CN102236020A

CN102236020A - Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit

Info

Publication number: CN102236020A
Application number: CN2010101524985A
Authority: CN
Inventors: 张小寒
Original assignee: SHANGHAI XINBO BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI XINBO BIOTECHNOLOGY CO Ltd
Priority date: 2010-04-20
Filing date: 2010-04-20
Publication date: 2011-11-09

Abstract

The invention discloses a hepatitis c virus antibody time-resolved fluoroimmunoassay and a kit; a multi-fragment HCV fusion recombinant antigen is selected as a coating antigen, and a sodium carbonate buffer solution diluted into 1-10 mug/mL is used as a coating solution; a matched HCV recombinant antigen labelled with biotin is used as a labelled antigen; streptavidin is marked with lanthanum series elements; during experiment, a reaction buffer solution is diluted according to a ratio of 1:21 for using; on a reaction plate with the coating antigen, 25-muL HCV negative and positive controls or samples to be tested, and the diluted biotin-labelled antigen are added orderly into each hole; after incubation and plate washing, the diluted streptavidin europium marker is added; and fluorescence detection is performed after incubation. The invention also provides a corresponding kit. The invention has high sensitivity, specificity and stability; and the analysis system has high automation, can increase the speed for clinical detection results, can greatly reduce artificial error and improve the reliability of the detected results.

Description

Antibody of HCV time-resolved fluoroimmunoassay and kit

Technical field:

The present invention relates to detecting reagent kit for antibody of hepatitis C virus, belong to the time-resolved fluoroimmunoassay detection range.

Background technology:

(Hepatitis C Virus is to cause NANB-PTH (Post-transfusion non-A non-B Hepatitis, main pathogen factor PT-NANBH) HCV) to hepatitis C virus.The symptom of HCV the infected acute stage is obvious not as HAV or the metainfective symptom of HBV, the jaundice ratio is lower, but HCV the infected infects easier chronicity than HBV, has HCV the infected of 50% to develop into chronic hepatitis approximately, and 20% patient wherein finally becomes cirrhosis and liver cancer.The whole world has the HCV the infected more than 1.7 hundred million at present approximately; China nearly 4,000 ten thousand is the high popular district of HCV, infection rate between 0.9%～5.1%, average out to 3.1%.

It is blood transfusion and input blood product that HCV propagates topmost approach, in addition, think that at present possible circulation way also comprises: needle sharing and syringe (as passing through the intravenous drug user), property contact and kinsfolk's close contact, mother and baby's vertical transmission, iatrogenic infection etc.; The main path that China HCV infects is to propagate by blood transfusion and blood product.

Hepatitis C virus (HCV) since being found in 1989, and scientists has been carried out big quantity research, has set up various detection methods.The anti-HCV reagent of China has progressively improved and generally is used for examination of blood source and clinical diagnosis since 1994 come out.Current HCV antibody diagnosing reagent is a third generation reagent, is all improving a lot than former generation reagent aspect sensitivity and the specificity, but is diagnosing in early days and low danger crowd's diagnosis aspect still has some shortcomings.

Time resolution immunofluorescence analysis (time-resolved fluoroimmunoassay, TRFIA) utilize lanthanide series with unique fluorescent characteristic, replace enzyme, isotope etc., as a kind of important method in the luminescence immunoassay, have highly sensitive, tracer stable, be not subjected to many advantages such as the interference of sample natural fluorescence, "dead" pollution.

Summary of the invention:

The purpose of this invention is to provide a kind of time-resolved fluoroimmunoassay and kit that is used for hepatitis C virus (HCV) antibody test, solve mainly that the sensitivity that prior art exists is low, poor stability, operation is loaded down with trivial details and technical matters such as contaminated environment.The present invention adopts europium label, and the europium labelling kit is provided, with the sensitivity and the stability of raising method.

Technical scheme of the present invention is: the antibody of HCV time-resolved fluoroimmunoassay comprises the following steps:

1. insolubilized antibody preparation: the HCV recombinant antigen is diluted to 1-10ug/mL as coating buffer with carbonate buffer solution, and bag is formed insolubilized antibody by reaction plate, and seals with confining liquid (bovine serum albumin(BSA) (BSA) of phosphate buffer and 2% (W/V));

2. the preparation of biotin labeling antigen: pairing antigen is carried out mark with biotin;

3. lanthanide ion labelled antibody preparation: streptavidin carries out the lanthanide ion mark with conventional method;

4. on the reaction plate with insolubilized antibody that 1. step makes, every hole adds contrast of HCV yin and yang attribute or testing sample, and hatches with the labelled antibody of reaction buffer dilution, after fluorescence enhancement solution dissociates, carries out fluorometric assay at last.

