CN107085111B - The board-like chemoluminescence method detection kit and preparation method of hepatitis B virus pre S 1 antigen - Google Patents
The board-like chemoluminescence method detection kit and preparation method of hepatitis B virus pre S 1 antigen Download PDFInfo
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- CN107085111B CN107085111B CN201710244167.6A CN201710244167A CN107085111B CN 107085111 B CN107085111 B CN 107085111B CN 201710244167 A CN201710244167 A CN 201710244167A CN 107085111 B CN107085111 B CN 107085111B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Abstract
The present invention relates to a kind of board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen, including reagent: the coated ELISA Plate of the former S 1 immune body of hepatitis virus type B solution of biotin labeling, anti-biotin antibodies, the former S 1 immune body of hepatitis virus type B solution of horseradish peroxidase-labeled, 1 negative control of hepatitis B virus pro-S, 1 positive control of hepatitis B virus pro-S, concentration washing lotion, substrate solution A, substrate solution B.Main design thought of the invention is that have anti-biotin antibodies-biotin system on the basis of chemiluminescence immune assay by introducing, while the detection of stable reagent box is specific, be added significantly to detection sensitivity and detection time.High-temperature oscillation formula anti-biotin antibodies coating technique is improved, to simplify reagent production procedure while reducing antibody/antigen usage amount, shorten the production cycle.
Description
Technical field
The invention belongs to immunoassay fields, more particularly to a kind of highly sensitive board-like chemoluminescence method quantitative detection serum
Hepatitis B virus pre S 1 antigen kit and preparation method thereof.
Background technique
Hepatitis type B virus (HBV) outer membrane protein includes S, preceding S2 and tri- kinds of ingredients of preceding S1.Pre-s1 protein is in Virus entry
It plays an important role during liver cell.Virus is attached on liver cell, and most important mediation position is the amino acid of pre-s1 protein
(AA) virus of 21-47 segment, variation is infectious as long as this section is intact.Albumen containing preceding S1 is primarily present in
On Dane particle and cast pellet.Pre-s1 protein is in terms of virus infection, assembly, duplication and stimulation body generate
Serve have it is particularly significant.
Pre-S 1 antigens of hepatitis B viruses Enzyme immunoassay kit formally pushes that clinical and nearly 2 years in Beijing, Sichuan, Zhejiang to
River, Henan, Shenzhen, Hainan Deng Jin various schools of thinkers hospital and hepatitis B " two double " detection use in conjunction, sufficiently show that the reagent exists
The clinical diagnosis of Type B viral hepatitis, the important value of guiding treatment etc..Pre S 1 antigen (Pre-S1Ag) detection is pair
The important supplement and reinforcement of hepatitis B " two double " especially e antigen and HBV-DNA measurement.
Both pre S 1 antigen and HBV-DNA recall rate meet, and pre S 1 antigen only detects in HBV-DNA positive serum.Before
S1 albumen disappears with HBeAg and is disappeared, and turns the time with yin and be positively correlated, in this way, pre S 1 antigen can be used as virus sweep and disease
The index that poison is turned out cloudy.The hepatitis B patient of the pre S 1 antigen positive propagates hepatitis type B virus than pre S 1 antigen feminine gender and asymptomatic
The risk of HBsAg carrier is bigger, thus illustrates that pre S 1 antigen can reflect hepatitis B replication and communicable index.
If pre S 1 antigen lasting masculin, indicate AHB to chronic transformation.Compare acute hepatitis B, chronic hepatitis B and HBsAg
Pre-s1 protein in positive patients serum, pre S 1 antigen yin turn more early, and the course for the treatment of of AHB patient is shorter, and prognosis is also better.Before explanation
The detection of S1 antigen and its antibody is the clinical diagnosis of acute hepatitis B, observation of curative effect and the good index for sentencing data prognosis.
