Establishment Of Detection Methods For The Immune Colloidal Gold Of TEM1 Type β-lactamase

β-lactamase is an extracellular enzyme secreted by theβ-lactam antibiotic-resistant bacteria, with which to generate the resistance for penicillins and cephalosporins.β-lactamase can decompound all the antibiotics after a certain period of time when added into the milk, thus covered up the presence of antibiotic in milk. The illicit use of the enzyme continued to exist in dairy breeding and milk production. Therefore, in order to ensure milk quality and the health of consumer the development of detection technology for monitoring theβ-lactamase is necessary.Many methods for detection ofβ-lactamase in milk have been established by now. Microbiological method is sensitive but time-consuming; others such as iodimetry method, HPLC are not suitable for screening a large number of samples because these methods are cost and highly-skilled. Gold Immunochromatographic assay, as a rapid, special, sensitive and safe biological method, could then provide a technical support for large-scale screening purpose.In this study, the monoclonal antibody (McAb) and the polyclonal antibody (PcAb) against TEM1 typeβ-lactamase were prepared. The McAb and PcAb were collected, puried and identified. The concentration of the McAb was 10mg/mL, and the PcAb was 6mg/mL. The titer of the McAb and PcAb were 1:320 000 and 1:80 000, respectively. Two antibodies were specific for TEM1 and showed no cross reactivity to cephalosporinase. The colloidal gold particle was obtained by reducing the gold chloride with sodium citrate and the diameter was 20nm. Then the gold particle was labeled with PcAb. The optimum pH for labelling is about 35μL volume of 0.1mol/L K2CO3, and the amount of PcAb is 72μg/mL. The colloidal gold-labeled PcAb was sprayed on the Ahlstrom8964 glass-fiber as detection reagent. The McAb and the goat anti-mouse IgG were drawn on a nitrocellulose membrane (NC) of Millipore 135 as test line and control line, respectively.The immunochromatographic test strip of the sample pad, NC membrane, gold standard pad material and associated fluid were optimized and assembled to prepare the immuno chromatography strip. The lowest detection limits were 6U/mL and 10U/mL in PBS and milk. The test strip was stable for three months at 4℃.In this paper, a simple, rapid and sensitive colloidal gold strip for detection of TEM1 typeβ-lactamase was developped. The sensitive, specific and stable strip could be used to detect TEM1 in milk.