CN101363853A - Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same - Google Patents
Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same Download PDFInfo
- Publication number
- CN101363853A CN101363853A CN 200710119983 CN200710119983A CN101363853A CN 101363853 A CN101363853 A CN 101363853A CN 200710119983 CN200710119983 CN 200710119983 CN 200710119983 A CN200710119983 A CN 200710119983A CN 101363853 A CN101363853 A CN 101363853A
- Authority
- CN
- China
- Prior art keywords
- antibody
- kit
- immunodeficiency virus
- human immunodeficiency
- hiv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention relates to the immunization analysis medical field, and particularly provides a kit for determining the human immunodeficiency virus (HIV) antigen/antibody by the chemoluminescent immunization analysis and a preparation method thereof. The kit comprises (1) a solid carrier coated by the HIV antigens and antibodies; (2) biotin-labeled HIV antibodies; (3) enzyme-labeled HIV antigens and streptavidin; (4) a chemoluminescent primer acting with the above enzyme; and (5) a contrast. The preparation method of the kit includes the following steps of (1) coating the solid carrier with the HIV antigens and antibodies; (2) labeling the HIV antigens with a biotin; (3) labeling the HIV antigens and the streptavidin with the enzyme; (4) preparing the chemoluminescent primer, (5) preparing the contrast; (6) separately packaging; and (7) assembling into a finished product. The kit has the advantages of simpliness, rapidness, sensitiveness, stability and the like.
Description
Technical field
The present invention relates to the immunoassay medical domain, particularly, the invention provides a kind of human immunodeficiency virus antigen/antibody and measure kit and preparation method thereof.
Background technology
Acquired immune deficiency syndrome (AIDS), be that (Acquired Immure Deficiency Syndrome AIDS), is by human immunodeficiency virus (human immunodeficiency virus to aids, HIV) cause the chronic progressive disease that still can not cure at present, case fatality rate is high.According to the difference of acquired immune deficiency syndrome (AIDS) clinical manifestation, it can be divided into four periods of acute infection period, latent period, pre-AIDS, typical AIDS stadium.The AIDS of acute infection period only shows as light symptoms such as heating, fash, enlargement of lymph nodes, weak, perspiration, nausea,vomiting,diarrhea, pharyngitis usually, most patients are after HIV1/2 infected for 2～6 weeks, HIV antibody can occur in the serum, can be used as the important indicator of early diagnosis and disorder in screening.Acute stage symptom continued after 3～14 days, and the asymptomatic latent period of (average 2～10 years) of a varying length appears in AIDS, and HIV virus continues breeding, has strong destruction, and the patient does not but have any clinical symptoms.Carry out examination widely, particularly hold blood transfusion and close, can reduce the probability that AIDS propagates greatly.After latent period, disease continues development and promptly enters pre-AIDS and typical AIDS stadium, and the infected causes various infection owing to immunodeficiency, the later stage of course of disease development, various opportunistic infections and malignant tumour appear in the infected, and serious consequence and high mortality ratio take place.
HIV virus is a kind of reverse transcription RNA viruses, and common is the HIV-1 type, and HIV-2 type virus is comparatively rare.HIV virus is spherical in shape under the Electronic Speculum, diameter 100～120nm, as seen the coniform core of a densification, include viral RNA molecule and enzyme (reverse transcriptase, integrase, proteinase), the outer cyst membrane of virus is the lipid bilayer protein film, wherein (HIV-1 type) is embedded with gp120 and gp41, forms furcella and transmembrane protein (the corresponding albumen of HIV-2 type is gp36) respectively.The cyst membrane inner face is the capsid that P17 albumen constitutes, and core protein (P24) parcel RNA is arranged in it.The HIV-1+2 type is mainly invaded the mankind and is had CD4
+The cell of acceptor, particularly CD4
+T assists lymphocyte, causes the human immunity system defect, final generator opportunistic infections and malignant tumour.HIV is to mononuclear macrophage, CD8
+T lymphocyte and bone-marrow-derived lymphocyte also have destruction, also play a part certain in disease generation, development, prognosis.
