CN103105491A - Kit and method for detecting beta-lactam antibiotic and tetracycline antibiotic - Google Patents
Kit and method for detecting beta-lactam antibiotic and tetracycline antibiotic Download PDFInfo
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- CN103105491A CN103105491A CN2011103569561A CN201110356956A CN103105491A CN 103105491 A CN103105491 A CN 103105491A CN 2011103569561 A CN2011103569561 A CN 2011103569561A CN 201110356956 A CN201110356956 A CN 201110356956A CN 103105491 A CN103105491 A CN 103105491A
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Abstract
The invention discloses a kit and a method for detecting beta-lactam antibiotic and tetracycline antibiotic. The kit comprises micropore strips, micropore reagents, test paper and micropore plugs, wherein the micropore reagents are lyophilized colloidal gold labeled cephalosporins and tetracyclines specific antibodies, the cephalosporins specific antibodies comprise cephalosporins monoclonal antibodies and cephalosporins polyclonal antibodies, the tetracyclines specific antibodies comprise tetracyclines monoclonal antibodies and cephalosporins polyclonal antibodies; the test paper comprises a sample absorption pad, a reaction film, a water absorption pad, a protection film and a base plate which are connected in sequence, the reaction film comprises two detection lines and a quality control line, each detection lines comprises cephalosporins-carrier protein conjugates and tetracyclines hapten-carrier protein conjugates, and the quality control line comprises antiantibodies. According to the invention, the method for detecting beta-lactam antibiotic and tetracycline antibiotic by using the kit is simple, rapid, visual, accurate, portable, wide in application range, low in cost and easy to popularize.
Description
Technical field
The present invention relates to a kind of kit and method that detects beta-lactam and TCs.
Background technology
Microbiotic is 20th century one of most important medical discovery, has brought into play great function to controlling the human infectious disease.But the increase of the pathogenic bacteria of drug-resistant that causes due to abuse of antibiotics in recent years not only makes medical expense increase, and makes the incidence of disease and the also corresponding increase of mortality ratio of infectious diseases.In addition, microbiotic be widely used in the agricultural with animal husbandry be also the reason of can not ignore that causes resistance.The treatment of the diseases such as antibiotic medicine effect domestic animals mammitis, hysteritis such as beta-lactam commonly used, Tetracyclines, also have the producer to add microbiotic in feed, to improve the feed conversion rate of livestock and poultry in dairy industry.The investigation of U.S. food and Drug Administration (FDA) shows: not noting the off-drug period, medication is unreasonable or uses forbidden drug, is the main cause that causes antibiotic residues in animal-derived food.The long-term absorption contains antibiotic food, can cause drug accumulation, after reaching finite concentration, will bring the series of negative impact to the health of human body, as producing allergic reaction, destroy the gastrointestinal bacterial flora balance and strengthening bacterial drug resistance etc.What is more important, microbiotic can be destroyed the normal flora environment of Healthy People by the excessive Healthy People enteron aisle of eating, and cause the reduction of body immunity, thereby endanger the healthy of consumer, also affect the export trade of milk and dairy products simultaneously.In the raw milk of China, beta-lactam and teracycline antibiotic residues are serious, and it is carried out monitor and detection is problems of concern very in the development of China dairy husbandry.In milk, the microbiotic problem obtains effective monitoring in order to make, meet the maximum residue limit standard, cattle farm, Milk Products Plant, government supervision department, entry and exit detect, large-scale Food circulation field etc. all making great efforts to seek accurately feasible detection method, and many large-scale chemical reagent and instrument company also are devoted to develop method and the instrument that beta-lactam and teracycline antibiotic residues detect.
At present, in milk and raw milk, the detection method of beta-lactam and teracycline antibiotic residues according to different detection principle, can be divided into three major types substantially: 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 the method testing cost valency is low, but sensitivity is low, the existence of length consuming time, drug-fast bacteria easily causes flase drop; The physics and chemistry detection method, it is the main method that detects at present beta-lactam and TCs, liquid phase chromatography, Chromatography/Mass Spectrometry coupling technique, vapor-phase chromatography etc. are arranged, degree of accuracy is high, but it exists that testing cost is high, equipment is complicated, to the having relatively high expectations of testing staff, detect the shortcomings such as length consuming time, the immune analysis method of commonly using at present, be mainly enzyme linked immunosorbent detection method and radio immunoassay, the testing cost that needs is still higher, is not suitable for enterprise and detects cheaply needs.
" method and the purposes of penicillin and carrier protein couplet product and the preparation of beta-lactam penicillin antibody " disclosed in Chinese invention patent 200810162588.5 (CN101429241A), the enzyme linked immunological kit of the Penicillin monoclonal antibody preparation in the method only can detect benzyl penicillin, ampicillin, three kinds of beta-lactam antibiotics of carbenicillin, the colloidal gold strip detection sensitivity of preparation is low, and the detection of drugs kind is few.Immunoassay field shortage can detect beta-lactam and the Tetracyclines antibiotic technology of this two class and application thereof simultaneously at present.Therefore, be necessary in practice to set up that a Species sensitivity 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.
Summary of the invention
An object of the present invention is to provide a kind of kit that detects beta-lactam and TCs.
