CN202631541U - Colloidal gold kit for detecting fluoroquinolones medicines in milk - Google Patents

Abstract

The utility model provides a colloidal gold kit for detecting fluoroquinolones medicines in milk. The colloidal gold kit comprises a micropore reagent strip lyophilized with a fluoroquinolones medicine monoclonal antibody-colloidal gold label, and test paper, wherein the test paper is composed of a soleplate, a sample absorption pad, a reaction film, a water absorption pad and a protective film; the sample absorption pad, the reaction film, the water absorption pad and the protective film are attached onto the soleplate and are connected tightly in sequence; and the reaction film is provided with a detection line blotting 'l' constituted by coating of a fluoroquinolones medicine-carrier protein conjugate and a quality control line blotting 'l' constituted by coating of an anti-goat anti-rat antibody. The kit disclosed by the utility model has the characteristics of good portability, high sensitivity, short detection time and the like, and can play an important role in detection of fluoroquinolones medicine residues in the milk.

Description

Detect the colloidal gold kit of FQNS in the milk

Technical field

The utility model relates to a kind of kit that detects FQNS, particularly detects the kit of FQNS in the milk, belongs to the immunology category.

Background technology

Comprecin (4-quinolones); Claiming pyridonecarboxylic acids or pyridone acids again, is one type of newer synthetic antibacterial drug, and the exploitation of such medicine can be traced back to 1962; Lesher isolates a kind of secondary product-acidum nalidixicum from synthetic antimalarial chloroquine; Quinolones is gone through 40 years of development, developed altogether four generation QNS, amount to more than 50 kind of medicine.

Comprecin is a target position with the DNA (DNA) of bacterium; Through optionally suppressing the DNA of bacteria gyrase and topoisomerase I V causes chromosomal irreversible lesion; And bacterial cell is no longer divided; Mainly act on the antibacterials of gram-negative bacteria, to a little less than the effect of gram positive bacteria (some kind has antibacterial action preferably to staphylococcus aureus).Become the animal doctor face examine with aquaculture in one of most important anti-infectives, be used for treatment, prevention and growth promotion in a large number pay close attention to because its drug resistance and potential carcinogenicity cause widely.In tissue; The sign residue of Enrofloxacin is Enrofloxacin and Ciprofloxacin; Wherein the highest with the residue concentration in liver organization and the renal tissue, secondly be muscle and the fatty skin histology that adheres to, its metabolic product Ciprofloxacin (CIF) of Enrofloxacin is biologically active still.It mainly is present in Ofloxacin (OFL) in the tissue with the form of original shape medicine; Metabolic rate is near 100% in vivo for Pefloxacin (PEF), and its metabolic product is Norfloxacin (NOR).

At present, the detection method of fluo quinolone drug residual substantially can be divided into three major types according to the different detection principle in milk and the raw milk: microorganism be obstructed detection, physics and chemistry detection method, immune analysis method.The microorganism detection method of being obstructed mainly contains inhibition zone test, turbidity test, color changing type and detects the gold marked reagent cassette method, and it is low that microorganism detection method detects cost price, but the existence of low, the consuming time length of sensitivity, drug-fast bacteria causes flase drop easily; The physics and chemistry detection method is to detect the FQNS main method at present, and liquid phase chromatography, chromatogram/mass spectrometric hyphenated technique, vapor-phase chromatography etc. are arranged; Physics and chemistry detection method degree of accuracy is high; But it exist to detect, and cost is high, checkout equipment is complicated, to the testing staff have relatively high expectations, detect shortcoming such as length consuming time, immune analysis method commonly used at present is mainly enzyme linked immunosorbent detection method and radio immunoassay; The testing cost that needs still is higher, is not suitable for enterprise and detects needs cheaply.Therefore, be necessary in practice to set up that a kind of susceptibility is high, simple to operate, cost is low, the detection of drugs kind is many, be fit to the detection method of large-scale promotion.

The utility model content

The purpose of the utility model provide a kind of highly sensitive, detect that the FQNS kind is many, cost is low, the kit of simple to operate, the detection FQNS that lacks detection time.

