CN201637746U - Immune colloidal gold reagent plate for detecting gentamycin - Google Patents

Abstract

The utility model relates to an antibiotic residual immune colloidal gold reagent plate, in particular to an immune colloidal gold reagent plate for detecting residual gentamycin in dairy products. The reagent plate is composed of an upper plastic template, a lower plastic template and a test paper strip. A sample pad, a colloidal gold combination pad, a nitrocellulose film and a water absorption pad are sequentially and closely stuck on a back lining of the test paper strip; and parts of 1 to 2cm between the above adjacent components are respectively overlapped. A combination substance of a gentamycin-resisting monoclonal antibody and colloidal gold is enveloped on the colloidal gold combination pad. A gentamycin-carrier protein coupling substance and goat anti-mouse IgG which are respectively adopted as a detecting line and a quality control line are sequentially enveloped on the nitrocellulose film in the direction from the sample pad to the water absorption pad. The residual gentamycin in a detected sample can be directly observed by a semi-quantitative reagent, and the entire detecting process needs 5 to 10 minutes without any experimental facility; therefore, the immune colloidal gold reagent plate is beneficial for screening samples in a large scale and is suitable for vast grass-roots departments to quickly detect gentamycin in the dairy products in a large scale.

Description

Detect the immune colloidal gold reagent plate of gentamicin

Technical field

The utility model relates to the residual immune colloidal gold reagent plate of a kind of detection of antibiotics, is specifically related to a kind of immune colloidal gold reagent plate that detects gentamicin medicament residue in the dairy products.

Background technology

Gentamicin belongs to aminoglycoside antibiotics, mainly acts on gramnegative bacterium, owing to its has a broad antifungal spectrum, good, cheap veterinary clinic and the animal feed additive of being widely used in of curative effect.But gentamicin easily produces toxic and side effect in application process, heavy dose of use lack of standardization causes the medicament residue in the animal food to exceed standard and can cause serious harm to health, produces renal toxicity, ototoxicity, muscle inhibition, a series of toxic and side effects such as allergic reaction.The residual limit standard of regulation gentamicin in dairy products is 100ng/mL in No. 235 bulletin of China Ministry of Agriculture " animal food herbal medicine maximum residue limit(MRL) ".

At present, the detection method for the gentamicin medicament residue mainly contains physico-chemical analysis method and immunoassay.The physico-chemical analysis method is commonly used high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry coupling analytic approach (LC-MS), and specificity is good, accuracy rate is high, is the prefered method that each big testing agency proves conclusively the detection sample.But the physico-chemical analysis method exists requirements such as equipment, environment, operative skills, is unfavorable for extensive screening sample, thereby is not suitable for department of vast basic unit.Immunoassay is commonly used enzyme linked immunosorbent assay (ELISA) and immunochromatographic method (GICA), ELISA method detection limit is big, operate simple relatively, be used to the detection of antibiotic residues in animal-derived food more and more, but whole operation still needs 1-2h, and need use special instruments and equipment, have certain limitation.GICA method sensitivity, quick, special, easy, extremely Chinese scholars is paid close attention in recent years, is the developing direction of gentamicin residue fast detecting from now on.

The utility model content

It is a kind of highly sensitive that the purpose of this utility model is to provide, and specificity is good, easy to be quick, the gentamicin immune colloidal gold reagent plate that production cost is low.

The utility model agent plate is by up and down two plastic formworks, backings and stick to the sample pad, collaurum pad, nitrocellulose filter, the adsorptive pads that closely link to each other successively on the backing and form.

Have between sample pad, collaurum pad, the adjacent each several part of nitrocellulose filter wherein that 1-2mm's is overlapping with adsorptive pads, its purpose is that the effect of assurance chromatography is carried out to the adsorptive pads position smoothly from sample pad on the one hand, be in order to make sample solution and sample be lined with sufficient reaction time on the other hand, buffer system on the sample pad can in and the potential of hydrogen of sample solution, guarantee that effective constituent in the sample solution and the golden labeling antibody on the collaurum pad react smoothly.

