A double-antibody sandwich method for the detection of Escherichia coli intracellular β-glucuronidase in food

Abstract

The invention discloses a double-antibody sandwich method for detecting beta-glucuronidase in escherichia coli cells of food, belonging to the technical field of immunoassay. The double-antibody sandwich method provided by the invention comprises the following steps of carrying out immune on seven-week BALB / c mice by using escherichia coli beta-glucuronidase (EC3.2.1.31), and fusing and screening to obtain ten monoclonal antibodies, respectively marking HRP and carrying out pairing; and establishing an sandwich ELISA analytical method by utilizing CGMCC No.7209 as a coating antibody, utilizing a CGMCC No.7211 as an enzyme labelled antibody, and utilizing the recombination expressive escherichia coli beta-glucuronidase as a standard substance. According to the double-antibody sandwich method provided by the invention, the beta-glucuronidase in escherichia coli ATCC 25922 detection cells is cracked, the escherichia coli is detected, and the LOD is 3.27*10^4cfu / mL; according to the method, the monoclonal antibody of a landmark of the escherichia coli, namely beta-glucuronidase, is prepared, the double-antibody sandwich method for detecting the escherichia coli is established, the method and salmonella, E.coli, enterobacter sakazakii, staphylococcus aureus and listeria monocytogenes do not have cross reaction, and the new detection method is provided for detecting the escherichia coli in food.

Description

technical field [0001] The invention relates to a double-antibody sandwich method for detecting intracellular β-glucuronidase of Escherichia coli in food based on a monoclonal antibody, which belongs to the technical field of immune analysis. Background technique [0002] Escherichia coli (Escherichia coli, E.coli), discovered by Escherich in 1885, is distributed in nature and mainly epiphytes in the intestinal tract of humans or animals. It is a normal flora. A small number of E. coli are toxic and can cause diseases. [0003] E.coli is often scattered in the environment with feces. As a representative of intestinal bacteria in warm-blooded animals, E. coli is often used as a hygienic standard for drinking water and food (or medicine). If this bacterium is detected in water and food, it can be considered as an indicator of feces contamination, so there may be the presence of enteropathogenic bacteria. Escherichia coli β-glucuronidase is an enzyme with a molecular weight of...

AI Technical Summary

Problems solved by technology

The culture method is the national standard method for detecting E.coli. Although it is authoritative and reliable, it generally takes 10 days to get the result, and the operation process is cumbersome, which cannot meet the requirements of rapid detection; the enzyme substrate method mostly uses E.coli specific β- The enzymatic activity of alkaline phosphatase or β-D-glucuronidase catalyzes the substrate to generate fluorescence, so that the detection of E.coli can be realized within 24 hours. The advantages are short detection time and simple operation. The disadvantage is the enzymatic activity It is easily affected by temperature, pH, and ionic strength. The quality of foreign products is relatively stable, but relatively expensive; PCR method, which detects E.coli-specific DNA fragments, has the characteristics of high sensitivity and short detection time. The disadvantage is that the cost is relatively high. High, requires high operating technology, and cannot avoid the enrichment process

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