Colloidal gold immunochromatography test strip for detecting furazolidone metabolites and preparation method and application of colloidal gold immunochromatography test strip

A technology of immunochromatography test paper and furazolidone, which is applied in the field of veterinary drug residue analysis and immunology, can solve the problems that are not suitable for rapid detection of large batches of samples, high requirements for experimental equipment and technology, and cumbersome operation, etc., and achieve easy promotion and use and applicable scope The effect of wide and simple method

Inactive Publication Date: 2017-03-08
BIOLOGY INST OF HEBEI ACAD OF SCI
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] Currently, the detection techniques for furazolidone residues mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS) and immunoassay techniques. The three methods are sensitive and accurate, but the operation is cumbersome and requires high experimental equipment and technology, so it is not suitable for rapid detection of large quantities of samples

Method used

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  • Colloidal gold immunochromatography test strip for detecting furazolidone metabolites and preparation method and application of colloidal gold immunochromatography test strip
  • Colloidal gold immunochromatography test strip for detecting furazolidone metabolites and preparation method and application of colloidal gold immunochromatography test strip
  • Colloidal gold immunochromatography test strip for detecting furazolidone metabolites and preparation method and application of colloidal gold immunochromatography test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Preparation of furazolidone metabolite antigen of the present invention

[0049] (1) Derivatization of furazolidone metabolites: take 0.25g AOZ, dissolve it with about 2mL of methanol at room temperature; take 0.15g of 3-carboxybenzaldehyde, dissolve it with 2mL of methanol, slowly drop into the above In the AOZ solution, stir and react at room temperature for 5 hours, filter, and dry the filter residue to obtain the furazolidone metabolite derivative CPAOZ;

[0050] (2) Synthesis of immunogen: Weigh 50 mg of bovine serum albumin (BSA) and dissolve it in 1 mL of 0.1 mol / L PBS with a pH value of 7.4, add 1 mL of N,N-dimethylamide (DMF), stir to dissolve ; Dissolve 8 mg of CPAOZ obtained in step (a) in 1 mL of DMF solution, add 14 mg of dicyclohexylimide (DCC) and 6 mg of N-hydroxysuccinimide (NHS) under stirring, and stir overnight at 4° C., 8000r Centrifuge at 1 / min for 15 minutes, collect the supernatant and put it into a dialysis bag, dialyze with 0.01mo...

Embodiment 2

[0052] Example 2 Preparation of monoclonal antibody to furazolidone metabolites according to the present invention

[0053] (1) Animal immunization: immunize 6-8 week-old female Blab / c mice with the immunogen whose carrier protein is bovine serum albumin, and immunize once every 2 weeks. , select the mouse with the best result to prepare for fusion;

[0054] (2) Cell fusion: Take mouse splenocytes and mouse myeloma SP2 / O cells for fusion, indirect ELISA method to measure the supernatant, select the wells with high positive, and subclone the positive wells by the limiting dilution method until the establishment A hybridoma cell line producing a single monoclonal antibody against a metabolite of furazolidone;

[0055] (3) Large-scale preparation of monoclonal antibodies: select large female Blab / c mice, use the method of in vivo induction of ascites, prepare a large amount of ascites, and purify the ascites by caprylic acid-ammonium sulfate precipitation, divide into small tube...

Embodiment 3

[0056] Example 3 Characteristic Identification of Furazolidone Metabolite Monoclonal Antibody According to the Present Invention

[0057] 1. Affinity determination

[0058] The affinity constant of the monoclonal antibody of furazolidone metabolite was determined by non-competitive ELISA, and the results were as follows: figure 1 As shown, the affinity constant Ka=1.6×10 9 L / mol.

[0059] 2. Subtype determination

[0060] The mouse monoclonal antibody subtype identification kit purchased from Sigma was used to determine the subtype, and the results are shown in figure 2 As shown, the subtype of the monoclonal antibody to the metabolite of furazolidone is IgG1.

[0061] 3. Potency determination

[0062] Coat the original coated detection plate with 1:40000 dilution, dilute the purified monoclonal antibody at 1:200, 1:400, 1:800, ... 1:800000, add it to the well of the microtiter plate, after reaction Add HRP-labeled goat anti-mouse secondary antibody, and finally use TMB...

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Abstract

The invention relates to a colloidal gold immunochromatography test strip for detecting furazolidone metabolites and a preparation method and application of the colloidal gold immunochromatography test strip, and belongs to the technical fields of immunology and veterinary drug residue analysis. The test strip comprises a base plate, a sample pad, a colloidal gold pad, a coating membrane and a water absorption pad; protective membranes are arranged at the two ends of the test strip, and the sample pad, the colloidal gold pad, the coating membrane and the water absorption pad are sequentially stuck to the base plate; the colloidal gold pad is coated with colloidal gold-labeled monoclonal antibodies which can recognize the furazolidone metabolites; the coating membrane is provided with an invisible detection line printed by a carrier protein solution coupled with the furazolidone metabolites and an invisible quality control line printed by a goat anti-mouse IgG solution. The colloidal gold immunochromatography test strip has the advantages of being convenient and rapid to operate, visual, high in timeliness and the like and is wide in application range, low in cost and convenient to apply and popularize.

Description

technical field [0001] The invention relates to a colloidal gold immunochromatographic test strip for detecting furazolidone metabolites and its preparation method and application, which is particularly suitable for the detection of furazolidone metabolite residues in animal tissues, and belongs to the technical field of immunology and the technical field of veterinary drug residue analysis . Background technique [0002] Furazolidone (FZD), also known as furazolidone, is a nitrofuran drug that has inhibitory effects on common Gram-negative and positive bacteria, acts on microbial enzyme systems, inhibits acetyl-CoA, and interferes with microbial Glucose metabolism, thereby playing an antibacterial role. Widely used in aquatic products and poultry to treat bacillary dysentery, enteritis, coccidiosis, etc., as well as scabies, red fin disease, ulcers, etc. of farmed fish. Because of its strong bactericidal ability, broad antibacterial spectrum, not easy to produce drug resi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/558
CPCG01N33/9446G01N33/558G01N33/577G01N33/68
Inventor 郝少彦李春生刘静静李玉静吴萌杜顺丰武孝利曹秀梅闫玉杰
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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