CN101949922A - Method for preparing reagent plate for detecting sulphadimidine - Google Patents
Method for preparing reagent plate for detecting sulphadimidine Download PDFInfo
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- CN101949922A CN101949922A CN2010102520163A CN201010252016A CN101949922A CN 101949922 A CN101949922 A CN 101949922A CN 2010102520163 A CN2010102520163 A CN 2010102520163A CN 201010252016 A CN201010252016 A CN 201010252016A CN 101949922 A CN101949922 A CN 101949922A
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Abstract
The invention relates to a method for preparing a reagent plate for detecting sulphadimidine residue in honey. The reagent plate consists of upper and lower plastic templates and a backing, wherein a sample pad, a colloidal gold bonding pad, a nitrocellulose film and an absorbent pad are tightly adhered to the backing. The nitrocellulose film is coated with a detection line and a quality control line in a direction from the sample pad to the absorbent pad in turn, and a reaction result can be characterized in megascopic color. The reagent plate has the advantages of capacity of performing semi-quantitative visual detection, only 20 to 30 minutes of the whole operation process, no need of expensive auxiliary experimental equipment, contribution to large-scale sample screening, and suitability for large-scale rapid detection of the sulphadimidine in raw materials and products in department of industry and commerce, inspection and quarantine mechanisms, beehouses and manufacturers of bee products.
Description
Technical field
The present invention relates to a kind of preparation method who detects the residual agent plate of sulfamethazine in the honey, be specifically related to a kind of preparation method who detects the immune colloid gold quick detection reagent plate of sulfamethazine.
Background technology
Sulfamethazine (Sulfamethazine) by diacetone directly and the sulphoamidine condensation make, be applicable to that treatment hemolytic streptococcus, meningococcus, pneumococcus etc. catch, as feed addictive, can prevent and treat the infection of staphylococcus and hemolytic streptococcus etc.Because its lasting medicine is remarkable, sulfa drugs commonly used carries out disease prevention and cure in bee rearing.
Yet unreasonable use, easily make sulfamethazine residual in honey, medicine can cause diseases such as blood urine, crystalluria, kidney damage after entering human body by food chain, health health is worked the mischief, and simultaneously sulfamethazine residual in honey causes obstacle also for the honey trade.
Many countries have all stipulated sulfamethazine and the sulfa drugs maximum residue limit(MRL) (MRL) in honey.China and European Union all in the regulation animal food MRL of total sulfanilamide (SN) and single sulfonamide be 100ppb.
At present, mainly contain physico-chemical analysis method and immunoassay for the residual detection method of sulfamethazine.The physico-chemical analysis method, as HPLC and LC-MS, have the advantages that specificity is good, accuracy rate is high, be that each big testing agency is to detecting the prefered method that sample is proved conclusively, but exist requirements such as equipment, environment, operative skills, required expense height is unfavorable for extensive screening sample, thereby is not suitable for department of vast basic unit.Immunoassay, as enzyme linked immunosorbent assay (ELISA), it is big to have detection limit, operate advantages such as simple relatively, be used to the detection of antibiotic residues in animal-derived food more and more, but the ELISA method whole operation time still needs 1-2h, and need use expensive special instruments and equipment microplate reader, has certain limitation.
Food security involves the interests of the state and the people, and develop a kind of detectability and meet the requirements, favorable reproducibility, detection time is short, is fit to field quick detection, and technical products or instrument and equipment cost are lower, and the quick detection reagent that operating cost is low is extremely urgent.
Summary of the invention
The object of the present invention is to provide a kind of highly sensitively, specificity is good, easy fast, the preparation method of the sulfamethazine immune colloid gold quick detection reagent plate that production cost is low.
Agent plate of the present invention is made up of up and down two plastic formworks and backing, is closely pasting sample pad, the golden pad of glue, nitrocellulose filter and adsorptive pads on the backing successively.What 1-2mm arranged between adjacent each several part is overlapping to guarantee chromatography effect carrying out smoothly from sample pad to the adsorptive pads position.
The each several part constituent and the function of agent plate of the present invention are as follows:
Plastic formwork plays fixedly backing and sign each functional areas (well, detection zone, control zone).
Backing, the toughness material that do not absorb water that is scribbled adhesive sticker by one side is made, and plays other ingredients of fixed support agent plate.