Present AIDS etiological diagnosis technology mainly comprises special viral antibody Anti-HIV detection, viral antigen detection, the separation of HIV virus, HIV virus load mensuration and HIV nucleic acid determination etc.Antibody test is the conventional sense method, comprises enzyme linked immune assay (ELISA), gelatin particle agglutination test (PA), immunofluorescent test (IFA) and Dot blot (chromatography or diafiltration) and western blot test (WB) etc.Western blot test (WB) is as confirmatory test, and other method is used for screening experiment more.Method beyond antiviral antibody detects generally is used in special circumstances, and for example, p24 Detection of antigen experimental applications infects in acute HIV.
The ELISA method that detects anti-HIV be use in the world the earliest, detection technique with fastest developing speed, that in the examination of blood source, generally use so far.ELISA the 3rd generation technique has adopted " dual-antigen sandwich method ", not only can detect anti-HIV IgG, and can detect the IgM antibody of early stage appearance, even can detect rare " O " hypotype HIV antibody.1998, the 4th generation ELISA reagent appears in the world, when detecting anti-HIV, also can detect p24 antigen.
(Chemiluminescence Immunoassay's chemiluminescence immune assay CLIA) came out in 1977, and first generation chemiluminescence immunoassay kit was succeeded in developing and put on market in 1985.Enter the nineties, the production of the development of chemical luminescence immune analysis reagent box and automatic measurement instrument has obtained breakthrough, thereby enters the high speed development stage.Chemiluminescence immune assay is a new immuno analytical method that grows up after fluorescence, radioactive isotope and EIA enzyme immunoassay, according to lot of experiment results and clinical practice data, from practicality, stability, accuracy and development prospect, chemiluminescence immune assay maintains the leading position in nonradioactive labeling's analytical technology, the direction and the trend of world today's development have been represented, it not only has immunoreactive specificity, and (detection limit can reach 10 to have the high sensitivity of chemiluminescence reaction
-15～10
-18Mol/L).Advantages such as that chemiluminescence immunoassay technology has is highly sensitive, quick, accurate, good reproducibility, the effect phase is long and safety non-toxic is pollution-free become the first-selection that replaces radiommunoassay and EIA enzyme immunoassay technology.
Chemiluminescence enzyme immunoassay is to carry out immune response with enzyme labeling bioactivator (as the antigen or the antibody of enzyme labeling), enzyme on the immune response compound remakes and is used for luminous substrate, luminous under the signal reagent effect, carry out luminescence assays with the luminous signal analyzer.Marker enzyme commonly used at present is horseradish peroxidase (HRP), the substrate that HRP is commonly used is luminol (the amino phthalylhydrazine of 3-, luminol) or derivatives thereof such as different luminol (the amino phthalylhydrazine of 4-) etc., the oxidation reaction of luminol is carried out in alkaline buffer, in the presence of peroxidase and active oxygen, generate the excited state intermediate, luminous when it gets back to ground state, its wavelength is 425nm.Luminous intensity depends on the concentration of enzyme in the enzyme immune reaction thing.
The sensitivity that biotin-avidin immunity amplifying technique can greatly improve detection method, and high specificity, stability is high, and applied widely, experimental cost is low.Biotin-avidin immunity amplifying technique has two kinds of fundamental types, one class is an intermediate with free Avidin, connect respectively and comprise macromolecular reaction system to be measured of biotin and mark biotin, after developed the avidin-biotin-complex technology on this basis; Another kind of is directly to connect biotinylation macromolecular reaction system with the mark Avidin to detect, and is called mark Avidin-biotin method.According to used in the reaction system to be measured be biotinylation first antibody or biotinylation second antibody, be divided into direct method and indirect method again.In indirect method, Avidin-biotin system can with the coupling of immunoassay detection architecture, amplify end reaction: will react simultaneously at the insolubilized antibodies of different antigenic determinants and biotinylated antibody and antigen (standard antigen and determined antigen) earlier, form the double-antibody sandwich immune complex at surface of solid phase carriers, adding Avidin again combines with biotin in the compound, reaction signal is amplified, thus the sensitivity that has improved immunoassay.