The concentration of HCV recombinant antigen is 1-10ug/mL in the 1. middle coating buffer of step, 2. middle pairing antigen of step and step 3. streptavidin need be used reaction buffer (the 50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) of pH 7.8, include 0.9%NaCl, 1%BSA, 0.5% casein, 0.08% tween (Tween) 20 and 0.1%NaN ₃(reaction buffer component % refers to W/V)) with volume ratio dilution in 1: 21.The lanthanide ion of step in 3. is preferably Eu ³⁺The reaction buffer of step in 4. with step 2., 4. step is finished on the time resolved fluoro-immunoassay instrument automatically, fluorescence enhancement solution is the beta diketone body.

Detecting reagent kit for antibody of hepatitis C virus, it is characterized in that kit comprises: the damping fluid of envelope antigen (carbonate buffer solution), confining liquid (BSA of phosphate buffer and 2% (W/V)), reaction buffer (the 50mmol/L Tris-HCl of pH 7.8, include 0.9%NaCl, 1%BSA, 0.5% casein, 0.08%Tween20 and 0.1%NaN ₃), work cleansing solution (phosphate buffer), fluorescence enhancement solution, as bag by the HCV recombinant antigen, the streptavidin of biotin labeling antigen and lanthanide ion mark.

The present invention uses double antibody sandwich method, and reactions steps comprises: reagent is prepared, application of sample reacts, washing pats dry, adds enhancing liquid, survey fluorescent value.

The invention has the beneficial effects as follows: sensitivity for analysis height (meeting or surpass the requirement of HCV country reference product), detection time short (60min), specificity is good, and is little with the cross reaction of interfering material.

Description of drawings:

Fig. 1 is reaction principle figure of the present invention

The present invention adopts dual-antigen sandwich method.HCV recombinant antigen bag is by the glimmering plate of exempting from 96 holes, and HCV antibody in yin and yang attribute contrast or the sample and envelope antigen and biotin labeling antigen are in micropore inside surface generation immune response; After the washing, add europium ion (Eu ³⁺) streptavidin of mark, after the immune response, the sandwich immunoassay compound of micropore surface separates by washing with the free label streptavidin.Eu in the micropore surface immune complex ³⁺Formed the stable fluorescence complex by the fluorescence enhancement solution back of dissociating, the proportional example of HCV antibody concentration in fluorescence intensity and yin and yang attribute contrast or the sample is by Cutoff value computing formula judgement yin and yang attribute result.

Fig. 2 is an operational flowchart of the present invention

The first step: melt biotin labeling antigen again, second step: dilution biotin labeling thing, the 3rd step: add yin and yang attribute contrast and testing sample, the 4th step: the biotin labeling antigen after the adding dilution, the 5th step: hatch, the 6th step: wash plate, the 7th step: add dilution back europium label, the 8th step: hatch the 9th step: wash plate, the tenth step: add enhancing liquid, the 11 step: detect.

Embodiment:

The invention will be further described by embodiment below with reference to Fig. 1,2.

Before carrying out time resolution immunofluorescence analysis detection method, need finish following preliminary work:

At first the preparation bag is by plate, and used bag is the antigen coated plate of reorganization by plate, and the preparation of HCV coated slab may further comprise the steps:

(1) the HCV recombinant antigen with purifying dilutes with carbonate buffer solution, adds then in each hole of coated slab, obtains HCV recombinant antigen coated slab after absorption, washing, sealing, drying;

The Aluminium Foil Package pack of (2) HCV recombinant antigen coated slab being packed into special-purpose seals and refrigerates standby.

Next is the preparation of HCV biotin labeling antigen, may further comprise the steps:

(1) gets antigen to be marked and add in the bag filter, put into pH 9.5 carbonate buffer solutions that prepare, room temperature dialysed overnight (pH 9.5 carbonate buffer solutions);

(2) next day, take out antigen, the antigen of dialysing is diluted to 0.2-5mg/mL with pH 9.5 carbonate buffer solutions, it in mass ratio 2: 1 ratio (biotin: antigen) add biotin while vibrating, on the vibration plate machine, vibrated at a slow speed 1 hour under the room temperature, remove back dialysed overnight in the phosphate buffer (phosphate buffer of pH7.5) of pH7.5 then;

(5) filtrator with aperture 0.2um filters;

(6) add the pure BSA of 0.3% (W/V) top grade, 2-8 ℃ of preservation.

Embodiment: antibody of HCV detects

1, biotin labeling antigen

With HCV recombinant antigen dialysed overnight, add biotin reaction afterwards 1 hour with carbonate buffer solution, take out dialysed overnight in the phosphate buffer of pH7.5.Packing is in+2～+ 8 ℃ of preservations.