Sample pad is made by glass fibre, works to absorb sample solution and buffering sample solution pH value.
Glue gold pad is made by polyester film, and the bond of anti-sulfamethazine monoclonal antibody and colloid gold particle is arranged on it.For the reaction of effective constituent in the sample solution and golden labeling antibody provides the place.
Nitrocellulose filter is sprayed with detection line and nature controlling line on from sample pad to the adsorptive pads direction successively, and reaction result is come out with macroscopic characterization.
Adsorptive pads is made by filter paper, and excessive solution in the course of reaction is absorbed.
Agent plate of the present invention has following beneficial effect:
(1) specificity is good.Agent plate of the present invention is 100% to the cross reacting rate of sulfamethazine, to all being lower than 1% as antibiotic cross reacting rates such as sulphadiazine, sulfamethyldiazine, madribons.As seen, reaction has the height selectivity to agent plate of the present invention to sulfamethazine.
(2) highly sensitive.Agent plate of the present invention to green molasses, concentrate that detecting of sulfamethazine is limited to 10ppb in the honey, meet country's requirement of limiting the quantity of.
(3) simple and quick.Most of raw material that agent plate of the present invention will be reacted required is incorporated in the PVC backing, after dripping sample, antigen-antibody reaction is carried out on immobilon-p fast, shorten 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 behind the sample.Detect implementation process and do not rely on any experimental facilities, the ordinary person all can operate, and does not need professional training.
(4) cost is low, easily promotes.Agent plate production technology of the present invention is simple, the flow process maturation.Low production cost requires less investment while yielding quicker results.
Description of drawings
Fig. 1 is a sulfamethazine immune colloid gold quick detection reagent backboard lining structure synoptic diagram, and wherein 1 is sample pad, and 2 is glue gold pad, and 3 is nitrocellulose filter, and 4 is detection line, and 5 is nature controlling line, and 6 is adsorptive pads, and 7 is adhesive sticker, and 8 is the PVC base plate.
Fig. 2 is a sulfamethazine 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 sulfamethazine 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 agent plate of the present invention comprises the preparation of sulfamethazine-carrier protein couplet thing, anti-sulfamethazine MONOCLONAL ANTIBODIES SPECIFIC FOR, the preparation of colloidal gold solution, the assembling of anti-sulfamethazine MONOCLONAL ANTIBODIES SPECIFIC FOR of colloid gold label and sulfamethazine immune colloid gold quick detection reagent plate.
1. the coupling of sulfamethazine and carrier protein
Adopt the diazonium method that sulfamethazine and carrier protein couplet are prepared immunizing antigen and envelope antigen.Take by weighing the 10mg sulfamethazine and be dissolved in the H of 10mL 12.5mol/L
2SO4 is with the NaNO of 20mL 20%
3Solution slowly splashes in the above-mentioned solution, and 4 ℃ are reacted 10min down.In the mixed solution that obtains, add the sodium carbonate buffer solution (pH 10.0) that 2mL is dissolved with 100mg bovine serum albumin (BSA) again, reaction is spent the night under 4 ℃, normal saline dialysis 72h, the sulfamethazine that makes-BSA carrier protein couplet thing is frozen standby in-20 ℃.
Ovalbumin (OVA) substitutes BSA, with preparing sulfamethazine-OVA conjugate with quadrat method.
2. anti-sulfamethazine MONOCLONAL ANTIBODIES SPECIFIC FOR
Get female Balb/c mouse in 6～8 ages in week, will be as immunogenic sulfamethazine-BSA conjugate and isopyknic Freund's complete adjuvant emulsification, by 100 a μ g/ dosage hypodermic injection, every 3 all booster immunizations 1 time, the full adjuvant that toos many or too much for use substitutes Freund's complete adjuvant and carries out lumbar injection 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 is selected 10mg/L, and sulfamethazine-OVA conjugate bag is 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 sulfamethazine that filters out-OVA conjugate bag quilt is blocked indirect ELISA.With cells and supernatant and 2 * 10
-3Mol/L sulfamethazine 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, pH 7.4) to substitute sulfamethazine solution in addition and compare, all the other steps are the same.If the OD value after the sulfamethazine 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
5ML
-1The time stop to change liquid, nutrient solutions are 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-sulfamethazine MONOCLONAL ANTIBODIES SPECIFIC FOR of colloid gold label
Get the 100mL colloidal gold solution that has prepared, transfer pH to 8.0 with the 0.1mol/L solution of potassium carbonate.Add the anti-sulfamethazine monoclonal antibody of 1.5mg while stirring, stir 20min, dropwise add 2mL 25mol/L Macrogol 2000 0 (PEG20000) again, stir 15min.20, the centrifugal 15min of 000rpm abandons supernatant, adds 10mL pH 7.4PBS damping fluid (containing 0.4mol/L PEG) and cleans 2 times.Precipitation is dissolved with the PBS damping fluid (pH 7.4) that 5mL contains 2%BSA, and after filtering with 0.22 μ m sterilizing filter, 4 ℃ of preservations are standby.
