CN101614738A - Diagnostic kit for quantitatively detecting hepatitis B virus surface antibody - Google Patents
Diagnostic kit for quantitatively detecting hepatitis B virus surface antibody Download PDFInfo
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- CN101614738A CN101614738A CN200810043550A CN200810043550A CN101614738A CN 101614738 A CN101614738 A CN 101614738A CN 200810043550 A CN200810043550 A CN 200810043550A CN 200810043550 A CN200810043550 A CN 200810043550A CN 101614738 A CN101614738 A CN 101614738A
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- hepatitis
- surface antibody
- europium
- hbs
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Abstract
The object of the invention is to provide a kind of kit of detection by quantitative anti-HBs, mainly solves the too high technical matters of import electrochemiluminescent immunoassay detection method cost.Kit is made up of by reaction plate, europium mark Purification of HBsAg, europium mark dilution, fluorescence enhancement solution, concentrated washing lotion hepatitis B surface antibody calibration object, Purification of HBsAg bag, use europium mark and time resolution immunofluorescence quantitative measurement technology, binding time is differentiated fluorescence detector, the anti-HBs in the detection by quantitative serum.Use the time resolution immunofluorescence technique, the result has advantages such as simple to operate, highly sensitive, that specificity is good, pollution-free accurately and reliably.
Description
Technical field:
The present invention relates to a kind of kit that is used for the detection by quantitative anti-HBs.
Background technology:
As everyone knows, hepatitis B is one of pathogen microorganism of serious threat China people's health, the general susceptible of crowd.China is the district occurred frequently of hepatitis B, and the total infection rate of crowd is up to 60%, and national HBsAg carrier has 1.2 hundred million at least, and wherein about 10% finally is converted into various chronic liver diseases, comprises chronic hepatitis, cirrhosis and liver cancer.After the adult infected HBV, 90%～95% was self limiting, and 5%～10% can become persistent infection or chronicity, and 50% in the HB-PTH is with chronicity.After perinatal period and neonate were infected by HBV, if do not carry out the vaccine blocking-up, 85%～90% can become asymptomatic HBsAg carrier.
Generally believe that hepatitis B surface antibody is unique protection antibody of hepatitis B, its existence is that the HBV infection is eliminated, and health produces the sign to hepatitis B virus immune power.In addition, along with the generally use of hepatitis B vaccine, also to whether produce surface antibody for the result of use of vaccine and judge by health.Therefore for the detection of hepatitis B surface antibody, can point out the recovery and the treatment situation of the hepatitis B state of an illness, health is to the immune situation of virus after the judgement vaccinate.
But for a long time, homemade HBV infects marker detection reagent and adopts enzyme linked immunological relatively backward on the methodology (ELISA) and radio-immunity (RIA) analytical reagent always, the then main dependence on import of more advanced electrochemiluminescent immunoassay detectable; Although the electrochemiluminescent immunoassay detection method of import is all having superiority aspect sensitivity, specificity, the term of validity and the measurement range, its expensive price is difficult for being born by domestic most of user.Thereby be badly in need of high-quality and inexpensive homemade diagnostic reagent on the market.
Summary of the invention:
The implication of abridging among the present invention:
HBsAg: hepatitis B surface antibody; Anti--HBs: hepatitis B surface antibody; DTTA-Eu: a kind of europium ion chelate N1-(P-isothiocyano benzyl)-diethylenetriamineteacidcetic acidcetic europium sodium; β-NTA: β-naphthoyl trifluoroacetone; Sephacryl S-200: a kind of gel chromatography column packing Sephacryl S-200; Triton X-100: the ninth of the ten Heavenly Stems-(ethylene glycol) benzene Octyl Ether; TOPO: trioctyl phosphine oxide; Ag: antigen; Ab: antibody, Ab-Eu: europium labelled antibody.
The purpose of this law invention provides a kind of simple to operate, highly sensitive, good, free of contamination diagnostic kit for quantitatively detecting hepatitis B virus surface antibody of specificity.Mainly solve the too high technical matters of import electrochemiluminescent immunoassay detection method cost.
For achieving the above object, the present invention passes through following scheme implementation:
Diagnostic kit for quantitatively detecting hepatitis B virus surface antibody, kit is made up of by reaction plate, europium mark Purification of HBsAg, europium mark dilution, fluorescence enhancement solution, concentrated washing lotion hepatitis B surface antibody calibration object, Purification of HBsAg bag.
Described Purification of HBsAg bag is by the preparation of plate: Purification of HBsAg is diluted proper proportion (0.75-6 μ g/ml) back with phosphate buffer add in the microwell plate, the every hole of 100 μ l, washing after spending the night, sealing, drying are then packed coated slab in the aluminium foil bag, refrigerate standby.
