Beta-lactam antibiotic detection card

A technology of lactams and antibiotics, applied in the field of inspection and quarantine, can solve the problems of unsuitable on-site rapid detection, non-detection of antibiotics, high cost, etc., and achieve the effect of ensuring the consistency of test results

Inactive Publication Date: 2014-12-17
吉林出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2001 and 2002, the Ministry of Agriculture successively announced "Non-Pollution Food Raw Milk" (NY / T5045-2001), "Pollution-Free Food Sterilized Milk" (NY5141-2002) and "Pollution-Free Food Pasteurized Milk" (NY5140-2002) and other industry standards stipulate that "antibiotics shall not be detected". The test method adopts the TTC (triphenyltetrazolium chloride) method in GB / T5049-85 "Testing Methods for Milk", but the detection limit of this method is Only 0.04U / mL, which is an order of magnitude different from the detection methods of European and American countries
However, the above method is costly, time-consuming, and requires professionals to operate, and is not suitable for on-site rapid detection of raw milk quality and other livestock and poultry products

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of β-lactam antibiotic immunogen

[0022] (1) Dissolve 50 mg of β-lactam antibiotics in 1.5 mL double distilled water to obtain liquid A.

[0023] (2) 40 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC for short) and 60 mg of N-hydroxysuccinimide (NHS for short) were dissolved together in 0.5 mL double-distilled water to obtain Liquid B.

[0024] (3) Add liquid B to liquid A under stirring, and react for 1 hour to obtain liquid C.

[0025] (4) Dissolve 60 mg ovalbumin in 8 mL double distilled water to obtain liquid D.

[0026] (5) Add solution D to solution C, stir and react at room temperature for 24 hours to obtain immunogen, dialyze with 0.02mol / LPBS buffer for 3 days, aliquot and freeze for later use.

Embodiment 2

[0027] Example 2 Preparation of β-lactam antibiotic capture monoclonal antibody

[0028] (1) BALB / c mice were immunized with the immunogen obtained in Example 1 at a dose of 150 μg / mouse to produce antiserum.

[0029] (2) Take the splenocytes of the above-mentioned immunized BALB / c mice and fuse them with SP2 / 0 myeloma cells at a ratio of 9:1 (quantity ratio), and screen to obtain cephalosporin monoclonal antibodies that stably secrete cephalosporin monoclonal antibodies Hybridoma cell lines.

[0030] (3) The hybridoma cells were placed in cell culture medium and cultured at 37°C, and the obtained culture medium was purified by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which were stored at -20°C. The cell culture medium is to add calf serum and sodium bicarbonate to the RPMI-1640 medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass percentage content), so that sodium bicarbonate The final conce...

Embodiment 3

[0031] Example 3 Preparation of polyclonal antibody coated with β-lactam antibiotics

[0032] (1) Using the immunogen prepared in Example 1, New Zealand big-eared rabbits with a body weight of 2 kg were used for the first immunization, and the immunization dose was 1 mL per rabbit, and injected subcutaneously at multiple points. Immunization was performed every 2 weeks, and blood was collected from the ear vein before immunization, and stored at -20°C. After 4 times of immunization, the serum polyantibody titer was determined by conventional indirect ELISA method.

[0033] (2) Separation of serum IgG. After the rabbit was anesthetized, blood was collected from the heart, placed at an angle of 4°C overnight, centrifuged at 3000r / min for 30min, and the serum was collected. The polyclonal antibody was purified by the AKTA protein purification system according to the caprylic acid-saturated ammonium sulfate method. Use a 30KD ultrafiltration centrifuge tube to wash the polyclo...

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PUM

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Abstract

The invention provides a beta-lactam antibiotic detection card which comprises a sample pad, a golden label pad containing beta-lactam antibiotic captured monoclonal antibody, a nitrocellulose membrane which sequentially comprises a detection line beta-lactam antibiotic coated polyclonal antibody and a quality control line rabbit antimouse IgG, an absorption pad and a lining plate. The invention also provides a performance detection and quality control method for the beta-lactam antibiotic detection card. The beta-lactam antibiotics can be accurately, simply and rapidly detected on the site, and the detection result consistency of the detection card is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of inspection and quarantine, in particular to a β-lactam antibiotic detection card. Background technique [0002] β-lactam antibiotics are the oldest antimicrobial drugs, and also the largest and most important class of antibiotics. Since the first application of penicillin in 1941, nearly a hundred kinds of β-lactam antibiotics have been developed. Penicillins, cephalosporins, cephamycins, carbapenems, oxycephalosporins, carbacephems, penems, monocyclic β-lactams and β-lactamase inhibitors, etc. . It is mainly used to treat various infections caused by Gram-negative bacteria, such as Staphylococcus, Pneumococcus, Streptococcus, Escherichia coli, Haemophilus, Salmonella, etc. In animal production, β-lactam antibiotics are widely used to control mastitis in dairy cows and treat urinary tract, gastrointestinal and respiratory tract infections in livestock and poultry. However, due to improper use methods o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/543
Inventor 刘金华罗雁非牟峻薜强邹明强
Owner 吉林出入境检验检疫局检验检疫技术中心
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