CN1325908A - Process for preparing artificial methamidophos antigen - Google Patents

Abstract

A process for preparing artificial methamidophos antigen is disclosed. Said antigen is prepared by direct phosphorylation of O,S-dimethyl thiophosphoryl chloride with the active radical on side chain of carrier protein to couple them together in alkaline condition. Its advantages include simple operation, one-step reaction, and easy screening of specific antibody.

Description

Process for preparing artificial methamidophos antigen

The present invention relates to utilize the method for protein phosphorylation; with acephatemet (O; S-dimethyl thiophosphoryl ammonia) a kind of very active precursor product O in the production process; the direct crosslinked method for preparing artificial methamidophos antigen of S-dimethyl thiophosphoryl chloride and carrier molecule (protein) belongs to the immunological technique field.

In field of immunology for to make the small-molecule substance of non-immunogenicity (often be called haptens, Hapten) can bring out generation antibody in animal body, to be artificial antigen often with this haptens and certain macromolecular carrier such as crosslinked be combined into such as protein or polypeptide, be injected into mouse again and bring out generation antibody, after the purification antibody, promptly can be used for immunoassay, it is one of most active fields during current clinical medicine detection and food residue are analyzed.

As everyone knows, acephatemet is both at home and abroad widely used a kind of severe toxicity, efficient organophosphorus pesticide, once is the agricultural chemicals that China produces the tonnage maximum, insect is had intensive is tagged and stomach poison function, people and animals are also had very big hazardness, and the country that has bans use of.There is expressly restriction in China to the use of methamidophos pesticide, forbids using on emergency crops, as forbids using on vegetable crop.But the phenomenon of the poisoning that causes because of the edible too high food of methamidophos residue still happens occasionally, so its safety monitoring work of necessary reinforcement.

Detection method to acephatemet mainly contains vapor-phase chromatography, high performance liquid chromatography, single-sweep oscillographic polarography, thin layer chromatography, spectrophotometry, dirext potentiometry etc. at present, these methods need be used the instrument of complex and expensive, and every kind of method all will pass through loaded down with trivial details pre-treatment, and requirement is detected at the scene that is difficult to reach quick, easy.Medically detect similarly gold-marking immunity or enzyme-linked immunoassay method such as early pregnancy, hepatitis B and AIDS if can adopt to resemble, then can reach needs quick, sensitive and the on-the-spot detection of energy methamidophos residue.

In order to set up its immune analysis method, at first must obtain the antibody of anti-acephatemet.The acephatemet molecular structure is simple, and molecular weight (141) is little, belongs to haptens, can not direct immunization produce antibody.Must at first itself and carrier protein coupling be prepared its complete antigen, just can bring out animal and produce antibody.We once repeatedly attempted utilizing the intramolecular amino group of acephatemet to come synthesis of methylamines phosphorus artificial antigen with classical carbodlimide method (EDC) or glutaraldehyde method, but all unsuccessful.Analyzing reason mainly is because acephatemet intramolecularly amino is phosphinylidyne ammonia, is not primary amine group, does not have the cause of reactive behavior.The artificial antigen that reports such as domestic Liu Feng power have synthesized acephatemet with the EDC method, and extrapolate with uv scan that the mole ratio of haptens and carrier proteins is more than 3000 in the artificial antigen, according to our experiment experience, think that this is impossible.

State Patent Office in June, 95 disclosed application number be in 94105042.4 patents, introduced a kind of artificial antigen cross-linking method of phosphorus compound, this patent relates to and utilizes bridge construction L-Methionin and O-ethyl dichloro phosphoric acid ester condensing agents such as (EDCP) with organo phosphorous compounds haptens and the crosslinked method for preparing artificial antigen of carrier protein.The artificial antigen immune mouse that we prepare in this way, test the specific antibody that almost detects less than anti-acephatemet with suppressing ELISA, illustrate that this method is not suitable for the synthetic of artificial methamidophos antigen, infer that reason may be that long lysine bridge has weakened the avidity of antibody to acephatemet because the acephatemet molecule is too simple.And intramolecular one of them group of acephatemet is a methylthio group, and therefore, the acephatemet molecule is not included in the disclosure patent and mentions within the phosphorus compound scope.