The kit of the utility model; It comprises that freeze-drying has the micropore reagent strip and the test strips of FQNS monoclonal antibody-colloid gold label thing; Test strips is by base plate, forms attached to the absorption of sample pad, reaction film, adsorptive pads and the diaphragm that closely link to each other successively on the base plate, has on the said reaction film to be coated with detection line trace " | " that FQNS-carrier protein couplet thing constitutes and the nature controlling line trace " | " that encapsulates sheep anti mouse antiantibody formation.Detection line is parallel with nature controlling line; Detection line is positioned at apart from absorption of sample pad one side 5～8mm, and said nature controlling line is positioned at distance detecting line 4～7mm, test strips two ends coated with protective film; Cover the handle end that is on the suction side; Cover the test side that is on the absorption of sample pad, the MAX mark line is arranged on the diaphragm of test side, have plug on the reacting hole of micropore reagent strip.

For the ease of transportation and use, kit also has holding appliance and is fixed in the reagent bucket that wherein is used for splendid attire micropore reagent strip and test strips, has gland bonnet on the said reagent bucket.

The test strips base plate can be the material that PVC base plate or other hard do not absorb water in the utility model kit; The absorption of sample pad can be suction strainer paper or filter paper for oil; Adsorptive pads is a thieving paper; Reaction film can be nitrocellulose filter or CAM; Diaphragm is a PE material diaphragm; The kit box body is a carton box; The reagent bucket is a plastics reagent bucket; Holding appliance is the rigid support material.

The utility model kit has following beneficial effect:

1. highly sensitive.The FQNS detection kit is that the basis is prepared from the monoclonal antibody of colloid gold label high-affinity; Priceless strong formation between gold grain and the antibody molecule in the gold labeling antibody; The two combines through the Van der Waals force between the charges of different polarity; Collaurum is very little to monoclonal antibody specificity and affinity influence, and has higher mark rate.FQNS monoclonal antibody wherein-colloid gold label thing freeze-drying in testing process, can make golden labeling antibody fully contact with sample liquid to be checked in the micropore reagent strip, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Therefore, the utility model kit has higher specificity and sensitivity.This kit is respectively 20 μ g/L, 20 μ g/L, 20 μ g/L, 20 μ g/L, 20 μ g/L, 20 μ g/L, 40 μ g/L, 20 μ g/L, 20 μ g/L, 40 μ g/L, 20 μ g/L to Enrofloxacin, sarafloxacin, two Flucloxacillin, Ofloxacin, Norfloxacin, Ciprofloxacin, Enoxacin, Pefloxacin, flumequine 、 oxolinic acid, Danofloxacin detection sensitivity.

2. easy and simple to handle quick.Need not any other reagent when using kit to detect, as long as sample solution to be checked is dripped in micropore reagent, behind the mixing, hatch 5min, it is downward that test paper is indicated MAX wire tag end, timing behind the micropore reagent after insertion is hatched, observations in the 5min.

3. show testing result image, accurately directly perceived.Test strip is to show that red line look " | " and " || " trace are as the positive and negative marker; Show on nitrocellulose filter that promptly the content that a red line " | " trace is illustrated in FQNS in the sample liquid to be detected is greater than or equal to the LDL of kit to FQNS; Article two, red line " || " trace is illustrated in that FQNS content is lower than the LDL of kit to FQNS in the sample to be detected; The result judges image, directly perceived, accurate, simple and clear, is not easy to occur result's erroneous judgement.

4. cost is low, small investment.Use the utility model kit, do not need to join in addition instrument and equipment and other reagent, the on-the-spot detection settled at one go, with low cost, small investment, instant effect.

5. be easy to apply on a large scale.Kit is simple to operate, can better satisfy different levels personnel's needs, like specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, dairy industry enterprise etc., has vast market prospect and bigger economical, societal benefits.

Description of drawings

Fig. 1 is the utility model test strips structural representation;

Fig. 2 is the utility model test strips vertical view;

Fig. 3 is the utility model micropore reagent figure;

Fig. 4 .a, Fig. 4 .b, Fig. 4 .c, Fig. 4 .d are the utility model test strips testing result process decision chart;

Fig. 5 is the utility model reagent barrel structure synoptic diagram;

Fig. 6 is the vertical view of the utility model holding appliance;

Fig. 7 is the side view of the utility model box body and holding appliance.