Kit provided by the present invention comprises capillary strip, micropore reagent, micropore plug and test strips, and test strips comprises base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm, and it connects successively; The cephalosporins medicine specific antibody that described micropore reagent is freeze-drying-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing; The cephalosporins medicine specific antibody can be cephalosporins medicine monoclonal antibody or cephalosporins medicine polyclonal antibody, and the tetracycline medication specific antibody can be tetracycline medication monoclonal antibody or tetracycline medication polyclonal antibody; Be coated with two detection lines and a nature controlling line on reaction film; When cephalo-type drug specificity antibody is the cephalosporins medicine monoclonal antibody, comprise cephalo-type monoclonal antibody-colloid gold label thing in micropore reagent, the Beta-lactam medicine detection line is coated with cephalosporins medicine-carrier protein couplet thing, and nature controlling line is coated with the sheep anti mouse antiantibody; When cephalo-type drug specificity antibody is the cephalosporins medicine polyclonal antibody, comprise cephalosporins medicine polyclonal antibody-colloid gold label thing in micropore reagent, the Beta-lactam medicine detection line is coated with cephalosporins medicine-carrier protein couplet thing, and nature controlling line is coated with goat-anti rabbit antiantibody; When the tetracycline medication specific antibody is the tetracycline medication monoclonal antibody, comprise tetracycline medication monoclonal antibody-colloid gold label thing in micropore reagent, the tetracycline medication detection line is coated with tetracycline medication hapten-carrier protein conjugate, and nature controlling line is coated with the sheep anti mouse antiantibody; When the tetracycline medication specific antibody is the tetracycline medication polyclonal antibody, comprise tetracycline medication polyclonal antibody-colloid gold label thing in micropore reagent, the tetracycline medication detection line is coated with tetracycline medication hapten-carrier protein conjugate, and nature controlling line is coated with goat-anti rabbit antiantibody; The tetracycline medication haptens is to utilize Mannich reaction by tetracycline and 4-piperidine carboxylic acid, has introduced N-methyl-resulting derivant of 4-piperidine carboxylic acid group on the amido of the acid amides of tetracycline, obtains the tetracycline medication haptens suc as formula shown in I:
(formula I)
Described diaphragm sticks on the test side on the absorption of sample pad, and the above has the MAX mark line.
Described tetracycline medication detection line is near absorption of sample pad one end, and nature controlling line is near adsorptive pads one end, and the Beta-lactam medicine detection line is between tetracycline medication detection line and nature controlling line.Wherein the tetracycline medication detection line represents with detection line (T), and the Beta-lactam medicine detection line represents with detection line (B), nature controlling line nature controlling line (C) expression.Take absorption of sample pad one end of test strips as top, take adsorptive pads one end of test strips as end, nature controlling line (C) is 5-8mm apart from the end that reaction film is connected with adsorptive pads top, detection line (B) is apart from nature controlling line (C) 4-6mm, and detection line (T) is apart from detection line (B) 4-6mm.
Described cephalosporins medicine-carrier protein couplet thing is to be obtained by cephalosporins medicine and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Described Tetracyclines hapten-carrier protein conjugate is that tetracycline medication haptens and the carrier protein couplet by formula I compound obtains, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Cephalosporins medicine specific antibody in described cephalosporins medicine specific antibody-colloid gold label thing is to prepare as immunogene with cephalosporins medicine-carrier protein couplet thing.
Tetracycline medication specific antibody in described tetracycline medication specific antibody-colloid gold label thing is to prepare as immunogene with tetracycline medication hapten-carrier protein conjugate.
Described base plate can be the material that PVC base plate or other hard do not absorb water; The absorption of sample pad can be suction strainer paper or filter paper for oil; Adsorptive pads is thieving paper; Reaction film can be nitrocellulose filter or cellulose acetate membrane; Adsorptive pads is thieving paper; Diaphragm is PE material diaphragm.
Another object of the present invention has been to provide a kind of method for preparing the mentioned reagent box, and it comprises step:
1) the cephalosporins medicine specific antibody-colloid gold label thing of preparation freeze-drying and the micropore reagent of Tetracyclines specific antibody-colloid gold label thing on capillary strip, and with the micropore gag on capillary strip;
2) reaction film of the nature controlling line of two of the preparations detection line that has respectively coated cephalosporins medicine-carrier protein couplet thing and a tetracycline medication hapten-carrier protein conjugate and a coated antiantibody;
3) with 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test strips;
4) with 1) and 3) freeze-drying for preparing has micropore reagent, test strips, capillary strip and the micropore plug of cephalosporins medicine specific antibody-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing to be assembled into kit.
Specifically, step comprises:
1, the preparation of the synthetic and antibody of Beta-lactam medicine antigen
1) with cephalosporins medicine and carrier protein couplet, form cephalosporins medicine-carrier protein couplet thing;
2) with cephalosporins medicine-carrier protein couplet thing immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of cephalosporins medicine monoclonal antibody or with cephalosporins medicine-carrier protein immunize rabbit, obtain the cephalosporins medicine polyclonal antibody;
3) extract mouse IgG or rabbit igg immune health goat, obtain sheep anti-mouse igg antibody or goat-anti rabbit antiantibody;
2, tetracycline medication antigen synthesizes and the antibody preparation
1) get the 4-piperidine carboxylic acid of 1.1-2.2g tetracycline, 0.37-0.75g and 2-5ml 37% formaldehyde in the middle mixing of 50-100ml ethanol, the preferred 1.1g of the consumption of tetracycline, the preferred 2ml of the consumption of 4-piperidine carboxylic acid, the preferred 2ml of the consumption of formaldehyde, the preferred 50ml of the consumption of ethanol, react 3-5min under 60% microwave intensity, reaction time is preferably 3min, and after desolventizing, reversed phase column chromatography separates, obtain yellow solid, be the tetracycline medication haptens;
2) with tetracycline medication haptens and carrier protein couplet, form tetracycline medication hapten-carrier protein conjugate;
3) with tetracycline medication hapten-carrier protein conjugate immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of tetracycline medication monoclonal antibody or with tetracycline medication hapten-carrier protein immunization rabbit, obtain the tetracycline medication polyclonal antibody;
4) extract mouse IgG or rabbit igg immune health goat, obtain sheep anti-mouse igg antibody or goat-anti rabbit antiantibody;
3, with trisodium citrate and gold chloride reaction preparation collaurum;
4, cephalosporins medicine specific antibody and the tetracycline medication specific antibody with preparation joins respectively in the collaurum of preparation, obtains the micropore reagent of cephalosporins medicine specific antibody-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing;
5, with after the micropore reagent freeze-drying of cephalosporins medicine specific antibody-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing is in capillary strip, the micropore plug will be covered on capillary strip;
6, bovine serum albumin(BSA) (final concentration of bovine serum albumin(BSA) in damping fluid is 0.5% (volumn concentration)), pH are 7.2 with containing, the 0.1mol/L phosphate buffer soaks 2h with the absorption of sample pad, dry 2h under 37 ℃;
7, stick in order absorption of sample pad, reaction film, adsorptive pads and diaphragm on base plate;
8, micropore reagent, test strips, capillary strip and the micropore plug for preparing is assembled into kit, preserved 12 months under 2-8 ℃ of condition.