Chemiluminescence immunoassay is a kind of than elder generation and then effective method in conjunction with biotin one Avidin immunity amplifying technique.Chemiluminescence detection needs certain luminescence enhancer, and is furnished with superoxide enzyme system separately.Mainly be that biotin combines with affinity element, peroxidase (or alkaline phosphatase), pass through chemiluminescence and humidification again, light quantity is amplified the back detect.
Biotin-Avidin immunity amplifying technique is applied in the sensitivity that can improve detection in the HIV immunoassay greatly in conjunction with chemiluminescence immunoassay technology.In addition, chemical luminescence immune analysis reagent box of the prior art is the luminous measuring system of closed full-automatic chemical, needs the expensive luminous measuring instrument of full-automatic chemical, promotes the use of thereby limited, and can't be widely used in clinical diagnosis and research work.Kit of the present invention adopts the microwell plate chemiluminescence immunoassay technology, both can be applicable to the open luminous measuring instrument of semi-automatic chemistry, also can be used for full automatic measuring system, can realize fast detection the in enormous quantities, and use cost is low, easier applying.
Summary of the invention
The present inventor proposes and has finished the present invention in order to address the above problem.
The purpose of this invention is to provide the kit of a kind of biotin-Avidin immunity amplifying technique in conjunction with chemiluminescence enzyme immunoassay technical measurement human immunodeficiency virus antigen/antibody.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit according to the present invention comprises:
1) human immunodeficiency virus antigen and antibody wrap the solid phase carrier of quilt simultaneously;
2) biotin labeled human immune defect virus antibody;
3) human immunodeficiency virus antigen of enzyme labeling and Streptavidin;
4) chemical luminous substrate that above-mentioned enzyme acted on; And
5) negative control thing, human immunodeficiency virus antigen and antibody positive tester.
Particularly, kit of the present invention can comprise:
1) human immunodeficiency virus HIV1+2 type antigen and HIV-1P24 antibody wrap the solid phase carrier of quilt simultaneously;
2) biotin labeled HIV-1P24 antibody;
3) the HIV1+2 type antigen and the Streptavidin of enzyme labeling;
4) chemical luminous substrate that above-mentioned enzyme acted on; And
5) negative control thing, HIV antibody HIV-1 P24 antigen positive tester.
According to kit of the present invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
According to kit of the present invention, wherein, described marker enzyme is horseradish peroxidase or alkaline phosphatase.
According to kit of the present invention, wherein, described chemical luminous substrate is luminol, different luminol or 1,2-two oxidative ethane analog derivatives.
Preferably, described 1 in the mentioned reagent box, 2-two oxidative ethane analog derivatives are (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.
The present invention provides a kind of chemical luminous substrate liquid of original creation simultaneously, and it is prepared from by following method:
A liquid: in distilled water, add the Tris-HCl damping fluid that Tris and dense HCl are made into 0.1M pH8.5.In this damping fluid, add Luminol, Tween20 and benzofluoranthrene (structure is as shown below), mix.
B liquid: in distilled water, add trisodium citrate and citric acid, be mixed with the citrate buffer solution of 0.1M pH4.6, in this solution, add 30% superoxol.
Before using A liquid being mixed the back with B liquid in the 1:1 ratio uses.
Thus, according to kit of the present invention, described chemical luminous substrate comprises A liquid and B liquid, wherein,
Based on the described chemical luminous substrate A of 100mL liquid, comprise 1.21g Tris, 295 μ L concentrated hydrochloric acids, 0.5g luminol, 20 μ L Tween20 and 0.1g benzofluoranthrene;
Based on the described chemical luminous substrate B of 100mL liquid, comprise the H of 0.73g trisodium citrate, 0.44g citric acid, 100 μ L30%
2O
2Solution, its pH value is 4.6.
The invention provides a kind of method for preparing the mentioned reagent box, may further comprise the steps:
1) wraps simultaneously by solid phase carrier with human immunodeficiency virus antigen and antibody;
2) with the biotin labeling human immune defect virus antibody;
3) with enzyme labeling human immunodeficiency virus antigen and Streptavidin;
4) the preparation chemical luminous substrate that above-mentioned enzyme acted on;
5) preparation negative control thing, human immunodeficiency virus antigen and antibody positive tester;
6) chemical luminous substrate that human immunodeficiency virus antigen and Streptavidin and this enzyme acted on of above-mentioned feminine gender of packing and positive control, biotin labeled human immune defect virus antibody, enzyme labeling; And
7) be assembled into finished product.
