CN109900896A - A kind of enzyme-linked immunosorbent assay (ELISA) kit and preparation method thereof - Google Patents
A kind of enzyme-linked immunosorbent assay (ELISA) kit and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of enzyme-linked immunosorbent assay (ELISA) kits and preparation method thereof, including the coated microwell plate of Avidin, the HbsAb of biotin labeling, the HCV gene recombinant antigens of biotin labeling, the HIV gene recombinant antigens of biotin labeling, mouse anti-human igg, the HIV gene recombinant antigens of horseradish peroxidase-labeled, substrate A liquid, substrate B liquid, concentrated cleaning solution and the terminate liquid of the HbsAb of horseradish peroxidase-labeled, horseradish peroxidase label；The present invention can detect HBsAg, HCV virus IgG antibody and inhibition of HIV antibody in sample simultaneously with a kit.Blood source screening enzyme-linked immunosorbent assay (ELISA) kit have the characteristics that quickly, easy, accurate and high sensitivity, the whole operation time only need 2 hours can interpretation result, it is applied widely, be very suitable to carry out the screening of blood source.
Description
Technical field
The present invention relates to kit of a kind of blood source screening and preparation method thereof, in particular to a kind of enzyme linked immunological
Method detection kit and preparation method thereof.
Background technique
In severe haemorrhage or when with diseases such as severe anemia, leukaemia, myelofibrosises, people have to receive
Blood transfusion.But the danger of infected Other diseases is also taken on while blood transfusion.It is led in Transfusion Transmission disease, HBV, HCV,
HIV is the main virus to constitute a serious threat to transfusion safety, and HBV infection rate in population of China is up to 10.3%, therefore,
Seriously threaten the transfusion safety in China.HCV infection rate in population of China is 1.0%~3.1%, due to technical reason, to confession
There is also certain omission factors by blood person, and causing 70% Transfusion Transmission hepatitis is caused by HCV.The route of transmission of HIV first is that
A possibility that menses are propagated, and the blood infection HIV with HIV is had input is estimated to exceed 90% (on the contrary, the risk of a sexual intercourse is hundred
/ it is several to less than 1%), primary blood transfusion bring into inhibition of HIV amount be it is very big, will soon after infecting in this way
Development is AIDS, and average time is 3-5 (children are about 2 years), and in global HIV infection person, about 5%~10% is menses sense
Dye, China should also pay attention to threat of the HIV to transfusion safety.Therefore how to efficiently control and prevent above-mentioned three kinds of viral diseases
Diffusion and propagation in crowd are to be related to human health and following major issue, while also having obtained national governments and the Ministry of Public Health
The great attention of door.Propagated since three of the above virus can all be taken viruliferous blood and blood product, and the whole world it is annual because
It blood transfusion and suffers from the event of three of the above viral disease using the blood product of pollution and happens occasionally.Therefore, how screening blood
And three of the above virus in blood product, it is the big of happiness that affect the livelihood of every family the problem of blood transfusion and blood product safety to increase
Thing and one of the key subjects of clinical laboratory medicine research.In short, HBV, HCV, HIV in stringent screening blood source and blood product,
It is to prevent three of the above virus to be transfused blood and propagate a most key link using pollution blood product.
Summary of the invention
The purpose of the present invention is to solve above-mentioned existing defects, and providing one kind can qualitative detection human serum or blood plasma
In HBsAg, HCV virus IgG antibody and the enzyme-linked immunosorbent assay (ELISA) kit of inhibition of HIV antibody and preparation method thereof.
To achieve the above object, technical scheme is as follows:
A kind of enzyme-linked immunosorbent assay (ELISA) kit, which is characterized in that including the coated microwell plate of Avidin, biotin labeling
HbsAb, biotin labeling HCV gene recombinant antigens, the HIV gene recombinant antigens of biotin labeling, horseradish peroxidase
The HbsAb of enzyme label, the HIV genetic recombination of the mouse anti-human igg, horseradish peroxidase-labeled of horseradish peroxidase label are anti-
Original, substrate A liquid, substrate B liquid, concentrated cleaning solution and terminate liquid.
The microwell plate is polystyrene micropore plate as a preferred technical solution,.
