CN101315365A - Clonazepam colloidal gold method detection test paper and preparation method thereof - Google Patents

Abstract

A clonazepam colloidal gold method test paper and a preparation method belong to the testing technology field of the biological immunity method. The test paper is composed of a sample pad, a combined release pad, a cellulose nitrate membrane, a water absorbing pad and a plastic base plate; the sample pad, the combined release pad, the cellulose nitrate membrane and the water absorbing pad are sequentially stuck to the plastic base plate; the combined release pad is coated with an anti-clonazepam multi-resistance colloidal gold label; the cellulose nitrate membrane is sequentially coated with clonazepam coupling antigen and goat anti-rabbit IgG antibody, wherein the clonazepam coupling antigen is taken as the test line and the goat anti-rabbit IgG antibody is taken as the quality control line. The clonazepam colloidal gold method test paper and the preparation method have the characteristics of convenient use, economy, rapidness, simple preparation and low cost.

Description

A kind of Clonazepam colloidal gold method detection test paper and preparation method

Technical field

A kind of Clonazepam colloidal gold method detection test paper and preparation method belong to the determination techniques field of biology immunization method.

Background technology

Clonazepam (Nitrazepam) is the Benzodiazepines sedative hypnotics, and chemical name is: 1, and 3-dihydro-7-nitro-5-(2-chloro-phenyl)-2H-1,4-Benzodiazepine-2-ketone.Once as animal feed additive,, had drowsiness few the moving of the animal of making, the effect of grow fast and the meat that changes as growth promoter.Also be used in butcher with animal quarantine before, or with animal shift butcher before, play sedation, to alleviate the animal tense situation, reduce animal injury and mortality ratio.This type of medicine is common drowsiness, as seen unable, headache, dizzy, nauseating, constipation, bad reactions such as accidental fash, hepatic lesion, bone marrow suppression.If with being intended to add this type of medicine in the feed, the medicine of accumulating enters human body by food chain, will cause huge harm.In the forbidden drugs list is listed this type of medicine by many for this reason countries.At present, the detection of relevant Clonazepam both at home and abroad mainly contains high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), thin-layered chromatography (TLC), gas-matter coupling method (GC-MS), liquid-matter coupling method (LC-MS) etc., but these methods not only need expensive instrument and equipment, to the requirement of sample also than higher, need further purification processes just can carry out, and can not adapt to the fast detecting at high flux, scene.Therefore realize having fast, many residue detection of the immunologic detection method of portable advantage have realistic meaning.

Summary of the invention

The purpose of this invention is to provide a kind of Clonazepam colloidal gold method detection test paper and preparation method, in order to overcome the defective of existing detection technique, provide a kind of portable, be fit to carry out immunochromatography chromatographic detection (the gold immunochromatography assay of on-the-spot chlorine detection nitrazepam fast; GICA).

Technical scheme of the present invention: a kind of Clonazepam colloidal gold method detection test paper, discharging pad, nitrocellulose filter, adsorptive pads and plastic bottom board by sample pad, combination forms, paste sample pad, combination release pad, nitrocellulose filter and adsorptive pads on the plastic bottom board successively, go up bag by anti-Clonazepam anti--colloid gold label thing how in conjunction with discharging pad, be coated with Clonazepam coupled antigen and goat anti-rabbit igg antibody on the nitrocellulose filter successively, with the Clonazepam coupled antigen as p-wire, with goat anti-rabbit igg antibody as nature controlling line.

Preparation process is:

(1) Clonazepam and bovine serum albumin molecule are carried out coupling, as the Clonazepam coupled antigen;

(2) how anti-obtain anti-Clonazepam with the immunity of Clonazepam coupled antigen;

(3) with the trisodium citrate reductive agent 20nm-40nm colloid gold particle is made in the gold chloride reduction;

(4) the 3mL collaurum is transferred to pH 9, stir and dropwise add the how anti-0.015mL of anti-Clonazepam that protein concentration is 0.18mg/mL down, place and to add 5% bovine serum albumin BSA solution behind the 30min to make final concentration be 1%, after placing at least 30min, 10000 leave heart 50min, remove after the supernatant resuspendedly with the borate buffer solution 3mL of the 0.002mol/L pH 9.0 that contains 5% sucrose, repeat 2 times, with the dissolving of 0.3mL damping fluid, obtain stable anti-Clonazepam and resist-the colloid gold label thing more at last;

(5) how anti--colloid gold label thing is coated on collaurum in conjunction with discharging on the pad will to resist Clonazepam, be coated on the nitrocellulose filter respectively Clonazepam coupled antigen and goat anti-rabbit igg antibody successively, the Clonazepam coupled antigen as p-wire and goat anti-rabbit igg antibody as nature controlling line, at 37 ℃ of oven dryings;

(6) assembling of test strips: sample pad, combination release pad, nitrocellulose filter, adsorptive pads are attached on the plastic bottom board successively by an end, promptly obtain being used for the Clonazepam colloidal gold method detection test paper of immune detection.