Embodiment

One, the assembling of FQNS detection kit preparation

1) test strips

Absorption of sample pad 1, reaction film 2, adsorptive pads 3 and diaphragm are pasted successively in order on said base plate 6, and the end of absorption of sample pad links to each other with reaction film top, and the end of reaction film links to each other with adsorptive pads; The top of absorption of sample pad aligns with the top of base plate; The end of adsorptive pads aligns with the end of base plate, and the test paper two ends are adhesive with diaphragm, and diaphragm 7-1 covers adsorptive pads left-hand seat pommel; Diaphragm 7-2 covers the test side of absorption of sample pad; On the diaphragm of test side, be printed on the MAX mark line, detection line 4 is coated with FQNS-carrier protein couplet thing, and nature controlling line 5 is coated with the sheep anti mouse antiantibody.

Base plate is the PVC base plate, and the absorption of sample pad is a suction strainer paper, and reaction film is a nitrocellulose filter, and adsorptive pads is a thieving paper, and diaphragm is a PE material diaphragm.

2) micropore reagent strip

Micropore reagent strip 8 has plug 9, and freeze-drying has FQNS monoclonal antibody-colloid gold label thing in the plate hole.

3) with 1) test strips and 2) the micropore reagent strip plastics reagent bucket 10 of packing into and having gland bonnet, plastics reagent bucket is positioned in the holding appliance 11 in the kit box body 12.

Two, the use of kit

Milk sample solution to be checked drips 200 μ l in micropore reagent, behind the mixing, hatches 5min, and it is downward that test paper is indicated MAX wire tag end, timing behind the micropore reagent after insertion is hatched, observations in the 5min.

Three, testing result analysis

The content of FQNS in sample is greater than or equal to kit its lowest detection is prescribed a time limit; FQNS monoclonal antibody-colloid gold label thing combines with FQNS; Antigen binding site on the gold labeling antibody is closed; Thereby in detection zone, because competitive reaction, can not combine and red stripes do not occur with FQNS-carrier protein couplet thing.Negatives owing to lack the antigen-antibody competitive reaction, will red stripes occur on detection line and nature controlling line in testing process.Shown in Fig. 4 .a, Fig. 4 .b, Fig. 4 .c, Fig. 4 .d.

Positive: when red stripes of nature controlling line (C) demonstration, and detection line (T) does not develop the color, and test strips is judged to the positive to show red line look " | ", shown in Fig. 4 .a figure.

Negative: when nature controlling line (C) shows a red stripes, detection line (T) also demonstrates a red stripes simultaneously, and detection line (T) color near or when being shallower than nature controlling line (C), test strips is judged to feminine gender, shown in Fig. 4 .b to show that the red line look is " || ".

Invalid: (C) do not demonstrate red stripes when nature controlling line, and then no matter whether detection line (T) demonstrates red stripes, and it is invalid that this test strips is judged to, shown in Fig. 4 .c, 4.d.

Four, the material preparation method of using in the detection kit

1, the synthetic and evaluation of FQNS-carrier protein couplet thing

FQNS is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.

1) the Norfloxacin derivant is synthetic

With Norfloxacin 1mmol, be dissolved in the 55ml methenyl choloride, add DCC 2mmol, the DMAP catalyzer is an amount of, p-ethyl benzene 1.5mmol, stirring at room 5h, TLC monitoring raw material disappears, and filters, with liquid phase washing, anhydrous Na S ₂O ₄Drying, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/5).Above-mentioned product is dissolved in the methyl alcohol, adds NaOH 0.76g, 60 ℃ of stirring at room 5h, TLC monitoring raw material disappears; The decompression desolventizing is dissolved in 1mol/L NaOH solution with the dope that obtains, and regulates pH3～5; Ethyl acetate extraction, drying, column chromatography purification (eluant, eluent; Ethyl acetate/petroleum ether, 1/1), get the quinolones haptens.