Another object of the present invention provides the detection method of beta-lactam and teracycline antibiotic residues in a kind of milk sample.
Detect with the above-mentioned arbitrary described kit of the present invention.
the detection principle of kit of the present invention: milk sample drop to be checked is added in the capillary strip that includes micropore reagent, after mixing, hatch 5min, to indicate MAX mark line end downward, insertion includes in micropore reagent capillary strip after hatching, sample liquid to be checked and golden labeling antibody in micropore in conjunction with after together with spread to reaction film, if in sample liquid to be checked, the content of beta-lactam or TCs is high, in diffusion process, the beta-lactam in analyte sample fluid or TCs can combine with golden labeling antibody, and then seal the antigen-combining site of beta-lactam antibiotic on golden labeling antibody or TCs fully, stop golden labeling antibody be combined with cephalosporins medicine on reaction film-carrier protein couplet thing or reaction film on the combination of tetracycline medication hapten-carrier protein conjugate, corresponding beta-lactam antibiotic detection line or TCs detection line can not develop the color, antiantibody can be combined with cephalo eka-gold labeling antibody or tetracycline eka-gold labeling antibody, the nature controlling line colour developing, if the low or nothing of the content of beta-lactam antibiotic or TCs in sample liquid to be checked, corresponding cephalo eka-gold labeling antibody or the antigen binding site on tetracycline eka-gold labeling antibody can not be closed, and then cephalo eka-gold labeling antibody can be combined with cephalosporins medicine on reaction film-carrier protein couplet antigen or tetracycline eka-gold labeling antibody can be combined by tetracycline medication hapten-carrier protein-coupled antigen on reaction film, corresponding Beta-lactam medicine detection line colour developing or the colour developing of Tetracyclines detection line, antiantibody also can be combined with cephalo eka-gold labeling antibody or tetracycline eka-gold labeling antibody simultaneously, the nature controlling line colour developing, if nature controlling line does not develop the color, test strips lost efficacy.
Negative: nature controlling line (C) and detection line (B) and detection line (T) all demonstrate red stripes, are judged to feminine gender, with "-" expression.Wherein detection line (B) represents the Beta-lactam medicine detection line, detection line (T) expression tetracycline medication detection line.
Beta-lactam medicine is positive: nature controlling line (C) and detection line (T) demonstrate red stripes, and detection line (B) does not develop the color, and is judged to the positive, with "+" expression.
Tetracycline medication is positive: nature controlling line (C) and detection line (B) demonstrate red stripes, and detection line (T) does not develop the color, and is judged to the positive, with "+" expression.
Beta-lactam and tetracycline medication all are positive: nature controlling line (C) demonstrates red stripes, and detection line (T) and detection line (B) all do not develop the color, and are judged to the positive, with "+" expression.
Invalid: (C) do not show red stripes when nature controlling line, and no matter whether detection line (T) and detection line (B) red stripes occurs, and test strips all lost efficacy.
Kit of the present invention can detect Beta-lactam medicine and comprise: benzyl penicillin, nafcillin, ampicillin, cefoperazone, Amoxicillin, ceftriaxone, oxacillin, Ceftiofur, adjacent penicillin, cefalonium, two penicillin, Cefquinome; Can detect tetracycline medication comprises: tetracycline, terramycin, fortimicin, duomycin.
Kit of the present invention has following beneficial effect:
1, highly sensitive: traditional test card all has the fixedly stationary installation that gets stuck of test strips, the stationary installation that gets stuck is fixing wherein with test strips tightly, to sample detection the time, tightr due to what get stuck and be combined with test strips, cause test sample solution can not fully be penetrated into one section, absorption of sample pad, and the speed of carrying out chromatography on test strips is very slow, cause the medicine in test sample fully to contact with coated coating antigen on detection line, cause the sensitivity of test card to descend.Kit of the present invention is higher than the detection sensitivity of traditional test card, because the test strips of kit of the present invention is naked strip, need not the housing fastener, test sample can directly contact absorption of sample pad one end of test strips, and the chromatography velocity ratio of sample on test strips is very fast, the coating antigen that medicine to be checked in sample can be coated with on detection line fully is combined, thereby has improved the detection sensitivity of test strips.Beta-lactam and TCs detection kit are prepared from as the basis take the monoclonal antibody of colloid gold label high-affinity, priceless strong formation between gold grain and antibody molecule in the gold labeling antibody, both combine by the Van der Waals force between the charges of different polarity, collaurum is very little on monoclonal antibody specificity and affinity impact, and has higher mark rate.Cephalosporins medicine monoclonal antibody wherein-colloid gold label thing and tetracycline medication monoclonal antibody-colloid gold label thing freeze-drying is in the micropore reagent strip, in testing process, golden labeling antibody is fully contacted with sample liquid to be checked, fully reaction, thereby the minimizing error, the reaction sensitivity of the whole system of increase.Therefore, kit of the present invention has higher specificity and sensitivity.This kit is respectively the Beta-lactam medicine detection sensitivity: benzyl penicillin 2 μ g/L, ampicillin 3 μ g/L, Amoxicillin 4 μ g/L, oxacillin 6 μ g/L, adjacent penicillin 6 μ g/L, two penicillin 6 μ g/L, nafcillin 20 μ g/L, Cefquinome 20 μ g/L, cefoperazone 40 μ g/L, ceftriaxone 50 μ g/L, Ceftiofur 90 μ g/L, cefalonium 10 μ g/L; To the tetracycline medication detection sensitivity be: tetracycline, terramycin, fortimicin, duomycin are respectively 80 μ g/L.