Pre S 1 antigen is primarily present in serum HBV surface.Just there is pre S 1 antigen containing HBV in prompt machine body.Preceding S1 is anti-
It is a highly important Viral replicative marker that former and HBV-DNA, HBeAg verification and measurement ratio height, which meets,.Prompt pre S 1 antigen can be made
The supplement and control detected for HBeAg and HBV-DNA.Anti- HBeAb (+) chronic hepatitis B (accounting for about 30% of patient or so) and
In HBV Chronic asymptomatic HBsAg carriers, pre S 1 antigen (+) can indicate the duplication of virus, prompt clinically only to detect " five indexes of hepatitis b "
Be it is inadequate, the measurement for supplementing pre S 1 antigen is highly important, HBeAg caused by can making up because of virus variation and other reasons
" accidentally the seeking " of (-).Virus is attached on liver cell, and most important mediation position is amino acid (AA) 21-47 piece of pre-s1 protein
Section, the virus of variation are infectious as long as this section is intact.
In order to improve pre-S 1 antigens of hepatitis B viruses diagnostic level and improve therapeutic effect, from detection angles for, at present
Clinically the method for measuring pre S 1 antigen mainly has radioimmunoassay technology, Enzyme-multiplied immune technique, time-resolved fluorescence to exempt from
Epidemic disease analysis method and chemiluminescence etc..Past measures by the human hepatitis B virus pre S 1 antigen of representative of radioimmunoassay technology
Limitation of the kit due to methodology, there is very big drawback, substantially moves back in sensitivity and anti-interference ability wretched insufficiency
Market out；At present, more applications are Enzyme-multiplied immune technique, time-resolved fluoroimmunoassay and chemiluminescences, wherein changing
It learn luminescence technology to be risen in the 80's of eighties of last century, be grown up after Enzyme-multiplied immune technique and radioimmunoassay technology
Emerging technology, since it is highly sensitive, high specific, while method is easy, quickly, and label conjugate is stablized, relative time
Resolved fluorometric immunoassay is low in cost, easy to operate, isotope that relative radioactivity immuno analytical method is "dead" damage and
The features such as pollution, is developed rapidly in recent years.
Summary of the invention
The object of the present invention is to provide a kind of kits of board-like chemoluminescence method detection serum hepatitis B virus pre S 1 antigen
And preparation method thereof, various key technologies relevant to serology immune response principle are researched and developed and optimize, to improve needle
To the intracorporal pre-S 1 antigens of hepatitis B viruses detection sensitivity of clinical patients and accuracy, it is good, raw to develop high sensitivity, stability
Produce convenient, easy to operate board-like chemiluminescence diagnostic kit.
Main design thought of the invention is resisted by introducing with antibiotin on the basis of chemiluminescence immune assay
Body-biotin system is added significantly to detection sensitivity while the detection of stable reagent box is specific.
The present invention solves the above problems used technical solution are as follows:
A kind of board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen, including reagent: biotin
The hepatitis B of the coated ELISA Plate of the former S 1 immune body of hepatitis virus type B solution of label, anti-biotin antibodies, horseradish peroxidase-labeled
Virus front S 1 antibody-solutions, 1 negative control of hepatitis B virus pro-S, 1 positive control of hepatitis B virus pro-S, concentration washing lotion, substrate solution A,
Substrate solution B.
The concentration of the former S 1 immune body of hepatitis virus type B solution of above-mentioned biotin labeling is 0.01~5.0 μ g/ml, the antibiont
The peridium concentration of the anti-biotin antibodies of the plain coated ELISA Plate of antibody is 0.1~5.0 μ g/ml, the horseradish peroxidase
The concentration of the former S 1 immune body of hepatitis virus type B solution of label is 0.03~5.0 μ g/ml.
Above-mentioned 1 negative control of hepatitis B virus pro-S does not include hepatitis B virus pro-S 1,1 positive control of hepatitis B virus pro-S
It is that 1 positive serum of hepatitis B virus pro-S is obtained with the Tris-HCl buffer that pH value is 7.4, dilution volume ratio is 1:
5000。
The preparation process of the former S 1 immune body of hepatitis virus type B solution of above-mentioned biotin labeling are as follows: the hepatitis B of biotin labeling
Former S 1 immune body is added in the buffer that pH value is 7.4, is adjusted and is arrived convenient multiple, is uniformly mixed so as to obtain.
