and infected person.Salmonella spp. causes the greatest number of food poisoning all around the world,which often leads to acute diarrhea, vomiting, abdominal pain, fever and septicemia.Thus, detecting Salmonella spp. in a fast and accurate way has importantsignificance.Traditional detection methods of Salmonella spp. would cost4~7days,and it is time consuming and laborious. At present, various rapid detection methodshave been developed for Salmonella spp., such as multiplex PCR, Microarray andEnzyme-linked immunosorbent assay. However, most of these methods need to useexpensive equipments or complex experiments, which make it difficult in promotingin basic level. An immunochromatographic assay based on colloidal gold as the tracermarker was studied to detect five kinds of common Salmonella spp.In this study, Salmonella anatum, Salmonella choleraesuls, Salmonellaenteritidis, Salmonella parathpyi A and Salmonella typhimurium were the targets.Twenty seven anti-Salmonella monoclonal antibodies were used to develop theimmunochromatographic assay based on double-antibody sandwich. Results showedthat Mab8H10-B3could couple with Mab5G6-D7well to detect Salmonella anatum,Salmonella enteritidis, Salmonella parathpyi A and Salmonella typhimurium, and theMab11D8-D4could couple with Mab9G6-A4well to detect Salmonella choleraesuls.The titers of four Mabs8H10-B3,5G6-D7,11D8-D4,9G6-A4were1:640000,1:2560000,1:2560000and1:1280000respectively. In this assay, Mabs8H10-B3and11D8-D4were used to lable the colloidal gold, the mix of Mabs5G6-D7and9G6-A4and the donkey-anti-mouse IgG were sprayed on nitrocellulose (NC) membranes asthe test line and control line, respectively. The optimal experimental conditions of thecolloidal gold based immunochromatographic strip were as follow. The concentrationof Mab8H10-B3and11D8-D4were15μg/mL and5μg/mL respectively and5%casein were used to block the rest sites on gold nanoparticles effectively. The mixantibodies (5G6-D7+9G6-A4) concentration of test line on NC membranes was(1.5+2.0) mg/mL, the donkey-anti-mouse second antibody concentration of control line was1mg/mL. The volume of gold-labelled antibody was both2μL for each ofdetection. The incubation time of gold-labelled antibody with bacteria was5min andthe testing time was15min. Under optimal conditions, the detection limits of thismethod was that the LOD of Salmonella anatum and Salmonella typhimurium were1×106CFU/mL, the LOD of Salmonella choleraesuls, Salmonella enteritidis andSalmonella parathpyi A were1×105CFU/mL. And the strips showed a bit ofcross-reactivity with Staphylococcus aureus and Escherichia coli. When applied inpure milk and raw egg samples with an inoculation amount of5~50CFU/25g, thetest strip could get positive result after19h enrichment.