CN101329338A - Clonazepam colloidal gold method detection test paper and preparation method thereof - Google Patents

Abstract

The invention provides a nitrazepam colloidal gold method detection test paper and a preparation method, belonging to the field of measurement technique of a biological immune method. The detection test paper of the invention consists of a sample pad, a bonded releasing pad, a nitrocellulose film, a water-absorbing pad and a plastic soleplate; the plastic soleplate is sequentially bonded with the sample pad, the bonded releasing pad, the nitrocellulose film and the water-absorbing pad; the bonded releasing pad is wrapped by an anti-nitrazepam multi-resistance-colloid golden marking; the nitrocellulose film is sequentially wrapped by nitrazepam coupling antigen and goat anti-rabbit IgG antibody; the nitrazepam coupling antibody is used as a testing line and the goat anti-rabbit IgG antibody is used as a quality control line. The preparation method of the invention has the advantages of convenience, economical and quick usage, easy fabrication 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

Nitrazepam (Nitrazepam) is the Benzodiazepines sedative hypnotics, and chemical name is: 1, and 3-dihydro-7-nitro-5-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 nitrazepam 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 on-the-spot immunochromatography chromatographic detection (the gold immunochromatography assay that detects 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-nitrazepam anti--colloid gold label thing how in conjunction with discharging pad, be coated with nitrazepam coupled antigen and goat anti-rabbit igg antibody on the nitrocellulose filter successively, with the nitrazepam coupled antigen as p-wire, with goat anti-rabbit igg antibody as nature controlling line.

Preparation process is:

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

(2) with the nitrazepam coupled antigen according to a conventional method immunity how anti-make anti-nitrazepam;

(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-0.02mL of anti-nitrazepam that protein concentration is 0.2mg/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/LpH 9.0 that contains 5% sucrose, repeat 2 times, with the dissolving of 0.3mL damping fluid, obtain stable anti-nitrazepam 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 nitrazepam, be coated on the nitrocellulose filter respectively nitrazepam coupled antigen and goat anti-rabbit igg antibody according to this, the nitrazepam 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-nitrazepam of colloid gold label more combining of competition form taken place, its consequence is, when the anti-nitrazepam of colloid gold label how anti-when excessive, unnecessary resist floats to p-wire more, combines with the nitrazepam coupled antigen and colour developing; And how anti-the anti-nitrazepam 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 nitrazepam residual quantity in half-quantitative detection serum or the urine sample, in 5-10min, detect sample rapidly and accurately and whether contain nitrazepam, to determine whether nitrazepam exceeds standard, can satisfy food security the nitrazepam 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: nitrazepam detects the preparation of test paper

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

(2) how anti-obtain anti-nitrazepam with the immunity of nitrazepam 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-0.02mL of anti-nitrazepam that protein concentration is 0.2mg/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 borate buffer solution (containing 5% sucrose) with 0.002mol/L pH 9.0 3mL is resuspended to separate, and repeat 2 times, with the dissolving of 0.3mL damping fluid, obtain stable anti-nitrazepam 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 nitrazepam, be coated on the nitrocellulose filter respectively nitrazepam coupled antigen and goat anti-rabbit igg antibody successively, the nitrazepam 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 the immuno-chromatographic test paper strip that is used to detect.

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 10,50, the nitrazepam 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 nitrazepam 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 5ng/mL.

(4) repeatability

With phosphate buffer (PBS:0.01mol/L, pH 7.4) respectively compound concentration be the nitrazepam standard solution of 10ng/mL, carry out 10 parallel laboratory tests, the reaction time 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, first centrifuging and taking supernatant or with distilled water 1: 1 dilution.

(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 nitrazepam 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 nitrazepam content of interpret sample is lower than 5ppb (5ng/mL).

Positive:

Two bands all develop the color, but the p-wire color is shallower than the nature controlling line color, illustrate that nitrazepam content surpasses 5ppb (5ng/mL) in the test sample.

P-wire does not develop the color, the nature controlling line colour developing, and the nitrazepam content in the interpret sample is higher than 10ppb (10ng/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-nitrazepam anti--colloid gold label thing how in conjunction with discharging pad, be coated with nitrazepam coupled antigen and goat anti-rabbit igg antibody on the nitrocellulose filter successively, with the nitrazepam 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) nitrazepam and bovine serum albumin molecule are carried out coupling, as the nitrazepam coupled antigen;

(2) with the nitrazepam coupled antigen according to a conventional method immunity how anti-make anti-nitrazepam;

(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-0.02mL of anti-nitrazepam that protein concentration is 0.2mg/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/LpH 9.0 that contains 5% sucrose, repeat 2 times, with the dissolving of 0.3mL damping fluid, obtain stable anti-nitrazepam 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 nitrazepam, be coated on the nitrocellulose filter respectively nitrazepam coupled antigen and goat anti-rabbit igg antibody according to this, the nitrazepam 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.