The preparation process of the above-mentioned coated ELISA Plate of anti-biotin antibodies are as follows: the anti-biotin antibodies coating plate is using high
Warm oscillatory type coating, coating temperature are 39 ± 1 DEG C, and the coating time is 1~3 hour, are coated with form using low-speed oscillation, selection
Shaker is that Ai Bensen scientific instrument Co., Ltd microplate oscillator, oscillation amplitude and mode: 3mm are turned using horizontal rotation
Speed is 200 ~ 500rpm, and closing and drying temperature are 37 ± 1 DEG C, and off-period is 1.5~3 hours, and drying time is 2~4 small
When；Using high-temperature oscillation formula coating technique, it is coated with plate preparation time and can be controlled in 5~7 hours.Universal 3~4 are coated with room temperature
Its preparation time is compared, and has apparent odds for effectiveness, while improving the sensitivity of reaction, reduces antibody/antigen usage amount
While, reagent production procedure is simplified, the production time is shortened.State's endoperidium plate technique using Streptavidin be coated with compared with
It is more, using anti-biotin antibodies there is not yet patent document is reported.In addition, low-speed oscillation coating method is coated with method compared to conventional room temperature
It has been obviously shortened the production time of coating plate, has improved reagent production efficiency.
The preparation process of the former S 1 immune body of hepatitis virus type B solution of above-mentioned horseradish peroxidase-labeled are as follows: horseradish peroxidase
The former S 1 immune body of hepatitis virus type B solution of enzyme label is added in the buffer that pH value is 7.4, is adjusted and is arrived convenient multiple, mixes
It arrives.
Coating buffer used in the above-mentioned coated ELISA Plate of anti-biotin antibodies be 0.01M carbonic acid buffer, pH9.6 ±
0.1, confining liquid used it is bright containing 0.3wt%BSA, 0.1 % Proclin300 (v/v), 1 wt % sheep blood serum, 1 wt % fish-skin
The 0.1M pH7.4 TBS buffer of glue, 2 wt % trehaloses and 10 wt % sucrose.For anti-biotin antibodies packet of the invention
By system, ELISA Plate largely unbonded position is shielded with a small amount of BSA using the carbonic acid buffer of 0.01M, and in confining liquid
Point eliminates nonspecific binding site with animal blood serum, ensure that the precision of coating plate under the synergistic effect of buffer system
And Low background is enhanced coating plate to the resistance of high temperature, drying, is protected using sucrose, trehalose and fishskin gelatin as stabilising system
The performance of anti-biotin antibodies coating plate is demonstrate,proved.
The detection method of kit of the present invention is that the hepatitis B virus front S 1 antibody of sample and biotin labeling is added
Into the coated ELISA Plate of anti-biotin antibodies, PreS1Ag and the antibody in sample, which are combined, forms immune complex, meanwhile,
The immune complex is fixed on ELISA Plate by the combination between anti-biotin antibodies and biotin.It is clear using washing lotion
ELISA Plate is washed, unbonded free ingredient is removed.The anti-HBs of horseradish peroxidase-labeled are added
(Anti-HBs), the enzyme labelled antibody is by immune response in conjunction with fixed immune complex.After cleaning microwell plate again, add
Enter substrate solution excitation chemiluminescence, measures relative light intensity RLU.Within the scope of a certain concentration, RLU value is with PreS1Ag in sample
The raising of content and linearly increase.Compared with negative control, it can be detected with the presence or absence of PreS1Ag in human serum sample, with sun
Property compares predictable PreS1Ag content.
The preparation method of the board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen of the invention, including
Following steps
One, the preparation of washing lotion is concentrated, steps are as follows:
1, KCl 60g, NaCl 300g are weighed in 1L container；
2,20.0g Tween-20 is weighed in 100ml container plus after 50ml water makes it completely dissolved, and pours into above-mentioned 1L
In container；
3, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L container；
4, appropriate purified water is measured in above-mentioned 1L container with graduated cylinder, be sufficiently stirred until being completely dissolved；
5, pH is adjusted, controls its range between 7.35~7.45；
6, it is finally settled to 1000ml, is filtered after being completely dissolved with 0.2 μm of filter to obtain the final product；
Two, the preparation of substrate solution
(1) preparation steps of substrate solution A
1, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
(2) preparation of substrate solution B
1, borax 11.44g, boric acid 4.948g, 500 μ l of urea peroxide 0.2g and PC300 are weighed in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
Three, the preparation of reference substance dilution, steps are as follows:
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range and exist
Between 7.35-7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in above-mentioned solution, it is spare as calibration object dilution；
Four, the preparation of negative control, positive control
The preparation of negative control: control dilution is taken, appropriate gauge is dispensed into.