easy and simple to handle fast: detect raw material milk in the past test card and raw material milk must be added dilution to dilute can to detect by point sample, reason is tightly test strips to be fixed in housing due to the fixing plastic casing of test strips, and raw material milk compares thickness, so directly raw material milk is carried out at the well of test card the words that point sample detects, be combined with test strips due to housing closely and due to the milk thickness, the raw material milk sample can't be penetrated into the absorption of sample pad fully and bond discharges in pad, and the chromatography velocity ratio on test strips is slower, to such an extent as to can't detect, therefore can detect after will adding dilution to dilute raw material milk.need not any other reagent when using kit of the present invention to detect, when detecting raw material milk, do not need raw material milk is diluted yet, reason is because the test strips in kit of the present invention is naked strip, namely need not with housing, it to be fixed, as long as directly being dripped in freeze-drying, sample solution to be checked has in the micropore of cephalosporins medicine monoclonal antibody-colloid gold label thing and tetracycline medication monoclonal antibody-colloid gold label thing, after mixing, hatch 5min, again test strips is indicated MAX wire tag end downward, in micropore reagent after insertion is hatched, test strips can fully contact with sample solution, observations after 5min.For the detection of milk sample, kit of the present invention is shorter detection time than traditional test card, because kit of the present invention need not sample is diluted, can directly detect sample.
Kit susceptibility of the present invention is high, the detection of drugs kind is many, cost is low, simple to operate, be easy to carry, detection time is short, be fit to various units uses, stores advantage simple, long shelf-life.Wherein, adopt cephalosporins medicine monoclonal antibody and the tetracycline medication monoclonal antibody of high specific, guaranteed the reliability of testing result; In capillary strip, in testing process, golden labeling antibody is fully contacted with sample liquid to be checked golden labeling antibody freeze-drying, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Kit of the present invention can detect beta-lactam and Tetracyclines two class microbiotic simultaneously, realized a test strips to the antibiotic detection of multiclass, has satisfied on market the demand that beta-lactam and TCs are detected simultaneously.Detect the method for beta-lactam antibiotic and TCs with kit of the present invention, easy, quick, directly perceived, accurate, applied widely, cost is low, easily promotes the use of.
Description of drawings
Fig. 1 freeze-drying has the capillary strip figure of golden labeling antibody.
Fig. 2 micropore plug vertical view.
Fig. 3 micropore plug cross-sectional view.
Fig. 4 test strips cross-sectional view.
The vertical view of Fig. 5 test strips.
Fig. 6 tetracycline medication haptens synthetic route chart.
Fig. 7 ELISA test strip method schematic diagram.
Fig. 8 ELISA test strip is process decision chart as a result.
Embodiment
The experimental technique that uses in following embodiment is conventional method if no special instructions.
The formation of the kit of embodiment 1, detection beta-lactam and TCs
One, freeze-drying has the capillary strip of golden labeling antibody
Micropore reagent is housed in described capillary strip, and wherein 1 is capillary strip, the 2 micropore reagent for the cephalosporins medicine specific antibody of freeze-drying-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing;
Has micropore plug 3 (Fig. 2 and Fig. 3) on the described capillary strip that includes micropore reagent;
The micropore reagent of the cephalo-type class anti-drug monoclonal antibody of freeze-drying in described capillary strip-colloid gold label thing and tetracycline medication monoclonal antibody-colloid gold label thing, the freeze-drying amount of cephalosporins medicine specific antibody-colloid gold label thing are 0.20-0.25 μ g/ml; The freeze-drying amount of tetracycline medication specific antibody-colloid gold label thing is 0.30-0.40 μ g/ml.
Two, test strips (Fig. 4 and Fig. 5)
Described test strips is comprised of base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm;
Described absorption of sample pad 4, reaction film 5, adsorptive pads 6 and diaphragm 7 are pasted successively in order on described base plate 8, the end of absorption of sample pad is connected with reaction film, the end of reaction film is connected with adsorptive pads, the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate;
Diaphragm 7 covers the test side on the absorption of sample pad, on the diaphragm of test side, the MAX printed words should be arranged.
There are two detection lines to be respectively detection line (T) 9-1 and detection line (B) 9-2 and a nature controlling line (C) 10 on described reaction film; article two, detection line is the ribbon vertical with the appearance of described test strips with a nature controlling line; article two, detection line is positioned at a side of the diaphragm that is bordering on the MAX mark line, and nature controlling line (C) is positioned at the side away from the diaphragm that the MAX mark is arranged.Nature controlling line (C) is 5-8mm apart from the end that the end of reaction film is connected with adsorptive pads, and detection line (B) is apart from nature controlling line (C) 4-6mm, and detection line (T) is apart from detection line (B) 4-6mm.Detection line (T) is coated with Tetracyclines hapten-carrier protein conjugate (tetracycline medication haptens-oralbumin), detection line (B) is coated with cephalosporins medicine-carrier protein couplet thing (conjugate of cephalosporins medicine-bovine serum albumin(BSA)), and nature controlling line (C) is coated with the sheep anti mouse antiantibody.
