CN100401067C - B type hepatitis virus antibody bridge type double-antigen sandwich ELISA detecting method - Google Patents

Abstract

The present invention relates to an antibody detection method of a hepatitis C virus (Hepatitis C virus which is short for HCV), which belongs to an ELISA detection method of antibodies in blood serum (pulp) in the technical field of disease diagnosis. An HCV antigen (which is short for HCVAg) and a section of peptide or protein (pontin protein) are connected by a gene engineering method or a chemical coupling method, wherein the pontin protein can react with an enzyme labelling object which has affinity with the pontin protein. The HCVAg antigen can form a compound of the HCV antigen, the pontin protein and the enzyme labelling object through the pontin protein and the enzyme labelling object to realize the indirect enzyme labelling of the HCVAg. The HCVAg antigen and the coating antigen can carry out the ELISA detection of a double-antigen sandwich method on HCVAb in the blood-serum (pulp).

Description

Antibody of HCV bridge-type double antigens sandwich ELISA detection method

The present invention relates to preparation method and labeling method that dual-antigen sandwich method detects antibody tense marker antigen in the serum (slurry).The ELISA detection method that belongs to the middle antibody of serum (slurry) in the medical diagnosis on disease field.

When known enzyme linked immunological solid phase assays (ELISA) dual-antigen sandwich method detects antibody, labelled antigen need carry out enzyme labeling, generally the method for Shi Yonging is: the aldehyde radical after the free amine group of labelled antigen and enzyme are activated carries out coupling reaction, or by bifunctional group such as glutaraldehyde the free amine group of labelled antigen and enzyme is carried out coupling reaction and form covalently bound enzyme-labelled antigen, enzyme-labelled antigen and envelope antigen are formed dual-antigen sandwich method reagent, can be to the IgG in the serum (slurry), IgM detects simultaneously, its advantage is: one, highly sensitive, serum (slurry) need not dilute or extension rate few (only needing usually 1: 1 or dilution in 1: 2); Two, recall rate height, IgM infecting early stage the appearance, can be realized early diagnosis usually, shorten the window phase that detects; Three, high specificity is compared with indirect ELISA, and the bag quilt is specific antigen with labelled antigen entirely, can reduce nonspecific reaction.Commercial kit with this method preparation has hepatitis B surface antibody dual-antigen sandwich method ELISA reagent, human immune defect virus antibody (I+II type) dual-antigen sandwich method ELISA reagent etc. at present.

But, up to the present, indirect elisa method is still used in the inspection-free survey of the enzyme of HCVAb, the enzyme labeling thing is that enzyme is marked anti-human IgG or enzyme is marked anti-people IgM, its main cause is that HCV core space antigen is rich in lysine and arginine (amino acid of free amine group is provided), if by HCVAg: the ratio of enzyme=1: 1 carries out mark, the coupling position overwhelming majority of HCVAg and enzyme occurs in the core space of HCVAg, rather than resemble stochastic distribution other antigen, enzyme combines the back and forms sterically hindered with the free amine group of HCVAg core space, hinder HCVAg core space antigenic determinant to combine with its corresponding this partial antibody, and the HCVAg core space is the dominant antigen determinant of HCVAg, like this, directly the dual-antigen sandwich method HCVAb ELISA detectable of mark HCVAg composition is understood omission HCVAg core space antibody positive sample usually, and HCVAb indirect ELISA reagent itself has unsurmountable shortcoming: at first, sensitivity is low, because being enzyme, the enzyme labeling thing marks anti-human IgG or enzyme is marked anti-people IgM, for fear of human IgG or IgM non-specific adsorption, serum (slurry) sample must dilute more than 1: 10 or 1: 20, reaction sensitivity and the dual-antigen sandwich method that does not need dilute serum (slurry) sample cause omission easily than hanging down 10～20 times naturally; The second, poor specificity, even serum (slurry) diluted through 1: 10～1: 20, IgG in indivedual samples or IgM are still had non-specific adsorption or bag are had specific adsorption by the foreign protein in the reaction plate by reaction plate bag.And anti-human IgG or anti-people IgM enzyme mark do not have resolution capability to the HCV antibody of specific adsorption and the human IgG or the IgM of alternate manner absorption, must cause a part of false positive; The 3rd, can not or be inconvenient to use a cover reagent to detect IgG and IgM simultaneously generally speaking, the commercialization HCVAb indirect elisa method detection kit overwhelming majority only detects IgG at present, might cause the part omission of IgM positive sample separately like this.