The preparation of positive control: by 1:5000(1:500~1:10000) Pre- of commercialization is added in control dilution
The Beijing S1(new master's silver is logical)；
Five, the coated ELISA Plate of anti-biotin antibodies
1 prepares the carbonic acid buffer of 0.01M, and pH is to pH9.6 ± 0.1 for adjusting, spare；
2, which are added anti-biotin antibodies solution in the carbonic acid buffer of above-mentioned 0.01M, stirs to final concentration of 0.1~5 μ g/ml
Mix 30 minutes to be uniformly mixed；
3 the solution after above-mentioned mixing is added in ELISA Plate by package amount for the every hole 100 μ L, using high-temperature oscillation formula packet
Quilt, coating temperature are 39 ± 1 DEG C, and the coating time is 1~3 hour, are coated with form using low-speed oscillation；
4 prepare 0.1M, and pH7.4TBS buffer contains 0.3wt%BSA, 1wt% sheep blood serum, 0.1% Proclin300 (v/
V), 1wt% fishskin gelatin, 2wt% trehalose and 10wt% sucrose are added in the coating plate after cleaning, closing amount as confining liquid
For the 150 every holes μ L, closure temperature is 37 ± 1 DEG C, and off-period is 2~5 hours；
5 sop up confining liquid, are placed in drying box, and at 37 ± 1 DEG C, drying time is 2~4 hours for drying temperature control,
It is vacuum-packed again with aluminium foil bag, labeling is spare；
Six, the preparation of the former S 1 immune body of hepatitis virus type B solution of horseradish peroxidase-labeled
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in beaker, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 4g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2um filter to obtain the final product；
(2) coupling of horseradish peroxidase (HRP) and anti-Pre-S1 antibody
1, former S 1 immune body of hepatitis virus type B 1mg is taken to be placed in 1ml glass tube；
2, the final concentration for taking 200 μ l DMSO lytic antibodies to make antibody reaches 5mg/ml, then mixes well；
3, the molar ratio that the disuccinimidyl suberate of 10mol is added according to 1mol antibody adds two amber of suberic acid
Amber imide ester reacts 1.5 hours in 37 DEG C of insulating boxs into above-mentioned 2 solution；
4, HRP is added into above-mentioned 3 solution according to the molar ratio that 1mol HRP is added in 3mol antibody, be then added
The pH of 1ml is the PB buffer that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5, the solution prepared above-mentioned 4 PD-10 column purification collects refined solution, adds certainly according to the volume of 1:3000
The enzyme reaction object dilution of system, control are uniformly mixed finally using concentration in 0.03-5.0 μ g/ml up to enzyme reaction object；
Seven: the preparation of the former S 1 immune body of hepatitis virus type B solution of biotin labeling
(1) preparation of biotin reaction object dilution
1, it takes Tris 4.846g, 2847 HCl μ l in flask, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 4g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product；
(2) take biotinylated antibody, with biotin reaction object diluted, control finally using concentration 0.01~
5.0 μ g/ml mix, biotin conjugate can be obtained.
Compared with prior art, the kit and its system of board-like chemoluminescence method of the invention detection change of serum C A242 antigen
Preparation Method has the coated ELISA Plate of anti-biotin antibodies by introducing, and improve on the basis of chemiluminescence immune assay
High-temperature oscillation formula anti-biotin antibodies coating technique improves the sensitive of reaction by anti-biotin antibodies-Biotin method system
Degree saves production cost and reagent production procedure is also simplified, when shortening production while reducing antibody/antigen usage amount
Between, detecting step is simplified, can provide that of high quality and at a reasonable price, reliable and stable, reproducible, difference between batch is small, accuracy is high-new for market
Generation detection examination.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be described, for embodiment be only to product of the present invention or method
Make generality illustration, helps to more fully understand the present invention, but be not limiting upon the scope of the invention.It is real described in following embodiments
Proved recipe method is unless otherwise specified conventional method；The material commercially obtains unless otherwise specified.
