Immuno-gold labeling card for general detection of fluoroquinolones and preparation method thereof

Abstract

The invention discloses an immuno-gold labeling card for the general detection of fluoroquinolones and a preparation method thereof. The immuno-gold labeling card comprises a colloidal gold pad and a chromatography membrane. The colloidal gold pad is sprayed with a monoclonal antibody protein generated by a hybridoma cell strain, wherein the hybridoma cell strain is labeled by colloidal gold and the preservation number of the hybridoma cell strain is CCTCC NO.C2015221. A detection line on the chromatography membrane is sprayed with a conjugate of enrofloxacin and cationized bovine serum albumin. A control line on the chromatography membrane is sprayed with a Goat Anti-Mouse secondary antibody IgG(H+L). According to the invention, a monoclonal antibody, which is high in sensitivity and strong in universality for ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, enoxacin and the like when measured through the indirect competition ELISA assay, is adopted. After that, the monoclonal antibody is applied to the immuno-gold labeling card for the detection of fluoroquinolones. Therefore, the prepared immuno-gold labeling card is still high in sensitivity and strong in universality for target drugs. In this way, the immuno-gold labeling card can be used for the rapid and qualitative screening of mainly fluoroquinolones in various fields.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a universal immune gold label card for detecting fluoroquinolones and a preparation method thereof. Background technique [0002] As synthetic antibiotics, fluoroquinolones are widely used in medicine (as first-line antibacterial drugs in clinical practice), agricultural In the production of by-water products (important antibacterial drugs used in the prevention and treatment of infectious diseases as livestock, poultry and aquatic animals), the current fluoroquinolone antibacterial drugs show a trend of multi-component mixing and high residue in food samples. What is more worrying is that related The diffusion of the above antibiotics in the environment in pharmaceutical companies, hospitals, and agricultural and sideline aquatic products production enterprises causes very small residues in soil and surface water sources, which is a huge potential threat to human health. Therefore, ...

AI Technical Summary

Problems solved by technology

[0005] 1. The detection limit is low: the single-component detection limit (T line completely disappears) of the commercialized immune gold standard drug fluoroquinolone is about 10ppb, while the Ministry of Agriculture’s minimum residue standard for the above-mentioned 8 kinds of drugs is: 30-100ppb, based on this The standard uses 5-10× buffer solution to prepare the homogenate supernatant of the sample to be tested. Under the premise of not enriching the target substance, its content is very likely to be lower than the detection limit, and the sample to be tested cannot be effectively screened qualitatively. Use less than 5× buffer solution to prepare the homogenate supernatant of the sample to be tested is not only not conducive to the full release of the target substance in the sample to be tested, affecting the detection accuracy, resulting in false negatives and missed detection, but also very likely to interfere with the antibody’s detection of the target substance due to the high content of the sample matrix. Identification affects detection specificity, and false positives lead to false detections. Therefore, the single-component detection limit of the immune gold standard card should be less than or equal to 3ppb to fully meet the actual needs of users;

[0006] 2. The versatility is not strong: Zhao Yinli and others reported in the literature that using self-prepared enrofloxacin strong specific monoclonal antibody as a labeled antibody to develop the detection limit of the immune gold label card (T line completely disappeared) is less than or equal to 3ppb, and chicken muscle As a model matrix, enrofloxacin external standard incorporation recovery test has obtained good results, but because there is no cross-reaction with other quinolones, it can only be used for strong specificity analysis of single-component residues of enrofloxacin. What is more unfavorable is that enrofloxacin Norofloxacin is partially converted to ciprofloxacin through in vivo metabolism. This card cannot be used to determine whether the mixed residues of enrofloxacin and ciprofloxacin in the body after enrofloxacin administration exceed the standard. Other fluoroquinolones such as norfloxacin There are also some sporadic reports on the immunoassay products of star, ofloxacin, pefloxacin and other targets, but most of them are suitable for single-component determination due to their strong specificity for their targets.

However, the residues of fluoroquinolone drugs in food samples tend to be multi-component mixed residues, so it is impossible to use a single antibody immunoassay product for qualitative or semi-quantitative analysis of multiple component residues at the same time

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