The objective of the invention is: develop HCVAb dual-antigen sandwich method ELISA reagent and substitute indirect ELISA reagent, the drawback that overcome that indirect elisa method sensitivity is low, poor specificity and IgG, IgM can not detect simultaneously.

The present invention is achieved in that by gene engineering method or chemical coupling method and obtains a kind of fusion, this fusion is made up of two parts: a part is HCVAg, can with serum (slurry) in the HCVAb specific reaction, a part is one section peptide or protein, be referred to as pontin protein, pontin protein is of a great variety, can be certain antigen (or antigenic determinant), also can be certain aglucon.The antibody of pontin protein or part are carried out marks such as horseradish peroxidase (HRP) or alkaline phosphatase can form the enzyme labeling thing.Because between antigen and the antibody, between part and the aglucon affinity is arranged, pontin protein and HCVAg are fusion, by a step or a multistep reaction, can form the compound of HCVAg-pontin protein-enzyme labeling thing, realize the indirect labelling of HCVAg, overcome the shortcoming of the HCVAg core space antibody omission that direct mark causes, above-mentioned compound can be formed HCVAb dual-antigen sandwich method ELISA detectable with the HCV envelope antigen.

Good effect of the present invention:

One, highly sensitive: serum (slurry) sample does not dilute or only needs dilution in 1: 1,1: 2, improves about ten times than indirect ELISA sensitivity.

Two, shortening window phase: IgM, IgG can detect simultaneously, and IgM generally appears at the early stage of infection or virus breeding active stage, generally occurs 7 to 10 days than IgG is early, and dual-antigen sandwich method can improve and detected the infected in 7 to 10 days.

Three, high specificity: because envelope antigen and bridge-type labelled antigen all are specific, indirect ELISA is some inevitable non-specific sample overwhelming majority can be excluded, and improves the accuracy of clinical diagnosis.

Four, social benefit is good: the present HCV infection rate of China promptly has up to ten million HCV carrier 1%～2%, and HCVAb is one of blood used in clinic or blood product essential items for inspection.Because the existence of the intrinsic drawback of indirect ELISA, even passed through the detection of HCVAb indirect ELISA, hepatitis C still happens occasionally after the blood transfusion that clinical blood transfusion (or blood product) causes.Dual-antigen sandwich method can greatly reduce this part hepatitis C.

Principle of the present invention, purpose, realizing route and positive effect more than have been described in detail in detail, because antigen (or antigenic determinant) and corresponding antibody, aglucon is extremely various with corresponding ligand species, even innumerable in theory, with following example in detail the present invention is described in detail:

One, the acquisition of envelope antigen:

The gene clone that contains HCVAg is in pET-22b (+) (U.S. Novagen company product) expression plasmid, called after pET-22b/HCV, with pET-22b/HCV transformed into escherichia coli BL21 (DE3), coating contains the LB flat board of ampicillin, 37 ℃ of growths, treat that bacterial plaque grows after, the single clone of picking is inoculated in the LB nutrient culture media test tube that contains ampicillin, 37 ℃, the 200rpm grow overnight.Second day, be inoculated at 1: 100 in the fresh LB nutrient culture media that contains ampicillin, 37 ℃ grow into OD ₆₀₀Between=0.6～0.9.Press final concentration 0.5mmol/L and add 1mol/L IPTG, 37 ℃ of 200rpm induced 4 hours, 5000rpm, 4 ℃ centrifugal 10 minutes, thalline multigelation three times, ultrasonic disruption.10000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, be precipitated as inclusion body, inclusion body is with containing 1% Triton-X ₁₀₀Washing once, centrifugal the same, with an amount of pH8.0 0.02mol/L PB 8mol/L urea buffer solution dissolving inclusion body, centrifugal the same, with pH8.00.02mol/L PB 8mol/L urea buffer solution balance CM cellulose ion exchange column, the inclusion body that has dissolved is crossed post, HCVAg is adsorbed, remove foreigh protein removing with the damping fluid washing that contains 0.1mpl/L NaCl pH8.0 0.02mol/L PB 8mol/L urea, with the buffer solution elution that contains 0.5mol/L NaCl pH8.0 0.02mol/L PB8mol/L urea, the Ni-metal affinity column damping fluid balance of 0.5mol/L NaCl H8.0 0.02mol/LPB 8mol/L urea, the HCVAg that merges CM post wash-out, last Ni-affinity column is removed foreign protein with the 0.5mol/LNaCl pH8.0 0.02mol/L PB 8mol/L urea buffer solution washing that contains the 25mmol/L imidazoles; Obtain pure HCVAg with the 0.5mol/L NaCl pH8.00.02mol/L PB 8mol/L urea buffer solution wash-out that contains the 100mmol/L imidazoles, be diluted to 0.5mg/ml with an amount of pH8.0 0.02mol/LPB8mol/L urea buffer solution ,-20 ℃ frozen.