The board-like chemoluminescence method detection kit main agents of the carbohydrate antigen 242 of the present embodiment include: biotin mark
The B-type hepatitis of the coated ELISA Plate of the former S 1 immune body of hepatitis virus type B solution of note, anti-biotin antibodies, horseradish peroxidase-labeled
Malicious former S 1 immune body solution, 1 negative control of hepatitis B virus pro-S, 1 positive control of hepatitis B virus pro-S, concentration washing lotion, substrate solution A, bottom
Thing liquid B.
The preparation method of kit
One, the preparation of washing lotion is concentrated, steps are as follows:
1, KCl 60g, NaCl 300g are weighed in 1L container；
2,20.0g Tween-20 is weighed in 100ml container plus after 50ml water makes it completely dissolved, and pours into above-mentioned 1L
In container；
3, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L container；
4, appropriate purified water is measured in above-mentioned 1L container with graduated cylinder, be sufficiently stirred until being completely dissolved；
5, pH is adjusted, controls its range between 7.35~7.45；
6, it is finally settled to 1000ml, is filtered after being completely dissolved with 0.2 μm of filter to obtain the final product；
Two, the preparation of substrate solution
(1) preparation steps of substrate solution A
1, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
(2) preparation of substrate solution B
1, borax 11.44g, boric acid 4.948g, 500 μ l of urea peroxide 0.2g and PC300 are weighed in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
Three, the preparation of reference substance dilution, steps are as follows:
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range and exist
Between 7.35-7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in above-mentioned solution, it is spare as calibration object dilution；
Four, the preparation of negative control, positive control
The preparation of negative control: control dilution is taken, appropriate gauge is dispensed into.
The preparation of positive control: by 1:5000(1:500~1:10000) Pre- of commercialization is added in control dilution
The Beijing S1(new master's silver is logical)；
Five, the coated ELISA Plate of anti-biotin antibodies
1 prepares the carbonic acid buffer of 0.01M, and pH is to pH9.6 ± 0.1 for adjusting, spare；
2, which are added anti-biotin antibodies solution in the carbonic acid buffer of above-mentioned 0.01M, stirs to final concentration of 0.3 μ g/ml
30 minutes to be uniformly mixed；
3 the solution after above-mentioned mixing is added in ELISA Plate by package amount for the every hole 100 μ L, using high-temperature oscillation formula packet
Quilt, coating temperature are 39 ± 1 DEG C, and the coating time is 1~3 hour, are coated with form using low-speed oscillation；
4 prepare 0.1M, and pH7.4TBS buffer contains 0.3wt%BSA, 1wt% sheep blood serum, 0.1% Proclin300 (v/
V), 1wt% fishskin gelatin, 2wt% trehalose and 10wt% sucrose are added in the coating plate after cleaning, closing amount as confining liquid
For the 150 every holes μ L, closure temperature is 37 ± 1 DEG C, and off-period is 2~5 hours；
5 sop up confining liquid, are placed in drying box, and at 37 ± 1 DEG C, drying time is 2~4 hours for drying temperature control,
It is vacuum-packed again with aluminium foil bag, labeling is spare；
Six, the preparation of the former S 1 immune body of hepatitis virus type B solution of horseradish peroxidase-labeled
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in beaker, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 4g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2um filter to obtain the final product；
(2) coupling of horseradish peroxidase (HRP) and anti-Pre-S1 antibody
1, former S 1 immune body of hepatitis virus type B 1mg is taken to be placed in 1ml glass tube；
2, the final concentration for taking 200 μ l DMSO lytic antibodies to make antibody reaches 5mg/ml, then mixes well；
3, the molar ratio that the disuccinimidyl suberate of 10mol is added according to 1mol antibody adds two amber of suberic acid
Amber imide ester reacts 1.5 hours in 37 DEG C of insulating boxs into above-mentioned 2 solution；
4, HRP is added into above-mentioned 3 solution according to the molar ratio that 1mol HRP is added in 3mol antibody, be then added
The pH of 1ml is the PB buffer that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5, the solution prepared above-mentioned 4 PD-10 column purification collects refined solution, adds certainly according to the volume of 1:3000
The enzyme reaction object dilution of system, control are uniformly mixed finally using concentration in 0.5 μ g/ml up to enzyme reaction object；
Seven: the preparation of the former S 1 immune body of hepatitis virus type B solution of biotin labeling
(1) preparation of biotin reaction object dilution
1, it takes Tris 4.846g, 2847 HCl μ l in flask, purified water is then added in flask, being sufficiently stirred makes
Reagent is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 4g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product；
(2) biotinylated antibody is taken, with biotin reaction object diluted, control is finally using concentration in 0.5 μ g/
Ml mixes, biotin conjugate can be obtained.