Be coated with the bond of anti-gentamicin monoclonal antibody and collaurum on the collaurum pad; Be coated with gentamicin-carrier protein couplet thing and sheep anti-mouse igg successively from sample pad to the adsorptive pads direction on the nitrocellulose filter, respectively as detection line and nature controlling line.The carrier protein of coupling gentamicin can be bovine serum albumin(BSA) (BSA), ovalbumin (OVA), hemocyanin (KLH) etc.Anti-gentamicin monoclonal antibody is for discerning immunoglobulin (Ig) or its fragment of gentamicin.

The each several part constituent and the function of the utility model agent plate are as follows:

Plastic formwork plays fixedly test strips and referential function district (well, detection zone, control zone)

Backing as the PVC plate, plays fixing other ingredients of test paper of supporting for simultaneously scribbling the toughness material that does not absorb water of adhesive sticker.

Sample pad is made by glass fibre, works to absorb sample solution and buffering sample solution pH value.

The collaurum pad is made by polyester film, is marked with the bond of anti-gentamicin monoclonal antibody and collaurum on it, is the place that effective constituent in the sample solution and golden labeling antibody react.

It is that reaction result is come out with macroscopic characterization that the cellulose nitrate membrane portions mainly acts on.

Adsorptive pads is a filter paper, and its effect is that the excessive solution that will move up absorbs as the suction part.

The utility model agent plate has following beneficial effect:

(1) specificity is good.The utility model agent plate is 100% to the cross reacting rate of gentamicin, microbiotic to other kinds comprises that the cross reacting rate of streptomysin, dihydrostreptomycin, neomycin all is lower than 1%, as seen, the utility model agent plate has the height selectivity to gentamicin.

(2) highly sensitive.The utility model agent plate is limited to 50ppb (ng/mL) to detecting of gentamicin in the former milk, can satisfy in No. the 235th, China Ministry of Agriculture bulletin " animal food herbal medicine maximum residue limit(MRL) " the regulation requirement to gentamicin maximum residue limit(MRL) 100ng/mL in dairy products.

(3) simple and quick, do not rely on any experimental facilities, do not need any professional training.Most of raw material that the utility model agent plate will be reacted required is incorporated in the reagent strip, antigen-antibody reaction is carried out on immobilon-p fast after dripping sample, shortened the sample time greatly, and sample need not special processing, can with the naked eye read the result by the detection line on the judgement nitrocellulose filter and the shade of nature controlling line in 5-10 minute, it is auxiliary that testing process need not specific apparatus, and the ordinary person all can operate, do not need professional training, very easily promote the use of.

(4) cost is low, and is profitable.The utility model agent plate mature production technology, flow process are simple, and low production cost requires less investment while yielding quicker results.

Description of drawings

Fig. 1 is a gentamicin immune colloid gold quick detection reagent plate structure synoptic diagram, and wherein 1 is sample pad, and 2 is the collaurum pad, and 3 is nitrocellulose filter, and 4 is detection line, and 5 is control line, and 6 is adsorptive pads, and 7 is adhesive sticker, and 8 is the PVC plate.

Fig. 2 is a gentamicin immune colloid gold quick detection reagent plate operation chart, and wherein S is a well, and C is the control zone, and T is a detection zone.

Fig. 3 really judges synoptic diagram for gentamicin immune colloid gold quick detection reagent hardens, and wherein C is the control zone, and T is a detection zone.

Specific implementation method

The preparation of the utility model agent plate comprises the preparation of gentamicin-BSA conjugate, anti-gentamicin MONOCLONAL ANTIBODIES SPECIFIC FOR, the preparation of colloidal gold solution, the assembling of anti-gentamicin MONOCLONAL ANTIBODIES SPECIFIC FOR of colloid gold label and gentamicin immune colloid gold quick detection reagent plate.