The method according to this invention, preferred, described bag be may further comprise the steps by the step of solid phase carrier:
I) bag quilt
The preparation coating buffer, comprise the natrium carbonicum calcinatum of 1.59g and the sodium bicarbonate of 2.94g based on the described coating buffer of 1000mL, the pH value of described coating buffer is 9.6, coating buffer with preparation mixes with the human immunodeficiency virus antigen and the antibody of debita spissitudo then, and the gained mixed liquor is carried on the solid phase carrier;
II) with PBST washing lotion washing above-mentioned solid phase carrier; And
III) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g BSA, 20g sucrose, 100mL NBCS and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
In said method, preferred, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
In said method, preferred, described enzyme is horseradish peroxidase or alkaline phosphatase.
In said method, preferred, described chemical luminous substrate is luminol, different luminol or 1,2-two oxidative ethane analog derivatives.
In said method, preferred, described 1,2-two oxidative ethane analog derivatives are (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.
Particularly, kit of the present invention comprises that the chemiluminescence bag that is coated with HIV1+2 type antigen and HIV-1P24 antibody simultaneously is by plate 48 or 96 holes, 2 bottles of labels, 2 bottles of chemical luminous substrate liquid, 1 bottle of cleansing solution, 1 bottle of negative control, 1 bottle of HIV antibody positive contrast, the contrast of P24 antigen positive 1 bottle and other auxiliary reagent.Wherein, particularly, label 1 contains biotin labeled P24 antibody; Label 2 contains the HIV antigen and the Streptavidin of enzyme labeling.Wherein, particularly, cleansing solution is 20 times of concentrated cleaning solutions, by 4g/LNaH
2PO
42H
2O, 58g/L Na
2HPO
412H
2O, 180g/L NaCl and 10ml/L Tween20 are mixed with.
Kit of the present invention can detect HIV antibody and the P24 antigen in HIV infected person anteserum or the blood plasma simultaneously, compares with the method for present detection HIV antibody, has further shortened the detection window phase again; Utilize biotin Avidin system, the sensitivity of P24 Detection of antigen is improved simultaneously greatly, this kit adopts chemiluminescence immunoassay technology, the detection sensitivity that has than ELISA, safe and reliable, easy and simple to handle, compare cheap with external chemiluminescence detection system and do not need the expensive luminous measuring instrument of full-automatic chemical, for clinical HIV antibody and HIV detection of antigens and blood safety examination provide a kind of new detection method.
What kit of the present invention was used is the enzymatic luminous substrate, by the chromogenic substrate in the light signal replacement enzyme-linked immuno assay that detects the luminous substrate generation, thereby have a specificity equal with enzyme-linked immuno assay, and sensitivity improves greatly, ratio Enzyme Linked Immunoadsorbent Assay sensitivity now improves about 10 times, and the diagnosis that can be HIV provides more special, quick, reliable foundation.
Kit of the present invention has adopted the biotin-avidin system, has amplified reaction signal, thereby has improved the sensitivity of immunoassay, and high specificity, and stability is high, and applied widely, experimental cost is low, can more effectively detect HIV.
Embodiment
Embodiment 1 preparation human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit of the present invention
Human immunodeficiency virus antigen/antibody chemiluminescence immune assay detection kit of the present invention contains following composition: wrap the pre-bag of quilt simultaneously by luminous plaque with HIV antigen and P24 monoclonal antibody; Enzyme mark HIV antigenic label and enzyme mark Streptavidin with horseradish peroxidase-labeled; Biotinylated P24 antibody; The cleansing solution (20 times of concentrated cleaning solutions) that contains the Tween20 phosphate buffer; The chemical luminous substrate liquid A that contains luminol, Tween20 and reinforcing agent benzofluoranthrene thereof; The chemical luminous substrate liquid B that contains hydrogen peroxide; The negative control of normal human serum; The HIV antibody positive contrast of NBCS dilution HIV antibody positive human serum; And the P24 antigen positive contrast of NBCS dilution P24 antigen positive human serum.