The objective of the invention is to seek a kind of suitable reagent, synthesize the artificial antigen of acephatemet, for the specific antibody that filters out it later on set up immune analysis method the basis is provided with simple, practical method.

The present invention selects the precursor product O of synthesis of methylamines phosphorus for use, amino group on S-dimethyl sulfuration phosphoryl chloride and the protein is in alkaline aqueous solution, under the magnetic agitation, directly phosphorylation and coupling, after dialysis and lyophilize, get artificial methamidophos antigen, the banded small molecules has haptenic structure on the product, does not at all destroy and changes haptenic configuration.Above-mentioned protein can be bovine serum albumin (BSA), ovalbumin (OVA), hemocyanin (KLH) or human serum protein (HSA).

The present invention can be achieved like this:

Protein is made into 1～15% concentration with distilled water, in the protein soln of the 1-15% concentration that is made into, add triethylamine, making the protein in the protein soln of 1-15% and the mol ratio of triethylamine is 1: 600-1000, place the ice bath magnetic agitation after 30 minutes in protein and triethylamine mixing solutions, dropwise add the O that doubly dilutes with chloroform or toluene 3-5 in advance, S-dimethyl thiophosphoryl chloride diluent, O, the add-on of S-dimethyl thiophosphoryl chloride diluent is above-mentioned protein and triethylamine mixing solutions cubic capacity 0.2-0.5 a times, continue ice bath magnetic agitation reaction 30 minutes, then reaction solution is changed in the separating funnel, leave standstill treat to take out after the layering in the middle of water layer put into dialysis tubing, in distilled water,, changed water once in per 4 hours in 4 ℃ of dialysis, last lyophilize promptly obtains the synthetic artificial methamidophos antigen.

The present invention has easy and simple to handle, and a step is finished reaction; Do not use bridge construction, can not produce the antibody of levying bridge, therefore, make things convenient for the advantages such as screening of specific antibody.

After adding the 1.0ml triethylamine in example: 8ml 10% bovine serum albumin (BSA), the ice bath lower magnetic force stirred 30 minutes, dropwise add the O of 2.7ml then with 4 times of dilutions of chloroform, the S-dimethyl thiophosphoryl chloride continued reaction after 30 minutes, and reaction solution changes in the little separating funnel, behind the standing demix water layer changed over to dialysis tubing (by molecular weight 10000), with distill water dialysis 3 days, changed water once in per 4 hours therebetween, at last the dialyzate lyophilize is promptly got synthetic artificial antigen (being called for short BSAM).Replace BSA to synthesize envelope antigen (being called for short OVAM) with ovalbumin (OVA) with method.

The evaluation of artificial antigen:

With the concentration of molybdenum antimony resistance colorimetric method mensuration phosphorus, the haptens in the calculating artificial antigen and the molecule coupling ratio of carrier proteins (BSA) are 26.

Artificial immunization antigen BSAM and original vector protein B SA infrared spectrogram relatively have more 1027cm in the artificial antigen BSAM infrared spectrogram ^-1And 789cm ^-1Two absorption peaks, these two peaks are respectively γ P=O (vs) and γ P-N (s) charateristic avsorption band, illustrate further artificial antigen and synthesize successfully.