The substrate A liquid is the citrate buffer containing 2% hydrogen peroxide as a preferred technical solution,.
The substrate B liquid is the citrate buffer containing 0.02%TMB as a preferred technical solution,.
The concentrated cleaning solution is containing 5%NaCl, 0.05%KCl and 5%Tween-20 as a preferred technical solution,
0.1mol/L phosphate buffer (pH6.3 ± 0.1).
The terminate liquid is 1M H as a preferred technical solution,2SO4Solution.
A kind of preparation method of enzyme-linked immunosorbent assay (ELISA) kit, which comprises the following steps:
A) preparation and encapsulation of plate are coated with；
B) preparation and packing of enzyme conjugates: by the HBsAb of horseradish peroxidase-labeled, mouse anti-human igg, HIV gene
Recombinant antigen is pressed with the enzyme mark dilution for containing 0.12 ‰ marennins with tack lid plastic bottle (blue) after quality inspection is qualified respectively
11ml/ bottles of packing, labeling are set 2~8 DEG C of semifinished product warehouses and are saved；
C) sample diluting liquid preparation and packing: sample diluting liquid is prepared by sample diluting liquid formula, is used after quality inspection is qualified
Tack lid plastic bottle (white) dispenses, labeling by 11ml/ bottles, sets 2~8 DEG C of semifinished product warehouses and saves；
D) preparation of substrate A liquid and packing: the citrate buffer containing 2% hydrogen peroxide is dripped after quality inspection is qualified with green lid
Bottle is set 2~8 DEG C of semifinished product warehouses and is saved by 6ml/ bottles of packing, labeling；
E) preparation of substrate B liquid and packing: the citrate buffer containing 0.02%TMB, with black lid drop bottle after quality inspection is qualified
(black) is set 2~8 DEG C of semifinished product warehouses and is saved by 6ml/ bottles of packing, labeling；
F) concentrated cleaning solution preparation and packing: the 0.1mol/L phosphoric acid containing 5%NaCl, 0.05%KCl and 5%Tween-20
Salt buffer (pH6.3 ± 0.1)；(transparent) by 30ml/ bottles of packing with tack lid plastic bottle, labeling sets 2~8 DEG C of semifinished product warehouses
It saves；
G) terminate liquid preparation and packing: 1M H2SO4Solution, with yellow lid drop bottle by 6ml/ bottles of packing, labeling sets 2~8 DEG C half
Warehouse for finished product saves.
H) in semifinished product warehouse coating plate and each encapsulation liquid carries out QA sampling and mounted box is saved into warehouse for finished product, into one
It lets pass after step QA sampling is qualified.
As the optimal technical scheme in above-mentioned preparation method, in the step a) preparation and encapsulation of coating plate include with
Lower step:
1) it is coated with: Avidin is diluted by determining working concentration with 0.05M carbonate buffer solution (pH9.6 ± 0.1).Through
It is coated with after examining qualified microwell plate to draw brown mark, 100 ± 5 hole μ l/ of package amount, stays overnight (16~26 in 33~37 DEG C of absorption
Hour)；
2) board-washing: continuous board-washing is not impregnated 2 times by 300 ± 50 holes μ l/ with working concentration washing lotion；
3) it closes: being closed 3~6 hours for 33~37 DEG C with confining liquid with 120 ± 5 hole μ l/；
4) dry and encapsulation: knockout plate, button is dry, dry；Immediately with compound film coiled material or aluminium after being dried confirmation and parching
Film bag adds desiccant to be packaged, and guarantees that aluminum foil bag envelope is closely knit, completely, and it is labelled, it sets 2~8 DEG C of semifinished product warehouses and protects
It deposits.
As the optimal technical scheme in above-mentioned preparation method, the confining liquid is made of following material in mass ratio: boron
Acid (H3BO3) 3.92g, borax (Na2B4O7.10H2O) 3.26g, sucrose 50g, BSA 10g, Proclin3001ml plus purified water
To 1000ml.
As the optimal technical scheme in above-mentioned preparation method, enzyme mark dilution is in mass ratio by following in the step b)
Material is made: boric acid (H3BO3) 3.92g, borax (Na2B4O7·10H2O) 3.26g, sucrose 50g, calf serum 200ml,
Proclin3001ml adds purified water to 1000ml.