One, wraps simultaneously with HIV antigen and P24 monoclonal antibody and wrapped by the chemiluminescence plate in advance
To wrap and be added to mixing in the carbonate buffer solution with HIV recombinant antigen and P24 antibody, add in the luminous plaque, every hole 100uL is incubated overnight, behind the phosphate buffer washing luminous plaque that contains Tween20 3 times, add the phosphate buffer that contains BSA again, after room temperature leaves standstill 2 hours, discard liquid in the hole, the finish-drying luminous plaque, use the aluminide-coating bag seal package, finish the HIV antigen/antibody and wrap the pre-bag of quilt simultaneously by the preparation of luminous plaque.
Two, the preparation of biotinylation P24 antibody
Biotin and P24 antibody are carried out mark, be prepared into biotinylation P24 antibody.
Three, horseradish peroxidase-labeled HIV antigen and labelled streptavidin
Sodium periodate method with improvement is in the same place HIV antigen and horseradish peroxidase, preparation HIV antigenic label; Streptavidin and horseradish peroxidase are in the same place the Avidin of preparation horseradish peroxidase-labeled with improvement sodium periodate method.
Four, the preparation of chemical luminous substrate liquid
A liquid: in the 100ml distilled water, add the Tris-HCl damping fluid that 1.21g Tris and the dense HCl of 295 μ L are made into 0.1M pH8.5.In this damping fluid, add Tween20 and the 0.1g benzofluoranthrene of Luminol, the 20 μ L of 0.5g, mix.
B liquid: in the 100ml distilled water, add trisodium citrate 0.73g and citric acid 0.44g, be mixed with the citrate buffer solution of 0.1MpH4.6, in this solution, add 100 μ L30% superoxols.
Using method: before using A liquid is mixed the back with B liquid in the 1:1 ratio and use.
Five, the preparation of other component:
Cleansing solution is the phosphate buffer that contains Tween20, the normal human serum of negative control for mixing, the contrast of HIV antibody positive is the HIV antibody positive human serum of NBCS dilution, and the contrast of P24 antigen positive is the P24 antigen positive human serum of NBCS dilution.
Six, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into the mensuration kit.Be assembled into also need inspect by random samples behind the kit and just can dispatch from the factory after qualified.
Embodiment 2～3 preparations human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit of the present invention
Except that respectively with plastic bead, magnetic-particle as the carrier, all the other all prepare kit of the present invention with the method identical with embodiment 1.
The using method of embodiment 4 kits of the present invention
One, adds sample, biotinylated antibody and tester at the chemiluminescence bag on by plate
Get HIV antigen-antibody bag by plate, balance is to room temperature, negative control 3 holes are established in each experiment, and the HIV antibody positive contrasts 2 holes, and the HIV-1P24 antigen positive contrasts 2 holes, every hole adds 100 μ l respectively, every hole adds sample 50 μ l in the sample well, adds biotinylation P24 antibody 50 μ l again, mixing, stick the shrouding film, put 37 ℃ of incubations 30 minutes.
Two, wash plate with cleansing solution
Get rid of dereaction liquid, wash plate 5 times, on clean thieving paper, buckle at last and do with the cleansing solution after the dilution.
Three, add the enzyme labeling thing
Except that blank well, every hole adds and contains the HIV1+2 type antigen of horseradish peroxidase-labeled and the Streptavidin enzyme labeling thing 100 μ l of horseradish peroxidase-labeled, sticks the shrouding film, leave standstill 37 ℃ 30 minutes.
Four, wash plate (method is identical with two) with cleansing solution
Five, mix substrate
Before using chemical luminous substrate liquid A and B were mixed with 1: 1, put room temperature lucifuge reaction 5 minutes.
Six, measure luminous intensity
On the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, 1 second/hole of Measuring Time.
Seven, read the result
Judge that according to the RLU value of sample to be tested and the ratio S/CO value of critical value if S/CO value 〉=1, then positive reaction illustrates and contains HIV antibody or P24 antigen in the sample; If S/CO value＜1, then negative reaction illustrates and does not contain HIV antibody or P24 antigen in the sample.