Base plate is the PVC base plate; The absorption of sample pad is suction strainer paper; Adsorptive pads is thieving paper; Reaction film is nitrocellulose filter; Described diaphragm is PE material diaphragm.
Above-mentioned test strips and micropore reagent, capillary strip and micropore plug are assembled into kit, store the term of validity 12 months in the environment of 2～8 ℃.
The preparation method of kit described in embodiment 2, embodiment 1
One, the preparation of kit
The preparation method of this kit mainly comprises the steps:
1) the cephalosporins medicine monoclonal antibody-colloid gold label thing of preparation freeze-drying and the micropore reagent of Tetracyclines monoclonal antibody-colloid gold label thing in capillary strip, and with the micropore gag on capillary strip.
2) preparation has the reaction film of the nature controlling line (C) of the detection line (B) of coated cephalosporins medicine-carrier protein couplet thing, the detection line (T) that is coated with tetracycline medication hapten-carrier protein conjugate and coated sheep anti mouse antiantibody;
3) with 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test strips;
4) with 1) and 3) freeze-drying for preparing has micropore reagent, test strips, capillary strip and the micropore plug of cephalosporins medicine monoclonal antibody-colloid gold label thing and tetracycline medication monoclonal antibody-colloid gold label thing to be assembled into kit.
Following substep is described in detail:
(1) preparation of each parts
1, the preparation of antigen
1), the synthetic and evaluation of cephalosporins medicine-carrier protein couplet thing
Cephalosporins medicine is small-molecule substance, only has immunoreactivity, there is no immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
A. immunogenic preparation-cephalosporins medicine and oralbumin conjugate are synthetic
Get the sodium salt 40mg of cefapirin, with the dissolving of 1.5ml distilled water, obtain (I) liquid; Get carbodiimides (EDC) 40mg and N-hydroxy-succinamide (NHS) 60mg obtains (II) liquid with the water-soluble solution of 0.5ml; In under stirring, (II) being added (I), reaction 1h obtains (III) liquid; Get oralbumin (OVA) 60mg with the water-soluble solution of 8ml, obtain (IV) liquid; (IV) added in (III), and stirring at room reaction 24h obtains immunogene, with 0.02mol/L PBS dialysis 3 days, packing, frozen.
B. preparation-the cephalosporins medicine of coating antigen and bovine serum albumin(BSA) conjugate are synthetic
Get the sodium salt 40mg of cefapirin, with the dissolving of 1.5ml distilled water, obtain (I) liquid; Get EDC 40mg and NHS60mg and obtain (II) liquid with the water-soluble solution of 0.5ml; In under stirring, (II) being added (I), reaction 1h obtains (III) liquid; Get bovine serum albumin(BSA) 100mg with the water-soluble solution of 8ml, obtain (IV) liquid; (IV) added in (III), and stirring at room reaction 24h obtains respectively coating antigen, with 0.02mol/L PBS dialysis 3 days, packing, frozen.
C. the evaluation of cephalosporins medicine-carrier conjugates
Carrier protein, cephalosporins medicine, cephalosporins medicine-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, cephalosporins medicine, cephalosporins medicine-carrier protein couplet thing with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of cephalosporins medicine and carrier protein couplet.
2), the synthetic and evaluation of tetracycline medication hapten-carrier protein conjugate
A. the tetracycline medication haptens synthesizes (Fig. 6)
Getting 1.1g tetracycline, 0.37g4-piperidine carboxylic acid and 2ml 37% formaldehyde mixes in the solution of 50ml ethanol, utilize Mannich reaction to react 3min under 60% microwave intensity, introduced N-methyl-4-piperidine carboxylic acid group on the amido of the acid amides of tetracycline, after desolventizing, reversed phase column chromatography separates, obtain yellow solid, be the tetracycline haptens, molecular structure is as follows:
B. immunogenic preparation-tetracycline medication haptens and BSA conjugate are synthetic
The tetracycline medication haptens of getting 10mg formula I compound obtains I solution with 1ml dimethyl formamide (DMF) dissolving, is cooled to 10 ℃, gets isobutyl chlorocarbonate 10 μ l and adds in I solution, and 10 ℃ of stirring reaction 30min obtain II solution.Get the Na that 40mg BSA uses 3ml 50mmol/L
2CO
3The dissolving and with II solution in 10 ℃ the reaction 4h, then 4 ℃ are spent the night.Dialysed 3 days under 4 ℃ with 0.01mol/L PBS, change dislysate every day 3 times, to remove unreacted small-molecule substance, packing saves backup in-20 ℃.
C. the preparation of coating antigen-tetracycline medication haptens and OVA conjugate are synthetic
The tetracycline medication haptens of getting 18mg formula I compound is dissolved in 2ml DMF, obtains I solution, gets to add in I solution after 25mg EDC fully dissolves with 0.2ml water, stirs 24h under room temperature, can obtain reactant liquor II solution.Take OVA36mg, make it fully to be dissolved in 8ml PBS (pH7.2), dropwise slowly be added drop-wise in protein solution II solution, and stir 24h under room temperature, dialysed 3 days under 4 ℃ with 0.01mol/L PBS, change dislysate every day 3 times, to remove unreacted small-molecule substance.Packing saves backup in-20 ℃.
The evaluation of d, tetracycline medication hapten-carrier conjugates
Carrier protein, tetracycline medication haptens, tetracycline medication hapten-carrier protein conjugate 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, tetracycline medication haptens, tetracycline medication hapten-carrier protein conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of tetracycline medication haptens and carrier protein couplet.