As the optimal technical scheme in above-mentioned preparation method, sample diluting liquid is in mass ratio by following in the step c)
Material is made: Na2HPO4·12H2O 3.0g、NaH2PO4·2H2O 4.2g, NaCl 8.5g, Proclin3001ml, purified water
Add to 1000ml.
Further, this kit uses enzyme-linked immunization, is coated with Avidin on microwell plate；It is affine in solid phase when detection
Element will combine HBsAb/HCV gene recombinant antigens/HIV gene recombinant antigens of biotin labeling；The HBsAb/ of biotin labeling
HCV gene recombinant antigens/HIV gene recombinant antigens virus IgG antibody/inhibition of HIV antibody combines, and it is anti-to form antigen/antibody-
Body/antigenic compound, by washing away other unbonded substances；It is anti-to add horseradish peroxidase-labeled HBsAb/ mouse
Human IgG/HIV gene recombinant antigens, by formation " sandwich " sandwich complex, by washing away other unbonded substances；
After substrate is added, substrate becomes color products by horseradish peroxidase enzyme catalytic；After concentrated sulfuric acid termination reaction is added, microplate reader is used
Absorbance (A value) is surveyed in 450nm or 450nm/630nm wavelength, so that the HBsAg, HCV virus IgG in qualitative detection sample are anti-
Body and inhibition of HIV antibody.
The invention has the benefit that HBsAg, the HCV virus IgG that can be detected in sample simultaneously with a kit are anti-
Body and inhibition of HIV antibody.Screening enzyme-linked immunosorbent assay (ELISA) kit in blood source has quick, easy, accurate and high sensitivity spy
Point, the whole operation time only need 2 hours can interpretation result, it is applied widely, be very suitable to carry out the screening of blood source.
Detailed description of the invention
Fig. 1 is a kind of production technological process of enzyme-linked immunosorbent assay (ELISA) kit of the present invention and preparation method thereof.
Specific embodiment
The effect of to make to structure feature of the invention and being reached, has a better understanding and awareness, to preferable
Examples and drawings cooperation detailed description, is described as follows:
As shown in Figure 1, a kind of enzyme-linked immunosorbent assay (ELISA) kit, including the coated microwell plate of Avidin, biotin labeling
HbsAb, biotin labeling HCV gene recombinant antigens, the HIV gene recombinant antigens of biotin labeling, horseradish peroxidase
The HbsAb of enzyme label, the HIV genetic recombination of the mouse anti-human igg, horseradish peroxidase-labeled of horseradish peroxidase label are anti-
Original, substrate A liquid, substrate B liquid, concentrated cleaning solution and terminate liquid.
In the present embodiment, microwell plate is polystyrene micropore plate.
In the present embodiment, substrate A liquid is the citrate buffer containing 2% hydrogen peroxide.
In the present embodiment, substrate B liquid is the citrate buffer containing 0.02%TMB.
In the present embodiment, concentrated cleaning solution is the 0.1mol/L phosphorus containing 5%NaCl, 0.05%KCl and 5%Tween-20
Phthalate buffer (pH6.3 ± 0.1).
In the present embodiment, terminate liquid is 1M H2SO4Solution.
A kind of preparation method of enzyme-linked immunosorbent assay (ELISA) kit, comprising the following steps:
A) preparation and encapsulation of plate are coated with；
B) preparation and packing of enzyme conjugates: by the HBsAb of horseradish peroxidase-labeled, mouse anti-human igg, HIV gene
Recombinant antigen is pressed with the enzyme mark dilution for containing 0.12 ‰ marennins with tack lid plastic bottle (blue) after quality inspection is qualified respectively
11ml/ bottles of packing, labeling are set 2~8 DEG C of semifinished product warehouses and are saved；
C) sample diluting liquid preparation and packing: sample diluting liquid is prepared by sample diluting liquid formula, is used after quality inspection is qualified
Tack lid plastic bottle (white) dispenses, labeling by 11ml/ bottles, sets 2~8 DEG C of semifinished product warehouses and saves；
D) preparation of substrate A liquid and packing: the citrate buffer containing 2% hydrogen peroxide is dripped after quality inspection is qualified with green lid
Bottle is set 2~8 DEG C of semifinished product warehouses and is saved by 6ml/ bottles of packing, labeling；
E) preparation of substrate B liquid and packing: the citrate buffer containing 0.02%TMB, with black lid drop bottle after quality inspection is qualified
(black) is set 2~8 DEG C of semifinished product warehouses and is saved by 6ml/ bottles of packing, labeling；
F) concentrated cleaning solution preparation and packing: the 0.1mol/L phosphoric acid containing 5%NaCl, 0.05%KCl and 5%Tween-20
Salt buffer (pH6.3 ± 0.1)；(transparent) by 30ml/ bottles of packing with tack lid plastic bottle, labeling sets 2~8 DEG C of semifinished product warehouses
It saves；
G) terminate liquid preparation and packing: 1M H2SO4Solution, with yellow lid drop bottle by 6ml/ bottles of packing, labeling sets 2~8 DEG C half
Warehouse for finished product saves.