The clinical trial comparison of embodiment 5 kits of the present invention
The clinical trial comparison of kit of the present invention, experimental result sees the following form:
| The clinical trial project | Experimental result | Interpretation of result |
| 1056 parts in normal human serum sample | 1054 parts of feminine genders | Specificity is 99.81% |
| 312 parts of HIV antibody positive blood serum samples | 312 parts of positives | Sensitivity is 100.00% |
| 132 parts of P24 antigen positive blood serum samples | 132 parts of positives | Sensitivity is 100.00% |
Claims (13)
1. a human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit is characterized in that, described kit comprises:
1) human immunodeficiency virus antigen and antibody wrap the solid phase carrier of quilt simultaneously;
2) biotin labeled human immune defect virus antibody;
3) human immunodeficiency virus antigen of enzyme labeling and Streptavidin;
4) chemical luminous substrate that above-mentioned enzyme acted on; And
5) negative control thing, human immunodeficiency virus antigen and antibody positive tester.
2. kit as claimed in claim 1 is characterized in that, described kit comprises:
1) human immunodeficiency virus HIV1+2 type antigen and HIV-1P24 antibody wrap the solid phase carrier of quilt simultaneously;
2) biotin labeled HIV-1 P24 antibody;
3) the HIV1+2 type antigen and the Streptavidin of enzyme labeling;
4) chemical luminous substrate that above-mentioned enzyme acted on; And
5) negative control thing, HIV antibody and HIV-1 P24 antigen positive tester.
3. kit as claimed in claim 1 is characterized in that, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
4. kit as claimed in claim 1 is characterized in that, described enzyme is horseradish peroxidase or alkaline phosphatase.
5. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate is luminol, different luminol or 1,2-two oxidative ethane analog derivatives.
6. kit as claimed in claim 5, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (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.
7. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
Based on the described chemical luminous substrate A of 100mL liquid, comprise 1.21g Tris, 295 μ L concentrated hydrochloric acids, 0.5g luminol, 20 μ L Tween20 and 0.1g benzofluoranthrene;
Based on the described chemical luminous substrate B of 100mL liquid, comprise the H of 0.73g trisodium citrate, 0.44g citric acid, 100 μ L30%
2O
2Solution, its pH value is 4.6.
8. method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
1) wraps simultaneously by solid phase carrier with human immunodeficiency virus antigen and antibody;
2) with the biotin labeling human immune defect virus antibody;
3) with enzyme labeling human immunodeficiency virus antigen and Streptavidin;
4) the preparation chemical luminous substrate that above-mentioned enzyme acted on;
5) preparation negative control thing, human immunodeficiency virus antigen and antibody positive tester;
6) chemical luminous substrate that human immunodeficiency virus antigen and Streptavidin and this enzyme acted on of above-mentioned feminine gender of packing and positive control, biotin labeled human immune defect virus antibody, enzyme labeling; And
7) be assembled into finished product.
9. method as claimed in claim 8 is characterized in that, described bag is adopted following method by the step 1) of solid phase carrier:
I) bag quilt
The preparation coating buffer, comprise the natrium carbonicum calcinatum of 1.59g and the sodium bicarbonate of 2.94g based on the described coating buffer of 1000mL, the pH value of described coating buffer is 9.6, coating buffer with preparation mixes with the human immunodeficiency virus antigen and the antibody of debita spissitudo then, and the gained mixed liquor is carried on the solid phase carrier;
II) with PBST washing lotion washing above-mentioned solid phase carrier;
III) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10gBSA, 20g sucrose, 100mL NBCS and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
10. method as claimed in claim 8 or 9 is characterized in that described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
11. method as claimed in claim 8 is characterized in that, described enzyme is horseradish peroxidase or alkaline phosphatase.
12. method as claimed in claim 8 is characterized in that, described chemical luminous substrate is luminol, different luminol or 1,2-two oxidative ethane analog derivatives.
