CN102234333A - Establishment of preparation and rapid detection method for polyclonal antiserum of DDT (dichlorodiphenyltrichloroethane) metabolite DDA (2,2-bis(4-chlorophenyl)acetic acid) - Google Patents

Abstract

The invention relates to a detection technology of organochlorine pesticide DDT (dichlorodiphenyltrichloroethane) metabolites, specifically relates to an anti-organochlorine pesticide DDT metabolite 2,2-bis(4-chlorophenyl)acetic acid (DDA) polyclonal antiserum prepared by synthesizing an antigen through protein coupling, and relates to a preparation method of a rapid detection kit capable of analyzing the DDA content in an environmental sample by utilizing the binding of a polyclonal antibody and a DDA antigen. An enzyme-linked immunosorbent assay kit for the rapid detection of organochlorine pesticide DDT metabolite 2,2-bis (4-chlorophenyl)acetic acid comprises a 96-well enzyme label plate detachable slat, an enzyme-labeled second antibody, a Tween-20-containing buffer salt washing liquid, a substrate liquid and a stopping liquid. A synthetic protein conjugate coated antigen is coated on the 96-well enzyme label plate by utilizing the prepared anti-serum containing anti-DDA-specific polyclonal antibody. The kit provided by the invention has the advantages of simple pre-treatment, easy operation process, low cost and price and the like, is suitable for on-site, rapid and large-batch sample detection, and has a detection limit less than 21 ng/ml.

Description

Polyvalent antibody preparation and the method for quick of dichlorodiphenyl trichloroethane metabolite DDA are set up

Affiliated technical field

The present invention relates to the detection technique of organochlorine pesticide dichlorodiphenyl trichloroethane metabolite, particularly relate to a kind of albumen coupling synthetic antigen that utilizes, prepare anti-organochlorine pesticide dichlorodiphenyl trichloroethane metabolite 2, the polyclonal antiserum of two (4-chloro-phenyl-) acetate (DDA) of 2-, and relate to and utilize polyclonal antibody to combine, and the method for making of the quick and convenient detection kit of the DDA content in the analyzing and testing environmental sample with DDA antigen.

Background technology

DDT was once used in the 1950's in a large number as broad spectrum pesticide.The organic synthesis sterilant of the chloride atom of this class, have high fat-soluble and stable, the very high concentration of enrichment in vivo, degraded slowly belongs to persistence organic pollutant in environment.The two kind main resolvents of DDT in settling are 2, two (4-the chloro-phenyl-)-vinylidene chlorides (DDE) and 2 of 2-, two (the 4-chloro-phenyl-s)-1 of 2-, 1-ethylene dichloride (DDD); DDT can slowly be degraded to 2 in animal tissues, two (4-chloro-phenyl-) acetate (DDA) of 2-.Itself does not accumulate DDA, but exposes the index that contacts as the human or animal with DDT, has reported that the content of DDA in the urine can expose the sensitive index that contacts DDT as people or other Mammalss.DDT and meta-bolites DDD, DDE etc. belong to environment incretion interferent matter, nerve and the reproductive system of infringement animal, long-term exposure has infringement to liver, therefore banned use of by developed country the seventies, but because it is cheap, it is remarkable to kill the mosquito effect, so far in some developing countries, comprise that indivedual areas of China are still being used; The hydroxyl homologue kelthane (Dicofol) of DDT is not disabled as yet as agricultural insecticide, and it contains and is not less than 5% DDT; Therefore in food, environmental sample in addition south poles still can detect DDT and its metabolite.The conventional method of analysis of organochlorine pesticide dichlorodiphenyl trichloroethane DDT and metabolite thereof at present mainly contains gas-chromatography in conjunction with mass spectrum or electron capture detector method (GC/MS, GC/FLD), this method not only needs the analyst of expensive plant and instrument and specialty, and the pre-treatment process very complicated of sample, be difficult for satisfying the check and analysis needs of batch samples, also be not suitable for on-the site analysis.The immuno analytical method that is rapidly developed in recent years, with its high specificity, highly sensitive, easy and simple to handle, detect rapidly and the low advantage of analysis cost is widely used in the analysis of various organic pollutants.Immuno-chemical method is to make specific antibody (polyclonal antibody or monoclonal antibody) by immune animal, utilize antibody to antigenic association reaction to be detected, make detection kit according to enzyme-linked immune analytic method, the analyzing and testing that is used for DDT and metabolite thereof, this method is easy, quick, can analyze batch samples simultaneously.Set up the Fast Detection Technique and the product of organochlorine pesticide dichlorodiphenyl trichloroethane metabolite,, particularly have very significant meaning for ensuring food safety to understanding the residual of organochlorine pesticide dichlorodiphenyl trichloroethane.

Summary of the invention

The objective of the invention is to overcome the deficiency of above-mentioned DDT and metabolite detection technique thereof, the enzyme-linked immunologic detecting kit of one of a kind of special, responsive and easy rapid detection DDT metabolite DDA is provided; For the scene that solves DDA, fast, the problem analysis of batch samples, the present technique invention provides a kind of anti-2, the preparation of the polyclonal antiserum of two (4-chloro-phenyl-) acetate of 2-, and utilize the indirect competitive enzyme-linked immunosorbent of the polyclonal antibody development of anti-DDA to analyze 2, the test kit of two (4-chloro-phenyl-) acetate of 2-, simple to operation, be suitable for the scene, fast, the detection of batch samples.