1. the coupling of gentamicin and carrier protein

Adopt carbodiimide (EDCHCl) method that gentamicin and carrier protein couplet are prepared immunizing antigen and envelope antigen.Take by weighing 20mgBSA, add 1mL distilled water.Use 1mL dissolved in distilled water 20mg EDCHCl and 20mg gentamicin in addition, join in the above-mentioned solution.Behind the solution mixing, place 4 ℃ of following lucifuges reactions 12 hours (during can put upside down mixing).Afterwards with PBS damping fluid dialysis 4 days, during every interval changed liquid in 8 hours 1 time.With solution centrifugal, collect supernatant again ,-20 ℃ frozen standby down.

2. anti-gentamicin MONOCLONAL ANTIBODIES SPECIFIC FOR

Get female Balb/c mouse in 6～8 ages in week, will be as immunogenic gentamicin-BSA conjugate and isopyknic Freund's complete adjuvant emulsification, press 100 a μ g/ dosage hypodermic injection, every 3 all booster immunizations 1 time, full adjuvant lumbar injection toos many or too much for use afterwards.The 3d reinforced immunological is 1 time before merging, and without adjuvant, dosage doubles.Fusion of Cells is carried out according to a conventional method: the Sp2/0 multiple myeloma cells is mixed in 1: 10 ratio with immune spleen cell, merge under the 50%PEG effect, the HAT nutrient culture media suspends, and divides kind in 96 well culture plates, cultivates in 37 ℃, 5%CO2 incubator.

After the fusion, treat that cell grows into 1/4 o'clock of culture hole area, adopt and divide step screening method screening hybridoma.Primary dcreening operation selects 10mg/L gentamicin carrier protein (OVA) conjugate bag by elisa plate, and measured hole adds culture supernatant, after hatching, cleaning, adds sheep anti-mouse igg-HRP (1: 1000), the OPD colour developing.The elisa plate of the positive Kong Zaiyong gentamicin that filters out-OVA bag quilt is blocked indirect ELISA.With cells and supernatant and 2 * 10 ^-3Mol/L gentamycin solution mixed in equal amounts, 1h is made in 37 ℃ of senses, adds to have wrapped in the ELISA Plate of quilt.Use PBS (0.01mol/L, pH7.4) to substitute gentamycin solution in addition and compare, all the other steps are the same.If the OD value after the gentamicin blocking-up is reduced to below 50% of control wells, then be judged to positive hole.Through the hole that 2～3 detections all are positive, carry out cloning with limiting dilution assay immediately.

In vitro culture: with the cell line enlarged culture of cloning, cell concentration reaches 5 * 10 ⁵Stop to change liquid during/mL, nutrient solution is collected in all dead back of cell.Induce ascites in the body: give the mouse peritoneal injection cloning cell line 10 of lumbar injection whiteruss after 10 days ⁷Individual cell extracted ascites after 7 days.

3. the preparation of colloidal gold solution

The mean size of colloid gold particle is 30nm, and its preparation method is to add 1mL 1% trisodium citrate in the 100mL deionized water, boils the back and adds 1mL 1% gold chloride rapidly, continues to boil 10min, after the cooling, preserves standby down for 4 ℃.

4. the anti-gentamicin MONOCLONAL ANTIBODIES SPECIFIC FOR of colloid gold label

Get the colloidal gold solution 100mL that has prepared, transfer pH to 8.0 with the 0.1mol/L solution of potassium carbonate.Add the anti-gentamicin monoclonal antibody of 2mg while stirring, stir 20min, dropwise add 2mL 25mol/L Macrogol 2000 0 (PEG 20000), stir 15min.20, the centrifugal 15min of 000rpm abandons supernatant, and the PBS damping fluid (containing 0.4mol/L PEG) that adds 10mL pH 7.4 cleans 2 times.Precipitation is dissolved with the PBS damping fluid (pH 7.4) that 10mL contains 2%BSA, and after filtering with 0.22 μ m sterilizing filter, 4 ℃ of preservations are standby.

5. the assembling of gentamicin immune colloid gold quick detection reagent plate

With reference to Fig. 1, with some film machine the gentamicin of debita spissitudo-carrier protein couplet thing and sheep anti-mouse igg are sprayed on the nitrocellulose filter, respectively as detection line and nature controlling line, 37 ℃ of oven drying 8h.In kind, the golden mark gentamicin monoclonal antibody for preparing is coated on the collaurum pad.