2, the preparation of the monoclonal antibody of cephalosporins medicine and tetracycline medication
(1) preparation monoclonal antibody
A. animal immune
Two kinds of immunogenes that step 1 is obtained are injected into respectively in the Balb/c Mice Body, and immunizing dose is 150 μ g/, make 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 respectively the cephalosporins medicine monoclonal hybridoma strain of stably excreting cephalosporins medicine monoclonal antibody and the tetracycline medication monoclonal hybridoma strain of secretion tetracycline medication monoclonal antibody.
C. cell cryopreservation and recovery
Hybridoma is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, cultivates under 37 ℃ of conditions, with sad-saturated ammonium sulfate method, the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in the RPMI-1640 nutrient culture media, making the final concentration of calf serum in cell culture medium is 20% (quality percentage composition), and making the final concentration of sodium bicarbonate in cell culture medium is 0.2% (quality percentage composition); The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse antiantibody
As immune animal, as immunogene, the pathogen-free domestic sheep is carried out immunity take mouse source antibody with sheep, obtain the sheep anti mouse antiantibody.
4, the preparation of monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized waters that boil off, 1% gold chloride (is purchased from sigma company, catalog number T09041) be diluted to 0.01% (quality percentage composition), put to stir on magnetic force heating rod stirrer and boil, every 100ml 0.01% gold chloride adds the 2.5ml1% trisodium citrate (to be purchased from Guangzhou Chemical Reagent Factory, catalog number BG11-AR-01KG), continue agitating heating and react stopped heating when taking on a red color to liquid, 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 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 100-150 μ g antibody/ml collaurum adds respectively above-mentioned cephalosporins medicine monoclonal antibody and Tetracyclines monoclonal antibody in colloidal gold solution, continue to stir and evenly mix 30min, adding the final concentration of 10%BSA to BSA in colloidal gold solution is 1% (volumn concentration), standing 30min.12000rpm, 4 ℃ of centrifugal 30min, abandon supernatant, precipitation is with redissolving the damping fluid washed twice, the redissolution damping fluid that is initial collaurum volume 1/20 with volume will precipitate resuspended, the concentration of the cephalosporins medicine monoclonal antibody that obtains-colloid gold label thing solution and tetracycline medication monoclonal antibody-colloid gold label thing solution is respectively 50 μ g monoclonal antibody/ml solution, put 4 ℃ standby.
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, monoclonal antibody-colloid gold label thing micropore reagent freeze-drying is in capillary strip
Add 50 μ l cephalosporins medicine monoclonal antibody-colloid gold label things and 50 μ l tetracycline medication monoclonal antibody-colloid gold label things in capillary strip, put into freeze drier, condenser temperature is under-70 ℃ of conditions, after pre-freeze 4h, freeze-drying 14h again, can take out, in the capillary strip that obtains, freeze-drying has the micropore reagent of cephalosporins medicine monoclonal antibody-colloid gold label thing and tetracycline medication monoclonal antibody-colloid gold label thing.The freeze-drying amount of cephalosporins medicine specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml; The freeze-drying amount of tetracycline medication specific antibody-colloid gold label thing is 0.30-0.40 μ g/ml.
6, the preparation of absorption of sample pad
The absorption of sample pad is placed in contains that BSA (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 standby.
7, the preparation of reaction film
Coated process: with phosphate buffer, cephalosporins medicine-bovine serum albumin(BSA) conjugate being diluted to 10mg/mL, is 1.0 μ g/cm with Biodot point film instrument with detection line (B) package amount that it is coated on nitrocellulose filter
2With phosphate buffer, tetracycline medication haptens-bovine serum albumin(BSA) conjugate is diluted to 10mg/mL, with Biodot point film instrument, it is coated in detection line (T) on nitrocellulose filter, package amount is 1.5 μ g/cm
2With 0.01mol/L, pH7.4PBS damping fluid, sheep anti-mouse igg antibody is diluted to 200 μ g/ml, with Biodot point film instrument, it is coated in nature controlling line (C) on nitrocellulose filter, package amount is 1.0 μ g/cm
2Coated good reaction film is placed in dry 2h under 37 ℃ of conditions, standby.
(2) assembling of each parts
1, the assembling of test strips
Described absorption of sample pad, reaction film, adsorptive pads, diaphragm are pasted successively in order on described base plate; The end of absorption of sample pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads, and the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate; Paste diaphragm at absorption pad one end that assembles test strips.
2, kit assembling
Above-mentioned steps 1 is obtained test strips, micropore reagent, capillary strip and micropore plug be assembled into kit, store the term of validity 12 months in the environment of 2～8 ℃.
Two, the sensitivity of kit and specificity check
Kit of the present invention can detect Beta-lactam medicine and comprise: benzyl penicillin, nafcillin, ampicillin, cefoperazone, Amoxicillin, ceftriaxone, oxacillin, Ceftiofur, adjacent penicillin, cefalonium, two penicillin, Cefquinome; Can detect tetracycline medication comprises: tetracycline, terramycin, fortimicin, duomycin.
(1) sensitivity test
With Beta-lactam medicine: benzyl penicillin, nafcillin, ampicillin, cefoperazone, Amoxicillin, ceftriaxone, oxacillin, Ceftiofur, adjacent penicillin, cefalonium, two penicillin, Cefquinome and tetracycline medication: tetracycline, terramycin, fortimicin, duomycin standard items (available from German Dr and China Veterinery Drug Inspection Office) are diluted to following variable concentrations: 1,2,4 μ g/L benzyl penicillins; 1.5,3,6 μ g/L ampicillins; 2,4,8 μ g/L Amoxicillins; 3,6,12 μ g/L oxacillins, adjacent penicillin, two penicillin; 10,20,40 μ g/L nafcillin, Cefquinome; 20,40,80 μ g/L cefoperazones; 25,50,100 μ g/L ceftriaxones; 45,90,180 μ g/L Ceftiofurs; 5,10,20 μ g/L cefaloniums; 40,80,160 μ g/L tetracyclines, terramycin, fortimicin, duomycin.Dilution used is that pH is 7.2, the phosphate buffer of 0.2mol/L.