2) immunogenic preparation-FQNS and bovine serum albumin(BSA) conjugate are synthetic

Get 15mg Norfloxacin haptens, be dissolved among the 1mL DMF, get 15mg EDC and fully dissolve in the back adding Norfloxacin lysate, stir 24h under the room temperature, can obtain reactant liquor A with 0.2ml water.Take by weighing BSA 40mg; Make it fully to be dissolved among the 3mL PBS (PH 7.2), A dropwise slowly is added drop-wise in the protein solution with reactant liquor, and under room temperature, stirs 24h; 3d changes dislysate 3 times every day with 4 ℃ of dialysis of 0.01mol/L PBS, to remove unreacted small-molecule substance.Packing, subsequent use in-20 ℃ of preservations.

3) preparation-FQNS of coating antigen and ovalbumin conjugate are synthetic

Get 20mg Norfloxacin haptens, be dissolved among the 1mL DMF, get 15mg EDC and fully dissolve in the back adding Norfloxacin lysate, stir 24h under the room temperature, can obtain reactant liquor A with 0.2ml water.Take by weighing OVA 30mg; Make it fully to be dissolved among the 3mL PBS (PH 7.2), A dropwise slowly is added drop-wise in the protein solution with reactant liquor, and under room temperature, stirs 24h; 3d changes dislysate 3 times every day with 4 ℃ of dialysis of 0.01mol/L PBS, to remove unreacted small-molecule substance.Packing, subsequent use in-20 ℃ of preservations.

4) evaluation of FQNS-carrier conjugates

Carrier protein, FQNS, FQNS-carrier protein couplet thing are made into the solution of 0.5mg/ml with the PBS of pH7.4; Return to zero with 0.01mol/L pH7.4PBS; In the interscan of wavelength 200-800nm scope, obtain the absorption curve of carrier protein, FQNS, FQNS-carrier protein couplet thing with ultraviolet spectrophotometer.The differing absorption curve appears in the three, shows the success of FQNS and carrier protein couplet.

2, FQNS MONOCLONAL ANTIBODIES SPECIFIC FOR

A. animal immune

The immunogene that step 1 is obtained is injected in the Balb/c mouse body, and immunizing dose is 150 μ g/, makes it produce antiserum.

B. Fusion of Cells and cloning

Get immune Balb/c mouse boosting cell, merge in 9: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, screening obtains the FQNS monoclonal hybridoma strain of stably excreting FQNS monoclonal antibody.

C. cell cryopreservation and recovery

Hybridoma is processed 1 * 10 with cryopreserving liquid ⁹The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.

D. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying

Increment cultivation: hybridoma is placed cell culture medium, under 37 ℃ of conditions, cultivate, the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations with sad-saturated ammonium sulfate method.

Said cell culture medium is in the RPMI-1640 nutrient culture media, to add calf serum and soda mint; Making the final concentration of calf serum in cell culture medium is 20% (quality percentage composition), and making the final concentration of soda mint in cell culture medium is 0.2% (quality percentage composition); The pH of said cell culture medium is 7.4.

3, the preparation of sheep anti mouse antiantibody

As immune animal, is that immunogene to pathogen-free domestic sheep carry out immunity with mouse source antibody with sheep, obtains the sheep anti mouse antiantibody.

4, the preparation of FQNS monoclonal antibody-colloid gold label thing

(1) preparation of collaurum

Boil off ionized water 1% gold chloride (is purchased the company in sigma with two; Catalog number T09041) is diluted to 0.01% (quality percentage composition); Put to stir on the magnetic force heating rod stirrer and boil, every 100ml 0.01% gold chloride adds 2.5ml 1% trisodium citrate (purchasing in Guangzhou Chemical Reagent Factory catalog number BG11-AR-01KG); Continue agitating heating and react and when liquid takes on a red color, stop heating, supply dehydration after being cooled to room temperature.The collaurum outward appearance for preparing is pure, bright, nothing precipitates and floating thing.

(2) preparation of FQNS monoclonal antibody-colloid gold label thing

Under magnetic agitation; Transfer the pH value to 7.0 of collaurum with 0.2mol/L sal tartari; Standard by 50～100 μ g antibody/ml collaurums adds above-mentioned FQNS monoclonal antibody in colloidal gold solution; Continue stirring and evenly mixing 30min, adding the final concentration of 10%BSA to BSA in colloidal gold solution is 1% (volumn concentration), leaves standstill 30min.12000rpm, 4 ℃ of centrifugal 30min; Abandon supernatant; Deposition is with redissolving the damping fluid washed twice; It is resuspended to use volume to be that the redissolution damping fluid of initial collaurum volume 1/20 will precipitate, and the concentration of the FQNS monoclonal antibody that obtains-colloid gold label thing solution is 50 μ g monoclonal antibody/ml solution, put 4 ℃ subsequent use.