H) in semifinished product warehouse coating plate and each encapsulation liquid carries out QA sampling and mounted box is saved into warehouse for finished product, into one
It lets pass after step QA sampling is qualified.
In the present embodiment, in step a) coating plate preparation and encapsulation the following steps are included:
1) it is coated with: Avidin is diluted by determining working concentration with 0.05M carbonate buffer solution (pH9.6 ± 0.1).Through
It is coated with after examining qualified microwell plate to draw brown mark, 100 ± 5 hole μ l/ of package amount, stays overnight (16~26 in 33~37 DEG C of absorption
Hour)；
2) board-washing: continuous board-washing is not impregnated 2 times by 300 ± 50 holes μ l/ with working concentration washing lotion；
3) it closes: being closed 3~6 hours for 33~37 DEG C with confining liquid with 120 ± 5 hole μ l/；
4) dry and encapsulation: knockout plate, button is dry, dry；Immediately with compound film coiled material or aluminium after being dried confirmation and parching
Film bag adds desiccant to be packaged, and guarantees that aluminum foil bag envelope is closely knit, completely, and it is labelled, it sets 2~8 DEG C of semifinished product warehouses and protects
It deposits.
In the present embodiment, confining liquid is made of following material in mass ratio: boric acid (H3BO3) 3.92g, borax
(Na2B4O7.10H2O) 3.26g, sucrose 50g, BSA 10g, Proclin3001ml plus purified water are to 1000ml.
In the present embodiment, enzyme mark dilution is made of following material in mass ratio in step b): boric acid (H3BO3)
3.92g, borax (Na2B4O7·10H2O) 3.26g, sucrose 50g, calf serum 200ml, Proclin3001ml, add purified water extremely
1000ml。
In the present embodiment, sample diluting liquid is made of following material in mass ratio in step c): Na2HPO4·12H2O
3.0g、NaH2PO4·2H2O 4.2g, NaCl 8.5g, Proclin3001ml, purified water add to 1000ml.
In the production process for carrying out above-described embodiment, it should be further noted that the determination to manufacturing condition,
Determination including (1) the coating time, temperature, package amount
Avidin is configured to coating buffer by determining use concentration, compares different coating times, temperature, package amount respectively
The height of lower five parts of inner quality controls A value determines optimal coating time, temperature, package amount.The coating time is finally set to 16-
26 hours, coating temperature be set to 33-37 DEG C, coating buffer liquid volume added be set to 100 ± 5 μ l.
(2) determination of formula of liquid is closed
In conjunction with the closing formula of liquid of company's existing product, using 5% sucrose, 2% liquid gelatin as quantitative, pass through reality
It tests and determines optimal buffer system, the additional amount of optimal casein sodium and aminopyrine.Finally the formula of confining liquid is set to
Boric acid (H3BO3) 3.92g, borax 3.26g, sucrose 50g, bovine serum albumin 10g, Proclin3000.5ml add purified water extremely
1000ml。
(3) determination of capping temperature and time
Coating plate after closing is individually positioned in refrigerator (2~8 DEG C), room temperature (23~28 DEG C), incubator (33~37 DEG C)
Capping is carried out, action time is all 18~20 hours, determines the Best Times reacted again after determining optimal reaction temperature；
Using internal quality-control product as research material, optimal capping temperature and time is determined with the height of internal quality-control product A value.Most
33~37 DEG C are determined eventually to close 3~6 hours.