The immune animal proof has produced the antibody of anti-acephatemet: the experiment of (1) mouse immune is made into above-mentioned synthetic BSAM with 0.8% physiological saline the solution of 1.0mg/ml, immune for the first time with 0.5ml complete Freund's adjuvant and the above-mentioned BSAM balanced mix for preparing, after fully emulsified, every mouse (Balb/c mouse, 6 ages in week) injection 0.2ml, be equivalent to 100 μ g protein, altogether 4 mouse of immunity.Every 2 weeks, use the same method of incomplete Freund's adjuvant booster immunization 3 times again instead.Mouse tail blood was got in the 4th immunity in back 15 days, the preparation antiserum(antisera).(2) ELISA competition inhibition test every hole in the enzyme mark bar in every 12 hole adds the envelope antigen of 100 μ l with the carbonate buffer solution dilution of pH9.6, includes 2 μ g OVAM, after 37 ℃ of constant temperature are hatched 1h, and 4 ℃ of refrigerator overnight of dislocation.Taking-up is given a baby a bath on the third day after its birth time with PBST, and (PBST:PBS 20nmol/L, 0.05%Tween-20 pH7.4), dry.Therefrom getting three is placed on the enzyme plate, add (the PBS preparation of 50 μ l, two anti-diluents in every preceding 1-2 hole (totally 6 holes), include 0.1% gelatin), the 3-4 hole adds the standard acephatemet liquid 50 μ l with two anti-diluent preparing, include 5 μ g standard specimen acephatemets, the 5-6 hole is with the 3-4 hole, include 10 μ g standard specimen acephatemets, the 7-8 hole includes 20 μ g standard specimen acephatemets, the 9-10 hole includes 100 μ g standard specimen acephatemets, the 11-12 hole also adds 50 μ l, two anti-diluents, every preceding 10 holes all add 1: 200 above-mentioned antiserum(antisera) 50 μ l with two anti-diluent preparing then, the 11-12 hole still adds 50 μ l, two anti-diluents, be placed on 37 ℃ of constant temperature water tanks after adding and hatch 1h, take out, it is inferior to give a baby a bath on the third day after its birth with PBST, every then hole adds 1: 5000 the sheep anti mouse ELIAS secondary antibody of 100 μ l with two anti-diluent preparing, 37 ℃ of constant temperature water tanks are hatched 1h, taking-up is given a baby a bath on the third day after its birth inferior with PBST, add O-Phenylene Diamine and the hydrogen peroxide substrate solution of 100 μ l with pH5.0 phosphoric acid-citric acid preparation, after 15min was placed in 37 ℃ of constant temperature water tank sealings, every hole added 50 μ l, 10% sulfuric acid, read the OD value with the 492nm wavelength then on microplate reader, it is as follows to get 6 hole average computation results:

Competition suppresses the ELISA experimental result

Hole count	????1-2	??3-4	??5-6	??7-8	?9-10	?11-12
							The OD value	???1.41	?1.05	?0.88	??0.69	?0.51	?0.08

Last table data declaration: the color that adds the acephatemet hole is all of light color than the hole that does not add acephatemet, comparison is according to dark, and along with increasing that the standard acephatemet adds, the color of enzyme-to-substrate reaction becomes more and more shallow, illustrate in the antiserum(antisera) exist can with the antibody of acephatemet reaction, thereby the proof mouse has produced the specific antibody of anti-acephatemet, proves that further artificial antigen synthesizes successfully.

Claims (3)

1. artificial methamidophos antigen synthetic method, it is characterized in that using O, the S-dimethyl thiophosphoryl chloride as amino based on this antigenic synthetic precursor and the protein because of in alkaline aqueous solution, under the magnetic agitation, directly phosphorylation and coupling synthesis of methylamines phosphorus artificial antigen, during preparation, protein is made into the 1-15% protein soln with distilled water, in protein soln, add triethylamine, making the protein in the 1-15% protein soln and the mol ratio of triethylamine is 1: 600-1000, protein soln magnetic agitation after 30 minutes in ice bath, dropwise add dilution 3-5 O doubly, S-dimethyl thiophosphoryl chloride diluent, its add-on be protein and triethylamine mixing solutions cubic capacity 0.2-0.5 doubly, continue ice bath magnetic agitation reaction 30 minutes, the dialysis of water intaking layer is 3 days behind the standing demix, again the dialyzate lyophilize is promptly got the synthetic artificial methamidophos antigen.

2. according to the said artificial methamidophos antigen synthetic method of claim 1, it is characterized in that carrier proteins can be bovine serum albumin (BSA), ovalbumin (OVA), hemocyanin (KLH) or human serum protein (HSA);

3. according to the said artificial methamidophos antigen synthetic method of claim 1, it is characterized in that haptens synthesizes precursor O, the S-dimethyl thiophosphoryl chloride can be made diluting solvent with chloroform or toluene.