Detectable consists of a PVC backing, is stained with sample pad, collaurum pad, nitrocellulose filter and adsorptive pads thereon in order.With cutting machine the kilocalorie that posts is cut into the wide bar of 4mm, make the detectable plate in the plastic formwork of packing into, put into the airtight storage of aluminium foil bag of band drying agent again.

6. gentamicin immune colloid gold quick detection reagent plate detects principle

After sample solution to be checked splashed into the agent plate well, sample solution spread to the other end because of the capillarity of cellulose nitrate membrane carrier.In the process that moves, the corresponding antigen antibody response can take place, and show by the color of immune colloid gold.If sample solution contains gentamicin residue, antibody response on gentamicin elder generation and the colloid gold particle, therefore when colloid gold particle diffused to detection line with sample solution, the avtive spot of antibody can't combine with gentamicin specific antigen on the detection line because of being occupied by the gentamicin in the sample solution on the colloid gold particle; When the gentamicin content in the sample surpassed the agent plate detection limit, the detection line colour developing on the agent plate was shallow even do not have colour developing than control line, is judged to be the positive.Otherwise when gentamicin content in the sample below the agent plate detection limit or during noresidue, the detection line colour developing on the agent plate is close with control line or partially deeply, is judged to be feminine gender.

7. gentamicin immune colloid gold quick detection reagent plate detects the embodiment method of operating

7.1 specimen preparation

Get the special-purpose PBST damping fluid of 400 μ L gentamicins and add in the clean 1.5mL centrifuge tube, draw 3 (about 100 μ L) former milk with dropper and add in the centrifuge tube, mix, it is to be checked to draw mixed solution.

7.2 detection step

Take out agent plate from packaging bag, draw solution to be checked with dropper, splash into 3 (about 100 μ L) in well, pick up counting behind the application of sample, the result should read at 3～5 minutes, and the other times interpretation is invalid.

7.3 the result judges

When reading as a result, the agent plate level is placed the observer front, shown in Fig. 2 right side.

Negative (-): the colour developing of T line shows that the gentamicin drug concentration is lower than 50ng/mL or does not have the gentamicin medicament residue in the sample than C line deeply or equally dark.Shown in Fig. 3 .a.

Positive (+): the colour developing of T line is more shallow than C line, or the T line do not have colour developing, shows that the gentamicin drug concentration is higher than 50ng/mL in the sample; The T line is more shallow more than C line, shows that the gentamicin drug concentration is high more in the sample.Shown in Fig. 3 .b.

Invalid: the C line do not occur, the improper or agent plate of possible operation lost efficacy.Should read instructions once more, and test again with the novel agent plate.Shown in Fig. 3 .c.

Claims (4)

1. immune colloidal gold reagent plate that detects the gentamicin medicament residue, by up and down two plastic formworks and test strips are formed, on the test strips backing, closely be pasted with sample pad successively, the collaurum pad, nitrocellulose filter and adsorptive pads, it is characterized in that sample pad, the collaurum pad, have between nitrocellulose filter and the adsorptive pads each several part that 1-2mm's is overlapping, be coated with the bond of anti-gentamicin monoclonal antibody and collaurum on the collaurum pad, be coated with gentamicin-carrier protein couplet thing and sheep anti-mouse igg successively from sample pad to the adsorptive pads direction on the nitrocellulose filter, respectively as detection line and nature controlling line.

2. agent plate according to claim 1 is characterized in that anti-gentamicin monoclonal antibody is for discerning immunoglobulin (Ig) or its fragment of gentamicin.

3. agent plate according to claim 1 is characterized in that the carrier protein of coupling gentamicin can be bovine serum albumin(BSA), ovalbumin or hemocyanin.

4. agent plate according to claim 1 is characterized in that backing is the PVC plate that one side scribbles adhesive sticker, and sample pad is made by glass fibre, and the collaurum pad is made by polyester film, and adsorptive pads is a filter paper.

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