Eight, optimization and verifying
Main performance index meets luminous detection reagent Technical Review specification.
1, negative match-rate
The clinical blood sample of 260 Abbott Laboratories' chemical illuminating reagents detection is detected, negative match-rate (-/-) is 260/260, is met
Rate 100%.
2, positive coincidence rate
The clinical blood sample of 215 parts of Abbott Laboratories' chemical illuminating reagents detection is detected, positive coincidence rate (+/+) is 212/215, is met
Rate is 98.6%.
3, repeated
Repeat detection 10 times with enterprise's precision reference material through National reference mark, coefficient of variation CV 1.60 ~
Between 2.75%, meet the industry standard (full-automatic instrument operation) no more than 10.0%.
4, difference between batch
Three lot number kits, interassay coefficient of variation are detected with enterprise's precision quality-control product through National reference mark
(CV) between 2.50 ~ 4.24%, meet requirement (full-automatic instrument operation) of the rower less than 15%.
It is as follows to analyze result:
| Accuracy quality-control product | Average value | Standard deviation | CV% |
| L | 9384.5 | 253.2 | 2.70% |
| M | 107235 | 4252.5 | 3.97% |
| H | 823305 | 14060.3 | 1.71% |
5, stability
Thermal stability: kit is detected after placing 7 days at 37 DEG C, and compared with the control, amplitude of variation is or not detection signal
More than 15%.
A kind of semi-automated instrument detection method of kit, detecting step include in embodiment
(1) it takes out kit and is placed in room temperature, make the equalized temperature of kit to room temperature (18~25 DEG C).
(2) sample should be uniformly mixed, if sample is freeze thawing sample, be detected again after balance to room temperature (18~25 DEG C).
(3) according to concentration washing lotion: distilled water=1:39 ratio is prepared washing lotion and is transferred in bottle for handling liquid toilet or cosmetic substance after mixing.
(4) detection hepatitis B virus pre S 1 antigen plate lath is taken out, if calibrating sample wells, measuring samples hole well.By remaining
Lath is put back in aluminium foil bag, is sealed up for safekeeping.
(5) by 1 sequence of table, semi-automatic sample-adding and operation are carried out.
The sample-adding of table 1 and the operation table of comparisons
Substrate solution A and B can also add 100 μ L in every hole after with preceding isometric mixing, use if substrate solution A and B are mixed,
Mixed liquor should be used in 30 minutes.
The half full-automatic instrument detection method of one kind of kit in embodiment, using Full-automatic chemiluminescence immunoassay analysis meter
ADC CLIA 200/300/400/500/600 and Smart3000/Smart300/ Smart3000S are detected, detection step
Suddenly include
(1) when using Full-automatic chemiluminescence immunoassay analysis meter (hereinafter referred to as " instrument "), instrument do be read over
The operation manual of device must be configured instrument according to corresponding operation explanation, examination and maintenance, to realize optimum detection
Energy.