5. the assembling of sulfamethazine immune colloid gold quick detection reagent plate
With reference to Fig. 1, with some film machine the sulfamethazine of debita spissitudo-BSA conjugate and sheep anti-mouse igg are sprayed on the nitrocellulose filter, respectively as detection line and control line, 37 ℃ of oven drying 8h.In kind, the golden mark sulfamethazine monoclonal antibody for preparing is coated on the glue gold pad.
Detectable consists of the 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. sulfamethazine immune colloid gold quick detection reagent plate detects the implementation and operation method
6.1 specimen preparation
Get honey 3mL and add in the 15mL scale freeze pipe, add sulfamethazine A liquid and each 1mL of B liquid more successively, in 60 ℃～80 ℃ hot water warm several minutes, dissolving honey, vibration mixing.Add 8mL ethyl acetate, leave standstill after spinning upside down vibration 5min., in 5mL scale freeze pipe, do with dropper transferase 45 mL upper strata organic solvent with 65 ℃ of nitrogen (sky) air-blowing.Dropwise 5 drips the special-purpose PBST damping fluid of sulfamethazine in this 5mL scale freeze pipe, fully dissolves residue on the inside pipe wall, and is to be checked.
6.2 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 3～5min, and the other times interpretation is invalid.
6.3 interpretation as a result
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 sulfamethazine concentration is lower than 10ppb or does not have sulfamethazine residual 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 sulfamethazine concentration is higher than 10ppb in the sample; The T line is more shallow more than C line, shows that sulfamethazine 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 (6)
1. preparation method who detects the agent plate of sulfamethazine, it is characterized in that agent plate is made up of up and down two plastic formworks and backing, have between the sample pad of closely pasting successively on the agent plate backing, glue gold pad, the adjacent each several part of nitrocellulose filter that 1-2mm's is overlapping with assurance chromatography effect carrying out smoothly from sample pad to the adsorptive pads position with adsorptive pads.
2. as the said preparation method of claim 1, it is characterized in that being coated with anti-sulfamethazine monoclonal antibody-colloid gold label thing on the collaurum pad on the nitrocellulose filter of agent plate, be sprayed with detection line and nature controlling line successively on from sample pad to the adsorptive pads direction, be coated with sulfamethazine-carrier protein couplet thing and sheep anti-mouse igg, reaction result is come out with macroscopic characterization.
3. as the said preparation method of claim 1, it is characterized in that being coated with anti-sulfamethazine monoclonal antibody and collaurum bond on the glue gold pad, the mean size of colloid gold particle is 30nm.
4. as the said preparation method of claim 1, it is characterized in that the carrier protein of coupling sulfamethazine can be bovine serum albumin, ovalbumin, hemocyanin.
5. as the said preparation method of claim 1, when it is characterized in that sulfamethazine content in the sample surpasses the agent plate detection limit, the detection line colour developing on the agent plate is shallow even do not have colour developing than control line, is judged to be the positive; Otherwise when sulfamethazine 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.
6. as the said preparation method of claim 1, it is characterized in that technological process comprises preparation sulfamethazine-BSA conjugate, the anti-sulfamethazine monoclonal antibody of preparation, preparation colloidal gold solution, the preparation anti-sulfamethazine monoclonal antibody of colloid gold label and assembling sulfamethazine immune colloid gold quick detection reagent plate.
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| CN2010102520163A CN101949922A (en) | 2010-08-11 | 2010-08-11 | Method for preparing reagent plate for detecting sulphadimidine |
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| CN2010102520163A CN101949922A (en) | 2010-08-11 | 2010-08-11 | Method for preparing reagent plate for detecting sulphadimidine |
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Application publication date: 20110119 |