13. method as claimed in claim 12, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710119983 CN101363853A (en) | 2007-08-06 | 2007-08-06 | Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710119983 CN101363853A (en) | 2007-08-06 | 2007-08-06 | Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101363853A true CN101363853A (en) | 2009-02-11 |
Family
ID=40390334
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710119983 Pending CN101363853A (en) | 2007-08-06 | 2007-08-06 | Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101363853A (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183636A (en) * | 2010-12-30 | 2011-09-14 | 王滔 | Diagnostic kit for anti-strep-A DNase B antibody with chemiluminescence immunoassay and using method thereof |
| CN102879566A (en) * | 2012-10-12 | 2013-01-16 | 武汉康苑生物医药科技有限公司 | Enzyme-linked immunosorbent assay (ELISA) kit for human immunodeficiency virus (HIV) P24 antigen |
| CN105527426A (en) * | 2015-12-31 | 2016-04-27 | 天津医科大学总医院 | A liquid-chip HIV-antigen antibody combined detection kit and a detecting method |
| CN106290864A (en) * | 2016-08-12 | 2017-01-04 | 江苏泽成生物技术有限公司 | A kind of human immunodeficiency virus antigen and TPPA test kit and preparation method |
| CN106501519A (en) * | 2016-06-30 | 2017-03-15 | 深圳市亚辉龙生物科技股份有限公司 | Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof |
| CN106546732A (en) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Human immunodeficiency virus antigen and antibody combined detection kit and application thereof and detection method |
| CN106883300A (en) * | 2016-12-29 | 2017-06-23 | 菲鹏生物股份有限公司 | HIV recombinant antigens, expressing gene, expression vector and HIV detection kits |
| CN107202889A (en) * | 2017-07-21 | 2017-09-26 | 苏州华益美生物科技有限公司 | Four pathogen synchronous detection reagent kits and its application and preparation |
| CN108896769A (en) * | 2018-08-08 | 2018-11-27 | 福建医科大学 | People's β2-microglobulin detection method and its kit based on gold nano cluster |
| CN109613240A (en) * | 2019-01-04 | 2019-04-12 | 广东菲鹏生物有限公司 | It is a kind of for detecting the kit of HIV |
| CN110346569A (en) * | 2019-06-28 | 2019-10-18 | 安徽恩禾生物技术有限公司 | A kind of thymidine kinase chemoluminescence method detection kit and preparation method thereof |
| CN110967493A (en) * | 2018-09-29 | 2020-04-07 | 深圳天辰医疗科技有限公司 | Vitamin B12 determination kit and preparation method thereof |
| CN111272997A (en) * | 2017-11-27 | 2020-06-12 | 科美诊断技术股份有限公司 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
| CN113125715A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Human immunodeficiency virus antibody detection kit and application thereof |
| CN113419059A (en) * | 2021-06-30 | 2021-09-21 | 迈克生物股份有限公司 | Blocking agent for chemiluminescence immune analysis method |
-
2007
- 2007-08-06 CN CN 200710119983 patent/CN101363853A/en active Pending
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183636A (en) * | 2010-12-30 | 2011-09-14 | 王滔 | Diagnostic kit for anti-strep-A DNase B antibody with chemiluminescence immunoassay and using method thereof |
| CN102879566A (en) * | 2012-10-12 | 2013-01-16 | 武汉康苑生物医药科技有限公司 | Enzyme-linked immunosorbent assay (ELISA) kit for human immunodeficiency virus (HIV) P24 antigen |
| CN106546732A (en) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Human immunodeficiency virus antigen and antibody combined detection kit and application thereof and detection method |
| CN105527426A (en) * | 2015-12-31 | 2016-04-27 | 天津医科大学总医院 | A liquid-chip HIV-antigen antibody combined detection kit and a detecting method |
| CN106501519A (en) * | 2016-06-30 | 2017-03-15 | 深圳市亚辉龙生物科技股份有限公司 | Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof |
| CN106290864A (en) * | 2016-08-12 | 2017-01-04 | 江苏泽成生物技术有限公司 | A kind of human immunodeficiency virus antigen and TPPA test kit and preparation method |
| CN106883300A (en) * | 2016-12-29 | 2017-06-23 | 菲鹏生物股份有限公司 | HIV recombinant antigens, expressing gene, expression vector and HIV detection kits |
| CN107202889A (en) * | 2017-07-21 | 2017-09-26 | 苏州华益美生物科技有限公司 | Four pathogen synchronous detection reagent kits and its application and preparation |
| CN111272997A (en) * | 2017-11-27 | 2020-06-12 | 科美诊断技术股份有限公司 | Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit |
| CN108896769A (en) * | 2018-08-08 | 2018-11-27 | 福建医科大学 | People's β2-microglobulin detection method and its kit based on gold nano cluster |
| CN110967493A (en) * | 2018-09-29 | 2020-04-07 | 深圳天辰医疗科技有限公司 | Vitamin B12 determination kit and preparation method thereof |
| CN109613240A (en) * | 2019-01-04 | 2019-04-12 | 广东菲鹏生物有限公司 | It is a kind of for detecting the kit of HIV |
| CN109613240B (en) * | 2019-01-04 | 2021-10-12 | 广东菲鹏生物有限公司 | Kit for detecting HIV |
| CN110346569A (en) * | 2019-06-28 | 2019-10-18 | 安徽恩禾生物技术有限公司 | A kind of thymidine kinase chemoluminescence method detection kit and preparation method thereof |
| CN113125715A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Human immunodeficiency virus antibody detection kit and application thereof |
| CN113419059A (en) * | 2021-06-30 | 2021-09-21 | 迈克生物股份有限公司 | Blocking agent for chemiluminescence immune analysis method |
| CN113419059B (en) * | 2021-06-30 | 2023-09-01 | 迈克生物股份有限公司 | Blocking agent for chemiluminescent immunoassay method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101363853A (en) | Human immunodeficiency virus antigen/antibody chemiluminescence immune assay determination kit and method for preparing same | |
| US4016043A (en) | Enzymatic immunological method for the determination of antigens and antibodies | |
| WO2021232712A1 (en) | Novel coronavirus igm/igg magnetic microparticle chemiluminescence immunoassay kit | |
| CN101196518B (en) | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method | |
| Dennehy et al. | Comparison of nine commercial immunoassays for the detection of rotavirus in fecal specimens | |
| JPH01150856A (en) | Simultaneous immunoassay of antigen and antibody | |
| CN108700584A (en) | Labeled complex and preparation method thereof, kit, application and detecting system | |
| WO2021232713A1 (en) | Enzyme-linked immunosorbent assay detection kit for novel coronavirus igg antibody | |
| JP2001124775A (en) | Flow-through assay for visually detecting presence of influenza type a and type b | |
| CN101377514A (en) | Insulin autoantibody chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| JPH04233461A (en) | Simultaneous assay method | |
| CN104697830B (en) | For acidic treatment agent, sample preprocessing method, kit and the detection method of HIV detections | |
| WO2021232714A1 (en) | Enzyme-linked immunosorbent assay test kit for novel coronavirus igm antibody | |
| CN101178404B (en) | Human immunodeficiency virus antibody chemiluminescence immune analyzing diagnose reagent box and method of producing the same | |
| US20230358757A1 (en) | Antigen for 2019 novel coronavirus and detection use thereof | |
| CN101363861A (en) | Hepatitis b virus surface antigen chemiluminescence immune assay determination kit and method for preparing same | |
| CN108535486A (en) | A kind of chloramphenicol immunofluorescence assay method based on europium label | |
| CN101498730A (en) | Improved double-antigen sandwiching immunity detection method | |
| CN101968483A (en) | One-step semi-double-antigen sandwich immunological detection method | |
| CN106501519A (en) | Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof | |
| US5391479A (en) | Method for determining total analyte concentration in a sample having both free and bound analyte | |
| CN102183636A (en) | Diagnostic kit for anti-strep-A DNase B antibody with chemiluminescence immunoassay and using method thereof | |
| CN101377494B (en) | Chemiluminescence immune analytic reagent kit for detecting tuberculosis antibody | |
| JPH03502248A (en) | Aqueous washing solution for assay, diagnostic test kit and method for measuring herpes simplex virus | |
| CN109073641A (en) | Use the Radioimmunoassay of vascular endothelial growth of sulfated polysaccharides class |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20111027 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20111027 Address after: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Applicant after: Beijing Kemei Biological Technology Co., Ltd. Address before: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Applicant before: Kemei Dongya Biological Technology Co., Ltd., Beijing |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20090211 |