(2) illustrate to configure reagent information according to the operation manual of instrument, and reference substance is set in " setting of microplate layout "
The position in hole and sample aperture.Particularly, please detection method is carried out according to parameter shown in table 2 at " method editor " interface of instrument to match
It sets.
(3) according to 2 full-automatic instrument parameter setting table of table, parameter setting and data are carried out
2 Full-automatic chemiluminescence immunoassay analysis meter parameter list of table
In addition to the implementation, all to use equivalent transformation or equivalent replacement the invention also includes there is an other embodiments
The technical solution that mode is formed should all be fallen within the scope of the hereto appended claims.
Claims (5)
1. a kind of board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen, it is characterised in that: including examination
Agent: the coated ELISA Plate of the former S 1 immune body of hepatitis virus type B solution of biotin labeling, anti-biotin antibodies, horseradish peroxidase mark
The former S 1 immune body of hepatitis virus type B solution of note, 1 negative control of hepatitis B virus pro-S, 1 positive control of hepatitis B virus pro-S, concentration washing lotion,
Substrate solution A, substrate solution B；
The preparation method of the kit includes the following steps
One, the preparation of washing lotion is concentrated, steps are as follows:
1, KCl 60g, NaCl 300g are weighed in 1L container；
2,20.0g Tween-20 is weighed in 100ml container plus after 50ml water makes it completely dissolved, and is poured into above-mentioned 1L and is held
In device；
3, Proclin-300 is measured into 2ml with pipettor, poured into above-mentioned 1L container；
4, appropriate purified water is measured in above-mentioned 1L container with graduated cylinder, be sufficiently stirred until being completely dissolved；
5, pH is adjusted, controls its range between 7.35~7.45；
6, it is finally settled to 1000ml, is filtered after being completely dissolved with 0.2 μm of filter to obtain the final product；
Two, the preparation of substrate solution
(1) preparation steps of substrate solution A
1, weigh borax 11.44g, boric acid 4.948g, luminol 2.0g and to iodophenol 0.2mg in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range in 7.95-
Between 8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
(2) preparation of substrate solution B
1, borax 11.44g, boric acid 4.948g, 500 μ l of urea peroxide 0.2g and PC300 are weighed in 1L beaker；
2, purified water is measured in 1L beaker with graduated cylinder, be sufficiently stirred until being completely dissolved, tune pH controls its range and exists
Between 7.95-8.05；
3, filtrate is collected by filtration with 0.2 μm of filter, is settled to 1000ml with purified water, after mixing to obtain the final product；
Three, the preparation of reference substance dilution, steps are as follows:
1,7 ml of Tris 12.11g and HCl is weighed in the container of 1L, is adjusted pH value of solution, is controlled its range in 7.35-
Between 7.45, it is settled to 1000ml；
2, calf serum 300ml is measured with graduated cylinder, be added in the solution that above-mentioned steps 1 obtain, it is spare as reference substance dilution；
Four, the preparation of negative control, positive control
The preparation of negative control: reference substance dilution is taken, appropriate gauge is dispensed into；
The preparation of positive control: 1 positive serum of hepatitis B virus pro-S is added in reference substance dilution by 1:500~1:10000；
Five, the coated ELISA Plate of anti-biotin antibodies
1 prepares the carbonic acid buffer of 0.01M, and pH is to pH9.6 ± 0.1 for adjusting, spare；
2 are added anti-biotin antibodies solution to final concentration of 0.1~5 μ g/ml, stirring in the carbonic acid buffer of above-mentioned 0.01M
30 minutes to be uniformly mixed；
3 above-mentioned solution after mixing is added in ELISA Plate by package amount for the every hole 100 μ L, using high-temperature oscillation formula packet
Quilt, coating temperature are 39 ± 1 DEG C, and the coating time is 1~3 hour, are coated with form using low-speed oscillation, the shaker of selection is Chinese mugwort
Benson scientific instrument Co., Ltd microplate oscillator, oscillation amplitude and mode: 3mm, using horizontal rotation, revolving speed is 200~
500rpm；