Two, pontin protein--the acquisition of iron thioredoxin (trx)

PET-32a (U.S. Novagen company product) transforms BL21 (DE3), and coating contains the LB flat board of ampicillin, 37 ℃ of growths, treat that bacterial plaque grows after, picking is single clones in the LB nutrient culture media test tube that contains ampicillin, 37 ℃, the 200rpm grow overnight.Second day, be inoculated in fresh contain in ampicillin LB nutrient culture media at 1: 100,37 ℃ grow into OD ₆₀₀Between=0.6～0.9.Press final concentration 0.5mmol/L and add 1mol/LIPTG, 37 ℃ of 200rpm, induced 4 hours, 5000rpm, 4 ℃ centrifugal 10 minutes, with pH8.00.02mol/L PB 0.5mol/L NaCl damping fluid once with the thalline washing, centrifugal the same, thalline multigelation three times, ultrasonic disruption, centrifugal back supernatant is crossed Ni-metal affinity column, removes foreign protein with the pH8.00.02mol/L PB 0.5mol/L NaCl damping fluid washing that contains the 50mmol/L imidazoles, obtain pure iron thioredoxin trx with the pH8.0 0.02mol/L PB 0.5mol/L NaCl buffer solution elution that contains the 100mmol/L imidazoles, after the packing-20 ℃ frozen.

Three, the acquisition of HCVAg-trx fusion

The gene clone that contains HCVAg transforms BL21 (DE3) in pET-32a (U.S. Novagen company product), coating contains the LB flat board of ampicillin, 37 ℃ of growths, treat that bacterial plaque grows after, picking is single clones in the LB nutrient culture media test tube that contains ampicillin, 37 ℃, the 200rpm grow overnight.Second day, be inoculated in fresh contain in ampicillin LB nutrient culture media at 1: 100,37 ℃ grow into OD ₆₀₀Between=0.6～0.9.Press final concentration 0.5mmol/L and add 1mol/L IPTG, 37 ℃ of 200rpm, induced 4 hours, 5000rpm, 4 ℃ centrifugal 10 minutes, with pH8.0 0.02mol/LpB 0.5mol/L NaCl damping fluid once with the thalline washing, centrifugal the same, thalline multigelation three times, ultrasonic disruption, centrifugal back supernatant is crossed Ni-metal affinity column, removes foreign protein with the pH8.0 0.02mol/LPB 0.5mol/L NaCl damping fluid washing that contains the 50mmol/L imidazoles, obtain pure HCVAg-trx fusion with the pH8.0 0.02mol/LPB 0.5mol/L NaCl buffer solution elution that contains the 100mmol/L imidazoles, after the packing-20 ℃ frozen.

Four, the preparation of goat-anti trx antibody

After trx behind the purifying dialyses to physiological water, it is mixed to get 0.2mg and equal-volume Fu Shi Freund's complete adjuvant, with the complete emulsification of emulsifier, one of the subcutaneous multiple spot immunity 5Kg left and right sides, back goat, after three weeks, use freund 's incomplete adjuvant instead, the subcutaneous immunity once more in 0.4mg trx back, after immune 4 weeks for the second time, get 2mg trx ear vein booster immunization, get blood from ear vein after 10 days, SRID is surveyed antibody titer, if tire greater than 32, then, obtain the trx polyvalent antibody, if tire less than 32 from the arteria carotis blood sampling, use booster immunization after two weeks again, until obtaining satisfied tiring with dosage trx.

