Background technology
Acquired immunodeficiency syndrome claims acquired immune deficiency syndrome (AIDS) (AIDS) again, is to infect caused a kind of immunodefiiciency disease by human immunodeficiency virus (HIV).Because this disease does not still have effective methods of treatment and prevention vaccine at present, just enlarge popular at present with the infected speed of about 1.4 ten thousand people.According to estimates, China HIV the infected is nearly 1,000,000, as not taking active and effective control measure, will reach 1,000 ten thousand to China HIV the infected in 2010, so form is very severe.HIV Infect And Diagnose and then the crowd who identifies early infection are the top priorities of AIDS prevention and control work.Therefore, set up responsive practical detection method for detection, diagnosis or blood screening, control the popular of acquired immune deficiency syndrome (AIDS) just seems extremely important.
The method that detects at present HIV has kind more than 100, wherein the serodiagnosis of HIV antibody test be develop the earliest, cheap and the simplest HIV infection analysis method.The method of the most frequently used detection HIV antibody has enzyme linked immune assay (ELISA), gelatin particle agglutination test (PA), immunofluorescence analysis (IFA) and Dot blot etc.Wherein ELISA is present general clinically primary dcreening operation detection method owing to have advantages such as screening simple to operate, quick, as to be fit to a large amount of samples and detection.Thereby but be restricted owing to its sensitivity is low, developed the higher chemiluminescence immune analysis method of sensitivity afterwards again.What these methods were used on the principle that detects HIV antibody mostly is dual-antigen sandwich method, and this method has excellent specificity, also has very big advantage on methodology.
But the method for current detection HIV antibody all be with the method direct coated of HIV recombinant antigen by physisorption at solid phase carrier, remove not in conjunction with the analyte or the sample substrate that get on by washing then.This direct coated method can have some influences to analytical parameters: (1) antigen on solid phase, can cause the inhomogeneity of antigen combination on solid phase by the physisorption direct coated, therefore, poor reproducibility, the incubation time is long; (2) different with the association reaction of Ag-Ab in liquid phase, be coated on the albumen (antibody or antigen) on the solid phase, therefore the more difficult quilt of its binding site is approaching, can cause the immunocompetence part inactivation of albumen, and is active sometimes even only reach about 10% of coating protein amount; (3) amount that is attached to antigen on the solid phase is not only relevant with character such as the hydrophobicity of antigen self, isoelectric point etc., also is subjected to the influence of external conditions such as the preparation of antigenic solution and preservation.And the HIV antigen of the present invention adopts anti--FITC antibody and FITC mark is attached to HIV antigen on the solid phase carrier, thereby overcome many shortcomings of physisorphtion direct coated HIV antigen.
The research of labelled immune analytical technology and application development are rapid over past ten years, have been widely used in each field of biomedical fundamental research and clinical disease diagnosis.Wherein the technical matters maturation has advance and practical, and what be easy to promote mainly contains: four kinds of radiommunoassay, EIA enzyme immunoassay, time resolved fluoro-immunoassay and chemiluminescence immune assays etc.The basic theories of these ultramicron detection techniques is identical substantially, and their common feature is that sensitivity is good, high specificity; Difference is that used tracer agent and the signal that sent are different.According to lot of experiment results and clinical practice data,, be followed successively by: chemiluminescence immune assay, time resolved fluoro-immunoassay, radiommunoassay and EIA enzyme immunoassay from practicality, stability, accuracy and development prospect.There are many shortcomings in radio immunoassay, as complicated operation, measurement result instability, reagent holding time weak point, radioactive contamination, instrument costliness etc.
Yet, chemical luminescence immune analysis reagent box of the prior art is the luminous measuring system of closed full-automatic chemical, need the expensive luminous measuring instrument of full-automatic chemical, promote the use of, can't effectively be widely used in clinical diagnosis and research work thereby limited.
Summary of the invention
Propose and finished the present invention in order to address the above problem.Particularly, the invention solves problems such as diagnostic kit homogeneity of the prior art, poor reproducibility.
The purpose of this invention is to provide a kind of human immunodeficiency virus (HIV) antibody chemical luminescence immune assay diagnostic kit.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
HIV antibody diagnosing reagent kit according to the present invention comprises following component:
1) solid phase carrier of anti--FITC antibody sandwich;
2) the HIV recombinant antigen of FITC mark;
3) the HIV antigen of enzyme labeling;
4) chemical luminous substrate that above-mentioned enzyme acted on; And
5) negative controls, described negative controls are normal human serum.