4 prepare 0.1M, and pH7.4TBS buffer contains 0.3wt%BSA, 1wt% sheep blood serum, 0.1% Proclin300 (v/v), 1wt%
Fishskin gelatin, 2wt% trehalose and 10wt% sucrose are added in the coating plate after cleaning as confining liquid, and closing amount is 150 μ L
Every hole, closure temperature are 37 ± 1 DEG C, and off-period is 2~5 hours；
5 sop up confining liquid, are placed in drying box, and at 37 ± 1 DEG C, drying time is 2~4 hours for drying temperature control, then with
Aluminium foil bag vacuum packaging, labeling are spare；
Six, the preparation of the former S 1 immune body of hepatitis virus type B solution of horseradish peroxidase-labeled
(1) preparation of enzyme reaction object dilution
1, it takes Tris 4.846g, 2900 HCl μ l in beaker, purified water is then added in beaker, being sufficiently stirred makes reagent
It is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 4g is weighed to pour into above-mentioned beaker；
4, last beaker is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product；
(2) coupling of horseradish peroxidase (HRP) and anti-Pre-S1 antibody
1, former S 1 immune body of hepatitis virus type B 1mg is taken to be placed in 1ml glass tube；
2, the final concentration for taking 200 μ l DMSO lytic antibodies to make antibody reaches 5mg/ml, then mixes well；
3, the molar ratio that the disuccinimidyl suberate of 10mol is added according to 1mol antibody adds two succinyl of suberic acid
In the solution that imines ester is obtained to above-mentioned steps 2, reacted 1.5 hours in 37 DEG C of insulating boxs；
4, according to 3mol antibody be added 1mol HRP molar ratio HRP is added into the solution that above-mentioned steps 3 obtain, then plus
The pH for entering 1ml is PB buffer that 7.4 concentration are 0.1M, is placed in 37 DEG C of insulating boxs and reacts 3 hours；
5, the solution for preparing above-mentioned steps 4 PD-10 column purification collects refined solution, adds certainly according to the volume of 1:3000
The enzyme reaction object dilution of system, control are uniformly mixed finally using concentration in 0.03-5.0 μ g/ml up to enzyme reaction object；
Seven, the preparation of the former S 1 immune body of hepatitis virus type B solution of biotin labeling
(1) preparation of biotin reaction object dilution
1, it takes Tris 4.846g, 2847 HCl μ l in flask, purified water is then added in flask, being sufficiently stirred makes reagent
It is completely dissolved；
2, pH is adjusted, controls pH in 7.35-7.45；
3, BSA 4g is weighed to pour into above-mentioned flask；
4, last flask is settled to 400ml, is filtered with 0.2 μm of filter to obtain the final product；
(2) biotinylated antibody is taken, with biotin reaction object diluted, control is finally using concentration in 0.01~5.0 μ
G/ml mixes, biotin conjugate can be obtained.
2. the board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen according to claim 1, special
Sign is: 1 positive control of hepatitis B virus pro-S is that 1 positive serum of hepatitis B virus pro-S is diluted with the reference substance that pH value is 7.4
What liquid was prepared, dilution volume ratio is 1:5000.
3. the board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen according to claim 1, special
Sign is: the preparation process of the former S 1 immune body of hepatitis virus type B solution of the biotin labeling are as follows: the hepatitis B of biotin labeling
Former S 1 immune body is added in the biotin reaction object dilution that pH value is 7.4, is adjusted and is arrived convenient multiple, is uniformly mixed so as to obtain.
4. the board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen according to claim 1, special
Sign is: the preparation process of the coated ELISA Plate of anti-biotin antibodies: off-period is 2~3 hours, drying time 2
~4 hours；The preparation time of the coated ELISA Plate of anti-biotin antibodies was controlled at 5~7 hours.
5. the board-like chemoluminescence method detection kit of hepatitis B virus pre S 1 antigen according to claim 1, special
Sign is: the preparation process of the former S 1 immune body of hepatitis virus type B solution of the horseradish peroxidase-labeled are as follows: in horseradish peroxidating
The enzyme reaction object dilution that addition pH value is 7.4 in the former S 1 immune body of hepatitis virus type B solution of object enzyme label, adjusts and arrives convenient multiple,
It is uniformly mixed so as to obtain.
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