Detect with kit.Drip 200 μ l samples in the capillary strip that includes golden labeling antibody micropore reagent, mixing after hatching 5min, inserts test strips and detects at every turn, and result is: drip the examination Beta-lactam medicine: 1 μ g/L benzyl penicillin; 1.5 μ g/L ampicillin; 2 μ g/L Amoxicillins; 3 μ g/L oxacillins, adjacent penicillin, two penicillin; 10 μ g/L nafcillin, Cefquinome; 20 μ g/L cefoperazones; 25 μ g/L ceftriaxones; 45 μ g/L Ceftiofurs; 5 μ g/L cefaloniums; Tetracycline medication: when 40 μ g/L tetracyclines, terramycin, fortimicin, duomycin, demonstrate macroscopic three red bar lines on test strips, be negative; When dripping examination 2,4 μ g/L benzyl penicillins; 3,6 μ g/L ampicillins; 4,8 μ g/L Amoxicillins; 6,12 μ g/L oxacillins, adjacent penicillin, two penicillin; 20,40 μ g/L nafcillin, Cefquinome; 40,80 μ g/L cefoperazones; 50,100 μ g/L ceftriaxones; 90,180 μ g/L Ceftiofurs; 10, during 20 μ g/L cefalonium, the colour developing of test strips nature controlling line, detection line (B) does not develop the color, and is positive; Look tetracycline medication when dripping: 80, when 160 μ g/L tetracyclines, terramycin, fortimicin, duomycin, the colour developing of test strips nature controlling line, detection line (T) does not develop the color, and is positive; Show, this kit is respectively the Beta-lactam medicine detection sensitivity: benzyl penicillin 2 μ g/L, ampicillin 3 μ g/L, Amoxicillin 4 μ g/L, oxacillin 6 μ g/L, adjacent penicillin 6 μ g/L, two penicillin 6 μ g/L, nafcillin 20 μ g/L, Cefquinome 20 μ g/L, cefoperazone 40 μ g/L, ceftriaxone 50 μ g/L, Ceftiofur 90 μ g/L, cefalonium 10 μ g/L.This kit to the Tetracyclines detection sensitivity is: tetracycline, terramycin, fortimicin, duomycin are respectively 80 μ g/L.
(2) specific test
Specificity cross reacting rate commonly used represents, refers to the ability of the antigenic determinant generation combination that antibody is different from structure.This kit is to Beta-lactam medicine: benzyl penicillin, ampicillin, Amoxicillin, oxacillin, adjacent penicillin, two penicillin, nafcillin, Cefquinome, cefoperazone, ceftriaxone, cefalonium, Ceftiofur detection sensitivity are respectively 2 μ g/L, 3 μ g/L, 4 μ g/L, 6 μ g/L, 6 μ g/L, 6 μ g/L, 20 μ g/L, 20 μ g/L, 40 μ g/L, 50 μ g/L, 10 μ g/L, 90 μ g/L; This kit is to Tetracyclines: tetracycline, terramycin, fortimicin, duomycin detection sensitivity are respectively 80 μ g/L.With the other drug (clenbuterol, Ractopamine, salbutamol, melamine, Trenbolone acetate, sulfamido, chloromycetin, macrolides, aminoglycoside, fluoroquinolones) of normal inspection in milk with pH be 7.2, the phosphate buffer of 0.2mol/L dilutes.Detect with the kit described in embodiment 1, result shows that clenbuterol, Ractopamine, salbutamol, melamine, Trenbolone acetate, sulfa drugs, chloromycetin medicine, macrolides, aminoglycoside, fluoroquinolones are when 500 μ g/L concentration, nature controlling line of test strips and two detection lines all develop the color, and can draw this kit not to these medicine generation cross reactions.
The application of embodiment 3, test strips
One, detect beta-lactam antibiotic and TCs in milk with kit described in embodiment 1
Kit of the present invention can detect milk sample.General detection method is as follows:
1, detection method
The micropore plug that covers on capillary strip is taken off, drip the sample solution that needs detection in the capillary strip that includes micropore reagent, obtain the mixed solution 11 of micropore reagent and sample solution, after mixing, hatch 5min, there is MAX wire tag end to insert in capillary strip down test strips 12, watches result in 5min, as shown in Figure 7.
2, testing result is judged
Beta-lactam antibiotic or (with) concentration is greater than or equal to the kit lowest detection in limited time in sample for tetracycline medication, colloidal gold antibody and beta-lactam antibiotic or (with) TCs is combined, thereby in detection zone because competitive reaction not can with the cephalosporins medicine conjugate or (with) the Tetracyclines conjugate be combined and do not occur red detection line (B) or (with) detection line (T), be positive.When beta-lactam antibiotic or (with) concentration is lower than the kit lowest detection in limited time in sample for tetracycline medication, in testing process owing to lacking the antibody antigen competitive reaction, detection line (B) or (with) detection line (T) and nature controlling line (C) will present red strip tape.As shown in Figure 8.
Negative: nature controlling line (C) and detection line (B) and detection line (T) all demonstrate red stripes, are judged to feminine gender, with "-" expression.As shown in Fig. 8 a.
Beta-lactam medicine is positive: nature controlling line (C) and detection line (T) demonstrate red stripes, and detection line (B) does not develop the color, and is judged to the positive, with "+" expression.As shown in Fig. 8 b.
Tetracycline medication is positive: nature controlling line (C) and detection line (B) demonstrate red stripes, and detection line (T) does not develop the color, and is judged to the positive, with "+" expression.As shown in Fig. 8 c.
Beta-lactam and tetracycline medication all are positive: nature controlling line (C) demonstrates red stripes, and detection line (T) and detection line (B) all do not develop the color, and are judged to the positive, with "+" expression.As shown in Fig. 8 d.