6) positive control solution, described positive control solution are that the positive of NBCS dilution is mixed slurry.
Wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.Described enzyme is alkaline phosphatase or horseradish peroxidase.The chemical luminous substrate that above-mentioned enzyme acted on is (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star, luminol or different luminol.In addition, described HIV recombinant antigen is gp41, gp120, gp36.
The present invention also provides a kind of method for preparing the mentioned reagent box, and this method may further comprise the steps:
1) with the solid phase carrier of anti--FITC antibody sandwich;
2), obtain the HIV recombinant antigen of FITC mark with FITC mark HIV recombinant antigen;
3), obtain the HIV antigen of enzyme labeling with enzyme labeling HIV antigen;
4) chemical luminous substrate that acted on of HIV antigen, this enzyme of the HIV recombinant antigen of the FITC mark of the above-mentioned acquisition of packing, enzyme labeling, mix slurry as the normal human serum of negative controls with as the positive of the NBCS dilution of positive control solution; And
5) be assembled into finished product.
Wherein, use the solid phase carrier of anti--FITC antibody sandwich by the following method:
1) will wrap and be added to mixing in pH=4.4~5.0, preferred 4.8 the citrate buffer solution with anti--FITC antibody, with gained mixed liquor bag by solid phase carrier;
2) washing step 1) the middle solid phase carrier that wraps quilt; And
3) use the solid phase carrier of bovine serum albumin(BSA) sealing through washing.
The method according to this invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.Described enzyme is horseradish peroxidase or alkaline phosphatase.The chemical luminous substrate that this enzyme acted on can be (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star, luminol or different luminol.
Kit of the present invention can adopt following concrete form, it comprises the opaque microwell plate in 96 holes or 48 holes of anti--FITC antibody sandwich, the gp41 of FITC mark, gp120 and gp36 recombinant antigen, the HIV recombinant antigen of horseradish peroxidase-labeled, the negative controls of normal human serum, sample diluting liquid is 0.1MTris-HCl damping fluid+0.1%BSA+0.1%Proclin300 of pH7.4, the positive of NBCS dilution is mixed slurry, 20 times of concentrated cleaning solutions are the 2M Tris-HCl damping fluid that contains the pH8.5 of 10% physiological saline, chemical luminous substrate A, B liquid is respectively luminol solution, hydrogen peroxide and special efficacy reinforcing agent (to iodophenol) solution thereof, each 1 bottle.
According to the present invention, when use two antibody system as FITC with anti--during FITC antibody system ankyrin, the immunocompetence of immobilized antigen albumen can obtain preservation to a great extent.Anti--FITC antibody molecule also can be in conjunction with 3~4 FITC molecules, and specificity and affinity are all very strong.Both are just very stable once combination.FITC-is anti--the FITC antibody forming system is during as solid phase, on solid phase pre-earlier bag by anti--FITC antibody, by FITC-anti--the FITC antibody response makes the HIV antigen immobilization of FITC mark.This method for coating not only can increase the antigen amount on the solid phase carrier, but also the binding site on the antigen is fully exposed.Therefore, use two antibody systems as solid phase, not only can improve the sensitivity of analysis, homogeneity and reappearance can also reduce the use amount of HIV recombinant antigen.And the anti--bag of FITC antibody be can be used as unified solid phase carrier by microwell plate and is applied to many analyzing and testing objects.
Kit according to the present invention adopts two antibody system as solid phase carrier, double antigens sandwich pattern principle, the chemical luminous system of highly sensitive luminol-hydrogen peroxide-horseradish peroxidase and reinforcing agent thereof is as the input system, a kind of highly sensitive, two anti-solid state chemistry luminescence immunoassay methods have cheaply been set up, and be prepared into the diagnostic kit of measuring human immunodeficiency virus (HIV) antibody in blood or the blood plasma, compare with the ELISA detection kit, sensitivity has improved about 100 times; Compare with existing chemiluminescence immune assay detection method, can improve the sensitivity of analysis, homogeneity and reappearance, the use amount of minimizing HIV recombinant antigen.This detection method can produce the antiviral antibody that the initial stage promptly detects denier at antibody, detects than traditional enzyme-linked method to shift to an earlier date a couple of days diagnosis, to reducing mortality ratio and reducing the transmission rate crucial meaning is arranged all.Use kit of the present invention to reduce cost, the limited health investment of people is used more effectively.