Five, the mark of the horseradish peroxidase of goat-anti trx IgG (HRP)

Goat-anti trx polyvalent antibody is with 50%, 33% ammonium sulfate precipitation secondary, precipitation is dialysed to 0.01mol/LpH8.00.1mol/L NaCl, hand over the post purifying with the DE52 ion, obtain pure anti-trx IgG, will resist trx IgG to be concentrated into 5-6mg/ml, to pH9.5 0.01mol/L carbonic acid buffer (CB) dialysed overnight, press IgG: HRP=1.5: 1 mass ratio, get an amount of HRP, after with sodium periodate method HRP being activated, to 1mmol/LpH4.0 acetate buffer solution dialysed overnight.Second day, press IgG: HRP=1.5: the HRP after with IgG and activation is mixed for 1 mass ratio, adjust potpourri pH=9.2 with an amount of pH9.5 0.2mol/L CB, adjust volume to IgG reaction density with pH=9.5 0.01mol/LCB and equal 2.0mg/ml, reacted 2 hours 25 ℃ of dark places, to react homomixture then and reduce to 4 ℃, press the amount adding 0.1mol/LNaBH that 1mg HRP adds 51ul ₄, 4 ℃ of reactions stopped coupled reaction in 2 hours.The dress bag filter changed liquid once in middle per 6 hours to pH7.0 0.02mol/L PB 0.2mol/L NaCl dialysis 48 hours.Add equal-volume glycerine mixing after dialysis is finished, deposit for-20 ℃, promptly obtain goat-anti trx-IgG-HRP enzyme labeling thing.

Six, the bridge-type dual-antigen sandwich method detects HCV Ab

0.5mg/ml 1: 1000 usefulness pH9.5 of HCVAg 0.05mol/L CB dilution, the 100ul/ hole adds enzyme reaction plate, 4 ℃ of bags are spent the night, and second day, cleansing solution (pH7.0 0.01mol/L PB 0.1mol/L NaCl0.1%tween-20) was washed plate once, add the pH7.0 0.01mol/LPB shrouding that contains 5% calf serum by the 115ul/ hole, 4 ℃ of sealings are spent the night, the shrouding liquid that exhausts then, 37 ℃ of dryings 2 hours, add drying agent and be packaged in the aluminium foil bag, bag is finished.The PBS that in wrapping, adds 50ul pH7.0 0.01mol/L PB0.1mol/L NaCl during detection earlier by reacting hole, add 50ul serum to be checked then, negative, positive control, 37 ℃ of incubations 20 minutes, wash plate five times with cleansing solution, pat dry, with pH7.0 PBS dilution in 1: 500 0.5mg/mlHCVAg-trx, the 100ul/ hole adds reaction plate, 37 ℃ of incubations 20 minutes, and it is the same to wash plate, pat dry 1: 3000 usefulness of back adding and contain goat-anti-trx-IgG-HRP that 20% calf serum PBS dilutes, 37 ℃ of incubations 20 minutes, it is the same to wash plate, adds developer A, each 1 of B liquid, 37 ℃ were developed the color 10 minutes, and developer A contains H ₂O ₂, developer B contains TMB, and after colour developing was finished, every hole added 1 2mol/L H ₂SO ₄Cessation reaction reads the result with microplate reader at the 450nm wavelength, and the person is positive for OD450nm 〉=2.1 * negative control OD mean value, and OD450nm＜2.1 * negative control mean value is negative.

Claims (1)

1. antibody of HCV enzyme-linked immune detection method, it is characterized in that labelled antigen is a fusion, fusion is formed by connecting by gene engineering method or chemical coupling method by HCVAg and pontin protein, pontin protein is one section peptide or protein, has affinity with the enzyme labeling thing, pontin protein has antigen or antigenic determinant or aglucon character, the enzyme labeling thing is to obtain by the antibody of enzyme labeling pontin protein or part, HCVAg forms following compound by pontin protein and enzyme labeling thing: HCVAg-pontin protein-enzyme labeling thing, thereby realize the indirect enzyme labeling of HCVAg, compound by HCVAg, can realize that the antibody in serum and the blood plasma is carried out dual-antigen sandwich method ELISA to be detected with bag.