2, operation steps

1.) kit is prepared: the damping fluid of envelope antigen (carbonate buffer solution), confining liquid (BSA of phosphate buffer and 2% (W/V)), reaction buffer (the 50mmol/L Tris-HCl of pH 7.8, include 0.9%NaCl, 1%BSA, 0.5% casein, 0.08%Tween20 and 0.1%NaN ₃), work cleansing solution (phosphate buffer), fluorescence enhancement solution (beta diketone body), as bag by the HCV recombinant antigen, the streptavidin of biotin labeling antigen and lanthanide ion mark.

A: the work cleansing solution: 40mL phosphate buffer (25 times of concentrates) is diluted to 1 with distilled water or deionized water, and 000mL is standby.

B: biotin labeling antigen working fluid: the freeze-dried powder of every bottle of biotin labeling HCV antigen adds 1,000 μ L pure water, leaves standstill 30 minutes, treats that it fully dissolves.To each bar 12 orifice plate, get 75 μ L and redissolve good biotin labeling HCV antigen (21 times of concentrates), add the 1.5mL reaction buffer, fully mixing (dilution in 1: 21).Test preparation in preceding 1 hour.

2) numbering: the corresponding micropore of sample is numbered according to the order of sequence, and every plate should be established each 2 hole of anti-HCV positive and negative contrast.

3) application of sample: in respective aperture, add anti-HCV negative control, positive control and testing sample 25 μ L respectively.

4) add biotin labeling antigen: every hole adds the good biotin labeling antigen working fluid 100 μ L of dilution.

5) first step is hatched: vibrate at a slow speed incubation 45 minutes of room temperature (20～25 ℃).

6. join Eu mark streptavidin working fluid: to each bar 12 orifice plate, get 75 μ L Eu mark streptavidins (21 times of concentrates), add the 1.5mL reaction buffer, fully mixing (dilution in 1: 21).Second step was hatched preparation in preceding 45 minutes.

7) washing: fully wash plate 5 times with the work cleansing solution, button is done.

8) add Eu mark streptavidin: every hole adds Eu mark streptavidin working fluid 100 μ L.

9) second step hatched: vibrate at a slow speed incubation 15 minutes of room temperature (20～25 ℃).

10) washing: fully wash plate 5 times with the work cleansing solution, button is done.

11) add fluorescence enhancement solution: every hole adds fluorescence enhancement solution 100 μ L, measures photofluorometer numerical value after room temperature (+20～+ 25 ℃) was vibrated at a slow speed 5 minutes.

12) measure photofluorometer numerical value: differentiate fluorescence detector with the time and measure photofluorometer numerical value.

Claims

1. the antibody of HCV time-resolved fluoroimmunoassay is characterized in that comprising the following steps:

1. insolubilized antibody preparation: the HCV recombinant antigen is diluted to 1-10ug/mL as coating buffer with carbonate buffer solution, and bag is formed insolubilized antibody by reaction plate, and seals with confining liquid;

4. on the reaction plate that 1. step makes with insolubilized antibody, every hole adds contrast of HCV yin and yang attribute or testing sample, and hatch with the labelled antigen of reaction buffer dilution, wash and add dilution behind the plate good europium mark streptavidin is hatched once more, carry out fluorometric assay at last.

2. according to the described antibody of HCV time-resolved fluoroimmunoassay of claim 1, it is characterized in that during step 2. pairing antigen and step 3. streptavidin be to dilute at 1: 21 with reaction buffer with volume ratio.

3. according to the described antibody of HCV time-resolved fluoroimmunoassay of claim 1, it is characterized in that the lanthanide ion during step 3. is Eu ³⁺

4. according to claim 1 or 2 described antibody of HCV time-resolved fluoroimmunoassays, it is characterized in that described reaction buffer is the 50mmol/L Tris-HCl of pH 7.8, includes 0.9%NaCl, 1%BSA, 0.5% casein, 0.08% polysorbas20 and 0.1%NaN ₃

5. according to the described antibody of HCV time-resolved fluoroimmunoassay of claim 1, it is characterized in that 4. step finish automatically on the time resolved fluoro-immunoassay instrument.

6. detecting reagent kit for antibody of hepatitis C virus, it is characterized in that kit comprises: the damping fluid of envelope antigen, confining liquid, reaction buffer, work cleansing solution, fluorescence enhancement solution, as wrapping by the HCV recombinant antigen streptavidin of biotin labeling antigen and lanthanide ion mark.

7. detecting reagent kit for antibody of hepatitis C virus according to claim 6 is characterized in that:

The damping fluid of envelope antigen: carbonate buffer solution,

Confining liquid: phosphate buffer and 2% BSA,

The 50mmol/L Tris-HCl of reaction buffer: pH 7.8 includes 0.9%NaCl, 1%BSA, 0.5% casein, 0.08% polysorbas20 and 0.1%NaN ₃,

The work cleansing solution: phosphate buffer,

Fluorescence enhancement solution: beta diketone body.