It is the capillary siphoning effect of utilizing sample pad to form that the present invention detects principle, detected material is at first resisted with the anti-Clonazepam of colloid gold label more combining of competition form taken place, its consequence is, when the anti-Clonazepam of colloid gold label how anti-when excessive, unnecessary resist floats to p-wire more, combines with the Clonazepam coupled antigen and colour developing; And how anti-the anti-Clonazepam of the colloid gold label that combines with the detection thing is, its V district detected material of binding site occupies, can only cross over p-wire and float to nature controlling line, carry out colorimetric and obtain testing result with p-wire with C position point and goat anti-rabbit igg antibody non-specific binding.

Beneficial effect of the present invention: the present invention uses one step of chromatography type competition law principle, the colorimetric that rolls off the production line in the test paper is come the Clonazepam residual quantity in half-quantitative detection serum or the urine sample, in 5-10min, detect sample rapidly and accurately and whether contain Clonazepam, to determine whether Clonazepam exceeds standard, can satisfy food security the Clonazepam residual quantity is detected demand, be applicable to meat producing plant and testing agency of government.

The present invention compared with prior art, its effect is self-evident, promptly have easy to use, economical quick, make characteristics easy, with low cost.

Description of drawings

The side view of Fig. 1 Clonazepam colloidal gold method detection test paper of the present invention.

Embodiment

Embodiment 1: Clonazepam detects the preparation of test paper

(1) with Clonazepam and the coupling of bovine serum albumin molecule, as the Clonazepam coupled antigen,

(2) how anti-obtain anti-Clonazepam with the immunity of Clonazepam coupled antigen;

(3) with the trisodium citrate reductive agent 20nm-40nm colloid gold particle is made in the gold chloride reduction;

(4) the 3mL collaurum is transferred to pH 9, stir and dropwise add the how anti-0.015mL of anti-Clonazepam that protein concentration is 0.18mg/mL down, place and to add 5% bovine serum albumin(BSA) (BSA) solution behind the 30min to make final concentration be 1%, after placing at least 30min, 10000 leave heart 50min, remove after the supernatant resuspended with borate buffer solution (the containing 5% sucrose) 3mL of 0.002mol/LpH 9.0, repeat 2 times, with the dissolving of 0.3mL damping fluid, obtain stable anti-Clonazepam and resist-the colloid gold label thing more at last;

(5) how anti--colloid gold label thing is coated on collaurum in conjunction with discharging on the pad will to resist Clonazepam, Clonazepam coupled antigen and goat anti-rabbit igg antibody is coated on respectively on the nitrocellulose filter, as p-wire and nature controlling line, at 37 ℃ of oven dryings.

(6) assembling of test strips: sample pad, combination release pad, nitrocellulose filter, adsorptive pads are attached on the plastic bottom board successively by an end, promptly obtain being used for the Clonazepam colloidal gold method detection test paper of immune detection.

Embodiment 2: the test paper requirement

(1) negative reference material coincidence rate

With phosphate buffer (PBS:0.01mol/L, pH 7.4) compound concentration is that the promethazine hydrochloride standard solution of 10ng/mL carries out 10 parallel detections, and with normal or correct the visual observation result, the reaction time is observations when 5min, should be negative.

With phosphate buffer (PBS:0.01mol/L, pH 7.4) compound concentration is that the barbital standard solution of 10ng/mL carries out 10 parallel detections, and with normal or correct the visual observation result, the reaction time is observations when 5min, should be negative.

(2) positive reference material coincidence rate

With phosphate buffer (PBS:0.01mol/L, pH 7.4) compound concentration be 25,50, the Clonazepam standard items of 100ng/mL carry out parallel detection, each concentration is carried out 10 parallel laboratory tests, the reaction time is observations when 5min, feminine gender must not occur.