Redissolution damping fluid: the 0.02mol/L of casein containing protein, Tween-80, the phosphate solution of pH7.2; Wherein the final concentration of casein in the redissolution damping fluid is 0.05-0.1% (volumn concentration), and the final concentration of Tween-80 in the redissolution damping fluid is 0.05-0.15% (quality percentage composition).

5, with FQNS monoclonal antibody-colloid gold label thing freeze-drying to micropore reagent

In micropore reagent microwell plate, add 100 μ l FQNS monoclonal antibody-colloid gold label things; Put into freeze drier; Condenser temperature is under-70 ℃ of conditions, behind the pre-freeze 4h, and freeze-drying 14h again; Can take out, obtain the micropore reagent that freeze-drying has FQNS monoclonal antibody-colloid gold label thing.

6, the preparation of absorption of sample pad

The absorption of sample pad placed contain that bovine serum albumin(BSA) (bovine serum albumin(BSA) is 0.5% (volumn concentration) at the final concentration of damping fluid), pH are 7.2, the 0.1mol/L phosphate buffer soaks 2h, 37 ℃ of baking 2h are subsequent use.

7, the preparation of reaction film

Encapsulate process: with phosphate buffer FQNS-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film appearance it is encapsulated the detection line on nitrocellulose filter, package amount is 1.0 μ g/cm ²With 0.01mol/L, pH 7.4PBS damping fluid sheep anti-mouse igg antibody is diluted to 200 μ g/ml, with Biodot point film appearance it is encapsulated the nature controlling line on nitrocellulose filter, package amount is 1.0 μ g/cm ²The reaction film that encapsulates is placed dry 2h under 37 ℃ of conditions, subsequent use.

Claims (9)

1. colloidal gold kit that detects FQNS in the milk; It is characterized in that comprising that freeze-drying has the micropore reagent strip and the test strips of FQNS monoclonal antibody-colloid gold label thing; Test strips is by base plate, forms attached to the absorption of sample pad, reaction film, adsorptive pads and the diaphragm that closely link to each other successively on the base plate, has on the said reaction film to be coated with detection line trace " ︱ " that FQNS-carrier protein couplet thing constitutes and the nature controlling line trace " ︱ " that encapsulates sheep anti mouse antiantibody formation.

2. colloidal gold kit as claimed in claim 1 is characterized in that said detection line is parallel with nature controlling line.

3. according to claim 1 or claim 2 colloidal gold kit is characterized in that described detection line is positioned at apart from absorption of sample pad one side 5 ~ 8mm, and said nature controlling line is positioned at distance detecting line 4 ~ 7mm.

4. colloidal gold kit as claimed in claim 1 is characterized in that, said test strips two ends coated with protective film, cover on the suction side for handle end, cover the test side that is on the absorption of sample pad, the MAX mark line is arranged on the diaphragm of test side.

5. colloidal gold kit as claimed in claim 1 is characterized in that having plug on the reacting hole of said micropore reagent strip.

6. inspection colloidal gold kit as claimed in claim 1; Said base plate can be the material that PVC base plate or other hard do not absorb water, and the absorption of sample pad can be suction strainer paper or filter paper for oil, and adsorptive pads is a thieving paper; Reaction film can be nitrocellulose filter or CAM, and diaphragm is a PE material diaphragm.

7. colloidal gold kit as claimed in claim 1 is characterized in that also having in the said kit holding appliance and the fixing reagent bucket that wherein is used for splendid attire micropore reagent strip and test strips.

8. like claims 7 described colloidal gold kits, it is characterized in that said kit box body is a carton box, the reagent bucket is a plastics reagent bucket, and holding appliance is the rigid support material.

9. colloidal gold kit as claimed in claim 8 is characterized in that having gland bonnet on the said reagent bucket.

Images