(4) determination re-closed after board-washing and confining liquid liquid volume added
The confining liquid of different liquid volume addeds is added in pre-coated plate；And it by using the hole washing lotion 300ul/, does not impregnate and continuously washes
It is re-closed after plate 2 times, it sets 33-37 DEG C and closes 3~6 hours, using inner quality control as research material, with the height of internal quality-control product A value
It is low to determine optimal confining liquid liquid volume added.It is final to determine that confining liquid liquid volume added is set to 120 ± 5 holes μ l/.
(5) determination of drying process
Using internal quality-control product as research material, closing is completed, and after plate button is dry, it is dry to be sent into dry plate room；In different dried strips
Under part it is dry after encapsulated with aluminum foil bag, after setting 37 DEG C of examinations 6 days, optimal drying is determined with the height of internal quality-control product A value
Time.It is final to determine that drying time is 5 to 26 hours.
(6) preparation of HBsAb, HCV gene recombinant antigens, HIV gene recombinant antigens of biotin labeling
The biotin of activation is mixed into rower with HBsAb, HCV gene recombinant antigens, HIV gene recombinant antigens respectively
Note, the unbonded biotin of dialysis removal, respectively obtains HBsAb, HCV gene recombinant antigens, the HIV gene weight of biotin labeling
Group antigen weight is former.
(7) preparation of horseradish peroxidase-labeled HBsAb, mouse anti-human igg, HIV gene recombinant antigens
Using Over-voltage protection, by the horseradish peroxidase of activation respectively with HBsAb, mouse anti-human igg, HIV gene weight
Group antigen is marked, the unbonded horseradish peroxidase of dialysis removal, and HBsAb, the mouse for respectively obtaining enzyme label are anti-human
IgG, HIV gene recombinant antigens
(8) determination of reaction temperature
At a temperature of differential responses, optimal reaction temperature is determined with the height of internal quality-control product A value.In order to guarantee to try
Reaction temperature is finally set to 37 DEG C by the sensitivity and specificity of agent box.
(9) determination in sample reaction time
Under the differential responses time, the optimal sample reaction time is determined with the height of internal quality-control product A value.In order to protect
Product quality is demonstrate,proved, is finally set to the sample reaction time 60 minutes.
(10) determination in enzyme conjugates reaction time
Under the differential responses time, the optimal enzyme conjugates reaction time is determined with the height of internal quality-control product A value.For
In the enzyme conjugates reaction time, be finally set to 30 minutes by guarantee product quality.
(11) determination of substrate A liquid and substrate B liquid liquid volume added and reaction time
Under the substrate A liquid and substrate B liquid difference liquid volume added, A, B liquid liquid feeding are determined with the height of internal quality-control product A value
Amount, is finally set to 50 μ l+50 μ l for the sample-adding amount of substrate A liquid and substrate B liquid.Then in substrate A liquid and substrate B liquid differential responses
Under time, the reaction time of A, B liquid is determined with the height of internal quality-control product A value, finally by the anti-of substrate A liquid and substrate B liquid
It is set to 15 minutes between seasonable.
(12) terminate liquid sample-adding amount and terminate after readings effective time determine
The terminate liquid of different liquid measures is added, terminate liquid is determined with the height of internal quality-control product A value, finally by terminate liquid
Sample-adding amount is set to 50 μ l.A value is read in different time respectively after adding terminate liquid, is come with the height of internal quality-control product A value true
Determine the effective time of readings after reaction terminating, in order to guarantee the accuracy of experimental result, should after termination readings in 15 minutes.