Embodiment
Embodiment 1 preparation HIV antibody diagnosing reagent kit of the present invention
HIV antibody diagnosing reagent kit of the present invention comprises:
1) anti--FITC antibody wraps the luminous microwell plate in 96 holes or 48 holes of quilt in advance;
2) gp41 of FITC mark, gp120, gp36HIV recombinant antigen;
3) the HIV recombinant antigen of horseradish peroxidase-labeled;
4) negative controls is a normal human serum;
5) positive control solution is the positive mixing slurry of NBCS dilution;
6) sample diluting liquid is 0.1M Tris-HCl damping fluid, 0.1%BSA and 0.1%Proclin300, pH7.4;
7) 20 times of concentrated cleaning solutions are the 2M Tris-HCl damping fluid that contains 10% physiological saline, pH=8.5;
8) chemical luminous substrate A, B liquid are respectively luminol solution, hydrogen peroxide and special efficacy reinforcing agent (to iodophenol) solution.
Prepare the mentioned reagent box by the following method:
1, will wrap quilt and be added to mixing in the citrate buffer solution (pH=4.8) with anti--FITC antibody, and add in the luminous micropore plate hole, every hole 100 μ L place 15h bag quilts for 4 ℃.After cleansing solution washing five times, use bovine serum albumin(BSA) (BSA) solution room temperature sealing 2 hours.Discard liquid in the hole, dry ELISA Plate, sealing is promptly finished pre-bag by the preparation of elisa plate.
2, with direct labelling method FITC and HIV recombinant antigen are carried out mark, promptly get the HIV recombinant antigen of FITC mark.
The direct labelling method concrete steps are as follows:
(I) A liquid: get the Na that recombinant protein HIV antigen 1 mg puts 50mM pH9.3
2CO
3-NaHCO
34 ℃ of dialysis 24h in the damping fluid, last volume is controlled within the 200 μ l.
(II) B liquid: get FITC (Beijing is glad through biotech company of section) 2mg and be dissolved in the Na of 1ml50mM pH9.3
2CO
3-NaHCO
3In the damping fluid.
(III) A liquid is placed magnetic stirring apparatus, get B liquid 25 μ l and slowly drop in the A liquid, between 5-8 minute, add, prevent bubble, put 4 ℃ then and stirred 12-16 hour.
(IV) reactant liquor is taken out in 10000 rev/mins speed centrifugal 20 minutes.Remove the microprecipitation thing, supernatant dress bag filter was put in the PBS solution of 10mMpH=7.4 dialysis 4 days, changed dislysate every day 2 times.
(V) the centrifugal sediment that goes is diluted to 1ml with label, adds an amount of NaN
3, packing is preserved down for-20 ℃.
3, utilize the sodium periodate method of improvement, mark is combined with HIV recombinant antigen and horseradish peroxidase, promptly get the HIV antigenic label of horseradish peroxidase-labeled.
The concrete steps of the sodium periodate method of improvement are as follows:
(I) taking by weighing 2mgHRP is dissolved in the 1ml distilled water.
(II) add the 200 μ l NaIO of the 0.1mol/L of configuration newly
4Solution stirred in the room temperature 20 minutes gently.Solution is brown-green.
(III) the sodium acetate buffer solution of the 0.001mol/L of usefulness pH=4.4 is in 4 ℃ of following dialysed overnight.
(IV) add 20 μ l0.2mol/LNa
2CO
3Damping fluid makes pH value of solution be promoted to about 9.0-9.5.
(V) use 0.01mol/L, the carbonate buffer solution of pH=9.5 is deployed into HCV antigen the concentration of 8mg/ml.And with in the isopyknic above-mentioned HRP solution of the slow adding of antigenic solution, stirring at room 2 hours.
(VI) add the dobell's solution of the 4mg/ml of fresh configuration, place 4 ℃ 2 hours.
(VII) in 4 ℃, dialysis is 24 hours in the sodium borate buffer liquid of 0.1mol/L, and exchange buffering liquid is 3 times therebetween.
(VIII) add equivalent 60% neutral glycerine solution, packing behind the mixing is preserved down for-20 ℃.
4, select best reference substance by experiment, negative controls is that normal human serum, positive control solution are the positive mixing slurry of NBCS dilution.
5, other components of reagent preparation box: 20 times of concentrated cleaning solution (2M Tris-HCl damping fluids that contain 10% physiological saline, pH=8.5), sample diluting liquid (0.1M Tris-HCl damping fluid, 0.1% hydrogel, 0.1%Proclin300, pH7.4), chemical luminous substrate A, B liquid be respectively luminol solution, hydrogen peroxide and special efficacy reinforcing agent (to iodophenol) solution thereof.