The present technique invention solves the technical scheme that its technical problem adopted: use the water-soluble carbodiimide method, press certain reaction ratio with 2, two (4-chloro-phenyl-) acetate (DDA) of 2-are coupled on the bovine serum albumin (BSA), and through dialysis purification conjugate, cryogenic freezing is preserved after the packing.30-100 μ g the time is got in immunity, and to add adjuvant fully emulsified, subcutaneous multiple spot intramuscular injection immunity New Zealand white rabbit, booster immunization for several times after, obtain the blood of immunize rabbit, the centrifugal anti-DDA polyclonal antiserum (containing the anti-DDA polyclonal antibody of specificity) that makes by heart; Wrap by 96 hole enzyme plates with synthetic DDA-ovalbumin (DDA-OVA), add DDA standard substance or sample, the DDA polyclonal antibody Pab-DDA that adds dilution again, envelope antigen DDA and the antibody of DDA competition freely Pab-DDA, DDA and antibody complex are by flush away freely, close with two resistive connections of horseradish peroxidase-labeled again with envelope antigen DDA bonded antibody Pab-DDA, develop the color through substrate, termination reaction, measure the light absorption value (OD) in each hole with microplate reader, the OD value is big more, and DDA content is few more freely in the sample, regressive equation of reference standards or curve, the content of DDA in the calculation sample.

The present technique beneficial effect of the invention is: utilize the antiserum(antisera) that contains the anti-DDA polyclonal antibody of specificity that makes, and with low cost, the low price of the enzyme-linked immunologic detecting kit of being developed, the detection limit of this test kit is less than 21ng/ml.

Compared with prior art, the present invention has following outstanding advantage: the enzyme-linked immunologic detecting kit of detection organochlorine pesticide DDT metabolite DDA of the present invention, has high specificity, detection sensitivity height (can reach 21ng/mL), detect fast, pre-treatment is simple, and it is low to detect cost, advantages such as easy grasp simple to operate.

Embodiment

Embodiment 1 (preparation embodiment)

The Antiserum Preparation method of the anti-DDA polyclonal antibody of specificity

1, the preparation of DDA-carrier protein couplet thing

With 2, two (4-chloro-phenyl-) acetate (DDA) of 2-are dissolved in the dimethyl sulfoxide (DMSO), add water-soluble carbodiimide (EDC), N-hydroxy-succinamide (NHS), 25 ℃ of reactions are after 2 hours, add the bovine serum albumin (BSA) or the ovalbumin (OVA) that are dissolved in (pH 9.6) in the 50mM carbonate buffer solution that contains KCL, 25 ℃ of reactions are stirred and are spent the night.With the PBS 3d that dialyses, 4 ℃, every 12h changes liquid 1 time with reaction mixture.After the dialysate packing ,-20 ℃ of preservations.Prepare DDA-BSA and the DDA-OVA conjugate is standby.

2, the preparation of anti-DDA polyclonal antiserum

Utilize 2 of preparation, two (4-chloro-phenyl-) acetate of 2--bovine serum albumin conjugate (DDA-BSA) is as immunogen, immune new zealand white rabbit.First immunisation adopts 400 μ L DDA-BSA and equal-volume complete Freund's adjuvant, after emulsification, takes ear, the back of the body and oxter multi-point injection method immunity.After this, after DDA-BSA that gets same dosage the 3rd, 5,7,9 weeks and the emulsification of equal-volume incomplete Freund's adjuvant, carry out the multi-point injection immunity respectively.Get rabbit ear blood and carry out the polyclonal antibody titration.Tire reach required value after, get subcutaneous multi-point injection booster immunization with the DDA-BSA of dosage, 3d places sedimentation after get blood by rabbit hearts, and is centrifugal, gets upper serum, standby in-20 ℃ of preservations after the packing.

Embodiment 2 (Application Example)

Detect 2, the enzyme linked immunological kit using method of two (4-chloro-phenyl-) acetate (DDA) of 2-

1, The pretreatment such as shellfish

Take by weighing shellfish homogenate 20g, add water 5ml (look its moisture content and add water, make the about 20ml of Total Water), add acetone 40ml, vibration 30min adds calcium chloride 6g, shakes up.Add sherwood oil 30ml, the 30min that vibrates again, standing demix.Get supernatant liquor 35ml through anhydrous sodium sulfate dehydration, in rotatory evaporator, be concentrated near doing, be settled to 5ml with sherwood oil, adding vitriol oil 0.5ml purifies, jolting 0.5min in the centrifugal 15min of 3000r/min, is concentrated near doing once more in rotatory evaporator, after dissolving extract fully with 5ml PBS, get supernatant liquor analysis.