Invalid: (C) do not show red stripes when nature controlling line, and no matter whether detection line (T) and detection line (B) red stripes occurs, and test strips all lost efficacy.As shown in Fig. 8 e1,8e2,8e3,8e4.
Following concrete example:
1, false positive rate is measured
50 parts of the negative milk samples of the LC-MS/MS that learnt from else's experience conclusive evidence are numbered 1#-50#, and sample is detected with test strips of the present invention, calculate its false positive rate.
Result: in 50 parts of negative milk samples were measured, the ELISA test strip result was total negative, and test strips false positive rate of the present invention is 0.
2, false negative rate is measured
160 parts of the negative milk samples of the LC-MS/MS that learns from else's experience conclusive evidence, difference detectability concentration is in accordance with regulations added benzyl penicillin, ampicillin, Amoxicillin, oxacillin, adjacent penicillin, two penicillin, nafcillin, Cefquinome, cefoperazone, ceftriaxone, cefalonium, Ceftiofur, tetracycline, terramycin, fortimicin, duomycin medicine, every kind of medicine adds each 10 duplicate samples, sample is detected with test strips of the present invention, calculate its false negative rate.
Result: in the mensuration of 160 parts of positive milk samples, the negative sample that ELISA test strip goes out is 0 part, and false negative rate is 0.The kit of detection beta-lactam antibiotic of the present invention and TCs can carry out fast detecting to beta-lactam and teracycline antibiotic residues simultaneously.
Claims (11)
1. kit that detects beta-lactam and TCs, it is characterized in that comprising test strips, capillary strip, micropore reagent and micropore plug, described test strips comprises reaction film, the absorption of sample pad, adsorptive pads, diaphragm, base plate, two detection lines and a nature controlling line are arranged on described reaction film, article two, detection line is coated with respectively cephalosporins medicine-carrier protein couplet thing and tetracycline medication hapten-carrier protein conjugate, nature controlling line is coated with antiantibody, the cephalosporins medicine specific antibody that described micropore reagent is freeze-drying-colloid gold label thing and Tetracyclines specific antibody-colloid gold label thing.
2. kit according to claim 1 is characterized in that described test strips is pasted on base plate successively by absorption of sample pad, reaction film, adsorptive pads, diaphragm to form, and has the micropore plug on the described capillary strip of claim 1.
3. kit according to claim 2, it is characterized in that: described diaphragm sticks on the absorption of sample pad of test strips, is the test side, and the above has the MAX mark line.
4. kit according to claim 1, is characterized in that described tetracycline medication detection line near absorption of sample pad one end, and nature controlling line is near adsorptive pads one end, and the Beta-lactam medicine detection line is between tetracycline medication detection line and nature controlling line.
5. kit according to claim 1, it is characterized in that: the tetracycline medication haptens is to utilize Mannich reaction by tetracycline and 4-piperidine carboxylic acid, has introduced N-methyl-resulting derivant of 4-piperidine carboxylic acid group on the amido of the acid amides of tetracycline.Molecular structure is suc as formula I:
6. kit according to claim 1, it is characterized in that: described cephalosporins medicine-carrier protein couplet thing is obtained by cephalosporins medicine and carrier protein couplet, described tetracycline medication hapten-carrier protein conjugate is obtained by tetracycline medication haptens and the carrier protein couplet of formula I compound, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
7. kit according to claim 1, it is characterized in that: the cephalosporins medicine specific antibody in cephalosporins medicine specific antibody-colloid gold label thing is to prepare as immunogene with cephalosporins medicine-carrier protein couplet thing, and described cephalosporins medicine specific antibody can be cephalosporins medicine monoclonal antibody or cephalosporins medicine polyclonal antibody; Tetracycline medication specific antibody in Tetracyclines specific antibody-colloid gold label thing is to prepare as immunogene with tetracycline medication hapten-carrier protein conjugate, and described tetracycline medication specific antibody can be tetracycline medication monoclonal antibody or tetracycline medication polyclonal antibody.
8. kit according to claim 1 is characterized in that: the micropore reagent of the cephalosporins medicine specific antibody of freeze-drying-colloid gold label thing and Tetracyclines specific antibody-colloid gold label thing is contained in described capillary strip bottom.
9. kit according to claim 1, it is characterized in that: described micropore plug can closely be filled in capillary strip.
10. method for preparing the described kit of claim 1-9 any one, it comprises step:
1) prepare respectively the micropore reagent that freeze-drying has cephalosporins medicine specific antibody-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing;
2) prepare respectively the reaction film with coated cephalosporins medicine-carrier protein couplet thing and nature controlling line of two detection lines that are coated with tetracycline medication hapten-carrier protein conjugate and coated antiantibody;
3) with 2) reaction film and absorption of sample pad, diaphragm, adsorptive pads, the base plate that prepare be assembled into test strips;
4) with 1) and 3) freeze-drying for preparing has micropore reagent, capillary strip, micropore plug and the test strips of cephalosporins medicine specific antibody-colloid gold label thing and tetracycline medication specific antibody-colloid gold label thing to be assembled into kit.
11. detection method that detects simultaneously beta-lactam antibiotic and teracycline antibiotic residues in milk sample, it is characterized in that directly test strips being inserted in the micropore of the mixed solution that includes test sample solution and micropore reagent and detect, it comprises step:
1) detect with the described kit of claim 1-9 any one;
2) analyzing and testing result.
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| CN105567645A (en) * | 2016-01-28 | 2016-05-11 | 江南大学 | Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof |
| CN106248894A (en) * | 2015-05-30 | 2016-12-21 | 北京艾旗斯德科技有限公司 | A kind of paper chip detecting Residue of Antibiotics in Milk |
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