(3) limit of identification

With phosphate buffer (PBS:0.01mol/L; pH 7.4) respectively compound concentration be 0,2,5,10,20,50, the Clonazepam standard solution of 100ng/mL; each concentration is carried out 10 parallel laboratory tests; reaction time is when 5min; with normal or rectification visual observation result, limit of identification is not higher than 10ng/mL.

(4) repeatability

With phosphate buffer (PBS:0.01mol/L, pH 7.4) respectively compound concentration be the Clonazepam standard solution of 50ng/mL, carry out 10 parallel laboratory tests; reaction time is when 5min; with normal or correct the visual observation result, unanimity as a result, colour developing degree homogeneous.

(5) stability test

Place after 10 days for 37 ℃, every index should meet above requirement.

Embodiment 3: the test paper using method

(1) specimen preparation

Serum: extract animal blood to be checked, centrifugal or leave standstill after get transparent supernatant and use; If serum has excessive haemolysis, serum is experimentized with behind one times of the distilled water diluting again, otherwise the analoids redness is too dark, influences test result.

Urine: directly test with urine, as muddiness, the dilution in 1: 1 of first centrifuging and taking supernatant or distilled water.

(2) operation

Test paper arrow end is immersed in the sample to be tested, does not surpass sample pad 1cm.Immerse the 30sec taking-up and set level, 10min reads the result.

(3) testing result

Negative:

Two bands all develop the color, and the p-wire color is deeper than the nature controlling line color, do not contain Clonazepam in the interpret sample.

Two bands all develop the color, and the p-wire color is identical with the nature controlling line color or approaching, and the Clonazepam content of interpret sample is lower than 10ppb (10ng/mL).

Positive:

Two bands all develop the color, but the p-wire color is shallower than the nature controlling line color, illustrate that Clonazepam content surpasses 10ppb (10ng/mL) in the test sample.

P-wire does not develop the color, the nature controlling line colour developing, and the Clonazepam content in the interpret sample is higher than 25ppb (25ng/mL).

Claims (2)

1, a kind of Clonazepam colloidal gold method detection test paper, it is characterized in that by sample pad, form in conjunction with discharging pad, nitrocellulose filter, adsorptive pads and plastic bottom board, paste sample pad, combination release pad, nitrocellulose filter and adsorptive pads on the plastic bottom board successively, go up bag by anti-Clonazepam anti--colloid gold label thing how in conjunction with discharging pad, be coated with Clonazepam coupled antigen and goat anti-rabbit igg antibody on the nitrocellulose filter successively, with the Clonazepam coupled antigen as p-wire, with goat anti-rabbit igg antibody as nature controlling line.

2, the preparation method of the described Clonazepam colloidal gold method detection test paper of claim 1 is characterized in that preparation process is:

(1) Clonazepam and bovine serum albumin molecule are carried out coupling, as the Clonazepam coupled antigen;

(2) how anti-obtain anti-Clonazepam with the immunity of Clonazepam coupled antigen;

(3) with the trisodium citrate reductive agent 20nm-40nm colloid gold particle is made in the gold chloride reduction;

(4) the 3mL collaurum is transferred to pH 9, stir and dropwise add the how anti-0.015mL of anti-Clonazepam that protein concentration is 0.18mg/mL down, place and to add 5% bovine serum albumin BSA solution behind the 30min to make final concentration be 1%, after placing at least 30min, 10000 leave heart 50min, remove after the supernatant resuspendedly with the borate buffer solution 3mL of the 0.002mol/L pH 9.0 that contains 5% sucrose, repeat 2 times, with the dissolving of 0.3mL damping fluid, obtain stable anti-Clonazepam and resist-the colloid gold label thing more at last;

(5) how anti--colloid gold label thing is coated on collaurum in conjunction with discharging on the pad will to resist Clonazepam, be coated on Clonazepam coupled antigen and goat anti-rabbit igg antibody on the nitrocellulose filter respectively, the Clonazepam coupled antigen as p-wire and goat anti-rabbit igg antibody as nature controlling line, at 37 ℃ of oven dryings;

(6) assembling of test strips: sample pad, combination release pad, nitrocellulose filter, adsorptive pads are attached on the plastic bottom board successively by an end, promptly obtain being used for the Clonazepam colloidal gold method detection test paper of immune detection.

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