2, detect

The DDA-OVA conjugate as envelope antigen, is added 96 hole enzyme mark versions (100 μ L/ hole) with carbonate buffer solution (pH 9.6) dilution back, and 4 ℃ are spent the night in the wet box; Discard raffinate, clean 3 times through washing lotion (PBS-Tween20), after patting dry, every hole adds the PBS solution 300 μ L that contain 1%PVA and hatch sealing 3h under 37 ℃, has sealed and has been placed on standby in 4 ℃ of refrigerators (can preserve several weeks).During mensuration, discard confining liquid, wash plate 3 times, pat dry, add the DDA reference material solution or the shellfish sample extraction liquid of 50 μ L serial dilutions, add the suitably anti-DDA polyvalent antibody of specificity of dilution of 50 μ L again, the jolting mixing is hatched 1h for 37 ℃; Wash plate, pat dry, add the suitably goat-anti rabbit second antibody of the HRP mark of dilution, every hole 100 μ L are hatched 40min for 37 ℃; Wash plate, pat dry, every hole adds substrate solution 100 μ L, and 37 ℃ of colour developing 12min are with 2mol/L H ₂SO ₄Stopped reaction (the every hole of 50 μ L/), 450nm wavelength measurement absorbance (OD).To absorbance drawing standard curve, the absorbance in the absorbance of sample and standard orifice and blank hole relatively draws the content of DDA with the concentration of DDA standard substance.

Embodiment 3 (Application Example)

Detect 2, the indirect competitive enzyme-linked immunosorbent The Application of Technology of two (4-chloro-phenyl-) acetate of 2-

1, DDA specificity polyvalent antibody is tired and the experiment of the suitableeest working concentration

After the immunity beginning, get blood from rabbit ear portion respectively, adopt direct ELISA method to measure serum titer (tiring) in fourth, fifth, six, seven weeks.After the immunity 4 times, tire and to reach more than 1,200,000.Through the square formation burette test, in direct ELISA method,, get absorbance 1.0～1.5 scopes according to the polyvalent antibody absorbance of different multiples dilution, the suitableeest thinning ratio that draws envelope antigen is 1: 2000; The suitableeest thinning ratio of goat-anti rabbit ELIAS secondary antibody is 1: 12000, and measuring the suitableeest thinning ratio of serum by direct ELISA method is 1: 10 ten thousand, and the work thinning ratio is 1: 5 ten thousand.

2, recovery test

In sample, add the DDA standard respectively, concentration after it is finally extracted is 20ng/ml, 200ng/ml, and each concentration is done 3 replicate(determination)s, carries out the DDA assay with the DDA indirect competitive enzyme-linked immunosorbent method of setting up, compare with the DDA amount of adding, calculate the rate of recovery.In 20～200ng/ml concentration range, the rate of recovery of DDA is 90.05%～106.38%.Can satisfy the requirement of quantitative analysis detection method fully.

Claims (6)

1. polyclonal antiserum.

2. discern organochlorine pesticide dichlorodiphenyl trichloroethane metabolite 2 specially for one kind, the polyclonal antibody of two (4-chloro-phenyl-) acetate (DDA) of 2-, it is contained by the described polyclonal antiserum of claim 1.

3. contain right and require 1 or 2 test kit, it is characterized in that, described test kit is to detect 2, the test kit of two (4-chloro-phenyl-) acetate of 2-.

4. rapid detection organochlorine pesticide dichlorodiphenyl trichloroethane metabolite 2, the enzyme-linked immunologic detecting kit of two (4-chloro-phenyl-) acetate (DDA) of 2-, comprise that the removable lath of 96 hole enzyme plates, enzyme labelling two resist, contain buffering salt washing lotion, substrate solution and the stop buffer of tween 20, is characterized in that: the envelope antigen that on the removable lath of 96 hole enzyme plates, has been coated with the synthetic protein conjugate.

5. the described a kind of rapid detection 2 of claim 4, the preparation method of the indirect competitive enzyme-linked immunosorbent detection kit of two (4-chloro-phenyl-) acetate (DDA) of 2-, its step comprises:

(1) with 2, two (4-chloro-phenyl-) acetate and the carrier protein couplets of 2-prepare 2, two (4-chloro-phenyl-) acetate of 2--carrier protein couplet thing;

(2) with 2, two (4-chloro-phenyl-) acetate of 2--bovine serum albumin conjugate immunity New Zealand white rabbit obtain resisting 2 the polyclonal antiserum of two (4-chloro-phenyl-) acetate of 2-;

(3) with 2, two (4-chloro-phenyl-) acetate of 2--ovalbumin conjugate are coated on the 96 hole enzyme plates, the confining liquid sealing;

(4) biological sample extraction liquid, blank damping fluid or DDA reference liquid are joined in the hole that (3) make, add the polyclonal antiserum that contains specific antibody that (2) make again;

(5) answer through incubation reaction, ELIAS secondary antibody mark, substrate color reaction and termination side, obtain described detection 2, the enzyme linked immunological kit of two (4-chloro-phenyl-) the acetate DDA of 2-.

6. each described test kit of claim 3-4 is detecting 2, the application among two (4-chloro-phenyl-) the acetate DDA of 2-.