In the present embodiment, the method for inspection of this kit includes the following steps,
Sample-adding: coating plate is taken out, 50 hole μ l/ of sample is added, it is anti-to add biotin labeling HBsAb/HCV genetic recombination
50 hole μ l/ of original/HIV gene recombinant antigens mixes；After covering sealing plate glue, sets 37 DEG C and incubate 60 minutes；
Board-washing: using cleaning solution board-washing 5 times, stands 1 minute every time, and button is dry；
Enzyme conjugate: HBsAb/ mouse anti-human igg/HIV gene recombinant antigens of horseradish peroxidase-labeled original are added
50 holes μ l/.After covering sealing plate glue, 37 DEG C of Incubation in dark are set 30 minutes；
Board-washing: ibid method is washed 5 times, and button is dry；
Colour developing and termination: adding each hole 50 μ l/ of substrate A, B liquid, and after 37 DEG C are protected from light colour developing 15 minutes, 50 μ l/ of terminate liquid is added
Hole；
Measurement: with microplate reader 450nm (Single wavelength) or 450nm/630nm (dual wavelength) with blank well school zero, each hole is measured
Absorbance (A) value.
Finally inspection result is determined, i.e., sample A value < critical value (Cut-off value) is judged to feminine gender；Sample A value >=
Critical value (Cut-off value) is judged to the positive.
The requirement further to test samples in the method for inspection is needed to be illustrated, i.e., serum sample is according to a conventional method by quiet
Arteries and veins acquisition；Plasma sample can be handled using heparin, sodium citrate, EDTA；The sample measured in 5 days can place 4 DEG C of guarantors
It deposits；Sample is placed on -20 DEG C and can at least save 3 months；Sample avoids haemolysis or multigelation；Sample that is muddy or having precipitating
Originally it to be detected again after clarification into after crossing centrifugation or filter.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and what is described in the above embodiment and the description is only the present invention
Principle, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these variation and
Improvement is both fallen in the range of claimed invention.The present invention claims protection scope by appended claims and its
Equivalent defines.
Claims (11)
1. a kind of enzyme-linked immunosorbent assay (ELISA) kit, which is characterized in that including Avidin coated microwell plate, biotin labeling
HbsAb, the HCV gene recombinant antigens of biotin labeling, the HIV gene recombinant antigens of biotin labeling, horseradish peroxidase
The HbsAb of label, the mouse anti-human igg of horseradish peroxidase label, the HIV gene recombinant antigens of horseradish peroxidase-labeled,
Substrate A liquid, substrate B liquid, concentrated cleaning solution and terminate liquid.
2. a kind of enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, it is characterised in that: the microwell plate is polyphenyl
Ethylene microwell plate.
3. a kind of enzyme-linked immunosorbent assay (ELISA) kit according to claim 2, it is characterised in that: the substrate A liquid be containing
The citrate buffer of 2% hydrogen peroxide.
4. a kind of enzyme-linked immunosorbent assay (ELISA) kit according to claim 3, it is characterised in that: the substrate B liquid be containing
The citrate buffer of 0.02%TMB.
5. a kind of enzyme-linked immunosorbent assay (ELISA) kit according to claim 4, it is characterised in that: the concentrated cleaning solution is
0.1mol/L phosphate buffer (pH6.3 ± 0.1) containing 5%NaCl, 0.05%KCl and 5%Tween-20.
6. a kind of enzyme-linked immunosorbent assay (ELISA) kit according to claim 5, it is characterised in that: the terminate liquid is 1M
H2SO4Solution.
7. a kind of preparation method of the enzyme-linked immunosorbent assay (ELISA) kit as described in claim 1~6 is any, which is characterized in that
The following steps are included:
A) preparation and encapsulation of plate are coated with；
B) preparation and packing of enzyme conjugates: by the HBsAb of horseradish peroxidase-labeled, mouse anti-human igg, HIV genetic recombination
Antigen is respectively with the enzyme mark dilution for containing 0.12 ‰ marennins, with tack lid plastic bottle (blue) after quality inspection is qualified, by 11ml/ bottles
Packing, labeling are set 2~8 DEG C of semifinished product warehouses and are saved；
C) sample diluting liquid preparation and packing: sample diluting liquid is prepared by sample diluting liquid formula, uses tack after quality inspection is qualified
Lid plastic bottle (white) dispenses, labeling by 11ml/ bottles, sets 2~8 DEG C of semifinished product warehouses and saves；
D) preparation of substrate A liquid and packing: the citrate buffer containing 2% hydrogen peroxide is pressed after quality inspection is qualified with green lid drop bottle
6ml/ bottles of packing, labeling are set 2~8 DEG C of semifinished product warehouses and are saved；
E) preparation of substrate B liquid and packing: the citrate buffer containing 0.02%TMB is (black with black lid drop bottle after quality inspection is qualified
Color) by 6ml/ bottles of packing, labeling is set 2~8 DEG C of semifinished product warehouses and is saved；
F) concentrated cleaning solution preparation and packing: the 0.1mol/L phosphate containing 5%NaCl, 0.05%KCl and 5%Tween-20 is slow
Fliud flushing (pH6.3 ± 0.1)；(transparent) by 30ml/ bottles of packing with tack lid plastic bottle, labeling is set 2~8 DEG C of semifinished product warehouses and is saved；
G) terminate liquid preparation and packing: 1M H2SO4Solution, with yellow lid drop bottle by 6ml/ bottles of packing, labeling sets 2~8 DEG C of semi-finished product
Library saves.