The preparation of described Purification of HBsAg europium label:: Purification of HBsAg is mixed with 1: 5 ratio with DTTA-Eu carbonate buffer solution dialysis back, left standstill 18 hours after Sephacryl S-200 separating column separates DTTA-Eu and Ag-Eu, obtain the Purification of HBsAg that the europium mark is crossed.
The preparation of described hepatitis B surface antibody calibration object: demarcate anti--HBs sheep blood serum with the national standard product 2mIU/ml of Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 5mIU/ml, 10mIU/ml, be diluted to the calibration object that content is respectively 5mIU/ml, 10mIU/ml, 40mIU/ml, 160mIU/ml, 640mIU/ml then, get final product with the bottled amount packing of 1ml/.
The preparation of described fluorescence enhancement solution with anhydrous alcohol solution β-NTA, TOPO, adds Potassium Hydrogen Phthalate and tri-distilled water again, after 40 ℃ of dissolvings, adds acetate, Triton X-100, transfers pH value to 3.2, and constant volume.
Described using method comprises following anti-HBs determination step:
1. the preparation of reagent
(1) cleansing solution: concentrated washing lotion of 40mL and 960mL deionized water are mixed in clean bottle for handling liquid toilet or cosmetic substance, standby as the work cleansing solution.
(2) europium label: accurately draw the 0.3ml deionized water and fully dissolve the label dried frozen aquatic products, and abundant mixing, use in last hour and also once use up (using disposable plastic containers to prepare) by 1: 50 times of dilution with europium mark dilution.
2. the micropore reaction bar of reagent and requirement is put room temperature (room temperature specially refers to 20-25 ℃ of scope, down together) balance.
3. draw 100ul anti--HBs calibration object and sample to be checked, be sequentially added in the micropore reaction bar aperture and stick on mounting.
4. micropore reaction bar was hatched 40 minutes with the slow jolting of shaker at ambient temperature.
5. after hatching end for the first time, carefully the mounting that micropore is reacted on the bar is taken off and is discarded, and micropore is reacted bar put into and wash that the plate machine blots each hole and cleansing solution 400ul is injected in every hole, blots each hole again, repeat above washing 4 times, for the last time micropore is reacted bar and pat dry.
6. add 100ul europium label working fluid in every hole, and stick on mounting.
7. micropore reaction bar was hatched 40 minutes with the slow jolting of shaker at ambient temperature.
8. after for the second time hatching end, carefully the mounting that micropore is reacted on the bar is taken off and is discarded, and micropore is reacted bar put into and wash that the plate machine blots each hole and cleansing solution 400ul is injected in every hole, blots each hole again, repeats above washing 6 times, for the last time micropore is reacted bar and pats dry.
9. add enhancing liquid 100ul (avoid in the application of sample process running into little bore edges or reagent wherein, avoid polluting as far as possible) in each hole, and stick on mounting.
10. micropore reacts bar at room temperature, with shaker jog 5 minutes (finishing mensuration in half an hour).
11. must use 6 calibration objects when using the hepatitis B surface antibody quantitative determination reagent kit, perhaps use the two-point calibration method.6 calibration objects that must use kit to provide when at every turn changing lot number are made curve (double-log), and use to guarantee accuracy in the multiple hole of calibration object.Anti--HBs concentration draws by calibration curve in the blood sample.Compare with existing anti-HBs detection kit, kit of the present invention has the following advantages:
A. be label with DTTA-Eu, the mode that adopts time-resolved fluorescence to detect makes detection sensitivity higher;
B. the content of hepatitis B surface antibody in the quantitative measurement serum is more accurate;
C. be unit with mIU/ml, testing result and international standard integrate with, and are convenient to promote.
Description of drawings:
Accompanying drawing is anti--HBs concentration calibration curve
Embodiment:
A kind of kit of detection by quantitative anti-HBs, kit is made up of by reaction plate, europium mark Purification of HBsAg, europium mark dilution, fluorescence enhancement solution, concentrated washing lotion hepatitis B surface antibody calibration object, Purification of HBsAg bag.
Described Purification of HBsAg bag is by the preparation of plate: Purification of HBsAg is diluted proper proportion (0.75-6 μ g/ml) back with phosphate buffer add in the microwell plate, the every hole of 100 μ l, washing after spending the night, sealing, drying are then packed coated slab in the aluminium foil bag, refrigerate standby.