6, identify accuracy, sensitivity, precision and the qualified stability of this method generate a reagent box.
7, the requirement according to kit is divided in these components in the bottle, is assembled into finished product.
Embodiment 2 preparations HIV antibody diagnosing reagent kit of the present invention
Is 4.5 outside as chemical luminous substrate and bag by the pH of citrate buffer solution used in the process as carrier, with alkali phosphatase enzyme mark HIV recombinant antigen, with AMPPD divided by plastic bead, prepares kit of the present invention with the method identical with embodiment 1.
The using method of embodiment 3 kits of the present invention:
1, take out kit in 4 ℃ of refrigerators, equilibrium at room temperature 20 minutes is got one bottle of concentrated washing lotion, and (1ml+19ml) is standby for adding distil water.
2, luminous plaque is taken out from sealing bag, the HIV recombinant antigen solution (1:2000) of 100 μ LFITC marks is joined in the ELISA Plate that is coated with anti--FITC antibody, 37 ℃ of following incubations 1 hour.
3, discard liquid in each hole, cleansing solution is filled with each hole, leave standstill 10-20 second, get rid of cleansing solution; Repeat to wash plate, on clean thieving paper, pat dry at last.
4, establish blank one hole, do not add sample solution, if negative control 3 holes, positive control 2 holes, get each 50 μ l of negative controls, positive control solution and sample solution in corresponding plate hole, add enzyme labeling thing 50 μ l again, the micro oscillator mixing that fully vibrates, with adhesive sticker sheet capping reaction plate, put 37 ℃ of incubations of reaction plate then 30 minutes.
5, carefully get rid of dereaction liquid, wash five subsynchronous rapid 3 then.
6, use preceding 5 minutes with luminous substrate A liquid with after B liquid 100:1 mixes, adding 50 μ L chemical luminous substrates in every hole successively, the room temperature lucifuge was reacted after 10 minutes, and luminous intensity is measured.
That 7, measures RLU value and critical value per sample relatively comes result of determination, the average RLU value of negative control hole of critical value=2.1 times, if sample RLU 〉=critical value, then positive reaction; If sample RLU<critical value, then negative reaction.
The Performance Detection of embodiment 4 kits of the present invention
We are to kit of the present invention and directly the antigen coated chemical luminescence immune analysis reagent box in the solid phase microwell plate of HIV has been carried out preliminary comparison from cost and two aspects of precision.The result is shown in table 1 and 2, and the result shows, uses two antibody system as solid phase, and the use amount of its HIV recombinant antigen can reduce about 4 times, and precision improves greatly.In addition, the microwell plate of anti--FITC antibody sandwich provides a general solid phase carrier, and they can be used for the analysis of many determinands, have avoided the loaded down with trivial details optimizing process of solid phase carrier condition.
The accuracy that kit of the present invention detects detects (as table 3) with up-to-date national standard reference material serum dish, investigate the coincidence rate of negative and positive reference material, according to national standard, require 20 parts of negative reference material negative rate to be not less than 18/20,20 parts of positive reference material positive rates are not less than 20/20, and No. 12 positive reference serum measured values〉No. 11 positive reference serum measured values.
Kit of the present invention has also been accepted clinical examination.With human immunodeficiency virus (HIV) antibody chemical luminescence immune assay diagnostic kit 223 examples are made a definite diagnosis aids patient serum and carried out antibody test, and contrast with electroselenium method and the Anti-HIVI/II-ELISA of AKZO company, the result shows that the positive recall rate of measuring of (table 4) kit of the present invention is better than the AKZO-HIVI/II-ELISA kit, far above HIVI/II electroselenium kit.
In addition, 210 routine HIV patients suspected of the HIVI/II electroselenium kit of learning from else's experience screening are negative blood sample all, rechecks through kit of the present invention and AKZO elisa kit, and positive routine number average is 12 examples.
We compare kit of the present invention and other detection methods from sensitivity and specificity, the result shows (table 5), and the sensitivity of this kit is better than additive method, and specificity is also better.
The comparison of table a kind of different solid-phase coating method HIV recombinant antigen use amounts
The comparison of two kinds of different solid-phase coating method chemical luminescence immune analysis reagent box precision of table 2
The testing result of this kit of table 3 country reference material serum
Table 4 kit of the present invention detects HIV blood serum sample and ELISA and electroselenium measurement result relatively
Table 5 kit of the present invention and ELISA and electroselenium sensitivity and specificity evaluation
We think that kit of the present invention is highly sensitive, specificity good, can be applicable to the clinical diagnosis to aids patient fully thus.