H) to the coating plate in semifinished product warehouse and respectively, simultaneously mounted box is saved into warehouse for finished product for encapsulation liquid progress QA sampling, to further QA
It lets pass after sampling is qualified.
8. the preparation method of enzyme-linked immunosorbent assay (ELISA) kit according to claim 7, it is characterised in that: the step a)
It is middle coating plate preparation and encapsulation the following steps are included:
1) it is coated with: Avidin is diluted by determining working concentration with 0.05M carbonate buffer solution (pH9.6 ± 0.1).Through examining
Qualified microwell plate is coated with after drawing brown mark, 100 ± 5 hole μ l/ of package amount, and in 33~37 DEG C of absorption, (16~26 is small overnight
When)；
2) board-washing: continuous board-washing is not impregnated 2 times by 300 ± 50 holes μ l/ with working concentration washing lotion；
3) it closes: being closed 3~6 hours for 33~37 DEG C with confining liquid with 120 ± 5 hole μ l/；
4) dry and encapsulation: knockout plate, button is dry, dry；Immediately with compound film coiled material or aluminum foil bag after being dried confirmation and parching
Add desiccant to be packaged, and guarantees that aluminum foil bag envelope is closely knit, completely, and it is labelled, it sets 2~8 DEG C of semifinished product warehouses and saves.
9. the preparation method of enzyme-linked immunosorbent assay (ELISA) kit according to claim 8, it is characterised in that: the confining liquid
It is made in mass ratio of following material: boric acid (H3BO3) 3.92g, borax (Na2B4O7.10H2O) 3.26g, sucrose 50g, BSA
10g, Proclin300 1ml, add purified water to 1000ml.
10. the preparation method of enzyme-linked immunosorbent assay (ELISA) kit according to claim 7, it is characterised in that: the step
B) enzyme mark dilution is made of following material in mass ratio in: boric acid (H3BO3) 3.92g, borax (Na2B4O7·10H2O)
3.26g, sucrose 50g, calf serum 200ml, Proclin300 1ml, add purified water to 1000ml.
11. the preparation method of enzyme-linked immunosorbent assay (ELISA) kit according to claim 7, it is characterised in that: the step
C) sample diluting liquid is made of following material in mass ratio in: Na2HPO4·12H2O 3.0g、NaH2PO4·2H2O 4.2g、
NaCl 8.5g, Proclin300 1ml, purified water add to 1000ml.
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| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof |
| CN111289745A (en) * | 2020-03-25 | 2020-06-16 | 中山生物工程有限公司 | Novel coronavirus IgM antibody enzyme-linked immunoassay kit and detection method thereof |
| CN112255413A (en) * | 2020-09-10 | 2021-01-22 | 中国人民解放军联勤保障部队第九0三医院 | Anti-transmembrane protein antibody microporous plate for detecting serum of HCV (hepatitis C Virus) infected person, chemiluminescence kit and detection method |
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| CN112255414A (en) * | 2020-09-10 | 2021-01-22 | 中国人民解放军联勤保障部队第九0三医院 | Chemiluminescence kit for detecting anti-nonstructural protein antibody in serum of HCV (hepatitis C Virus) infected person and detection method |
| CN112255411A (en) * | 2020-09-10 | 2021-01-22 | 中国人民解放军联勤保障部队第九0三医院 | Chemiluminescence kit for detecting anti-nucleocapsid protein antibody in serum of HCV (hepatitis C Virus) infected person and detection method |
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