The preparation of described Purification of HBsAg europium label:: Purification of HBsAg is mixed with 1: 5 ratio with DTTA-Eu carbonate buffer solution dialysis back, left standstill 18 hours after Sephacryl S-200 separating column separates DTTA-Eu and Ag-Eu, obtain the Purification of HBsAg that the europium mark is crossed.
The preparation of described hepatitis B surface antibody calibration object: demarcate anti--HBs sheep blood serum with the national standard product 2mIU/ml of Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 5mIU/ml, 10mIU/ml, be diluted to the calibration object that content is respectively 5mIU/ml, 10mIU/ml, 40mIU/ml, 160mIU/ml, 640mIU/ml then, get final product with the bottled amount packing of 1ml/.
The preparation of described fluorescence enhancement solution with anhydrous alcohol solution β-NTA, TOPO, adds Potassium Hydrogen Phthalate and tri-distilled water again, after 40 ℃ of dissolvings, add acetate, Triton X-100,, transfer pH value value to 3.2, and constant volume.
Described using method comprises following anti-HBs determination step:
1. the preparation of reagent
(1) cleansing solution: concentrated washing lotion of 40mL and 960mL deionized water are mixed in clean bottle for handling liquid toilet or cosmetic substance, standby as the work cleansing solution.
(2) europium label: accurately draw the 0.3ml deionized water and fully dissolve the label dried frozen aquatic products, and abundant mixing, use in last hour and also once use up (using disposable plastic containers to prepare) by 1: 50 times of dilution with europium mark dilution.
2. the micropore reaction bar of reagent and requirement is put room temperature (room temperature specially refers to 20-25 ℃ of scope, down together) balance.
3. draw 100ul anti--HBs calibration object and sample to be checked, be sequentially added in the micropore reaction bar aperture and stick on mounting.
4. micropore reaction bar was hatched 40 minutes with the slow jolting of shaker at ambient temperature.
5. after hatching end for the first time, carefully the mounting that micropore is reacted on the bar is taken off and is discarded, and micropore is reacted bar put into and wash that the plate machine blots each hole and cleansing solution 400ul is injected in every hole, blots each hole again, repeat above washing 4 times, for the last time micropore is reacted bar and pat dry.
6. add 100ul europium label working fluid in every hole, and stick on mounting.
7. micropore reaction bar was hatched 40 minutes with the slow jolting of shaker at ambient temperature.
8. after for the second time hatching end, carefully the mounting that micropore is reacted on the bar is taken off and is discarded, and micropore is reacted bar put into and wash that the plate machine blots each hole and cleansing solution 400ul is injected in every hole, blots each hole again, repeats above washing 6 times, for the last time micropore is reacted bar and pats dry.
9. add enhancing liquid 100ul (avoid in the application of sample process running into little bore edges or reagent wherein, avoid polluting as far as possible) in each hole, and stick on mounting.
10. micropore reacts bar at room temperature, with shaker jog 5 minutes (finishing mensuration in half an hour).
11. must use 6 calibration objects when using the hepatitis B surface antibody quantitative determination reagent kit, perhaps use the two-point calibration method.6 calibration objects that must use kit to provide when at every turn changing lot number are made curve (double-log), and use to guarantee accuracy in the multiple hole of calibration object.Anti--HBs concentration draws by calibration curve in the blood sample, with reference to accompanying drawing.
Claims (5)
1. diagnostic kit for quantitatively detecting hepatitis B virus surface antibody is characterized in that: kit by hepatitis B surface antibody calibration object, purifying hepatitis B surface antibody bag by reaction plate, europium mark purifying hepatitis B surface antibody, europium mark dilution, fluorescence enhancement solution, concentrate washing lotion and form.
2. anti-HBs quantitative measurement diagnostic kit according to claim 1, it is characterized in that described purifying hepatitis B surface antibody bag is by the acquisition of plate: it is to add in the microwell plate behind the 0.75-6 μ g/ml that the purifying hepatitis B surface antibody is diluted to concentration with phosphate buffer, the every hole of 100 μ l, washing after spending the night, sealing, drying, then coated slab is packed in the aluminium foil bag, refrigerate standby.
3. anti-HBs quantitative measurement diagnostic kit according to claim 1, it is characterized in that the acquisition of described purifying hepatitis B surface antibody europium label: the purifying hepatitis B surface antibody is mixed with 1: 5 ratio with N1-(P-isothiocyano benzyl)-diethylenetriamineteacidcetic acidcetic europium sodium carbonate buffer solution dialysis back, left standstill 18 hours after Sephacryl S-200 separating column separates N1-(P-isothiocyano benzyl)-diethylenetriamineteacidcetic acidcetic europium sodium and europium labelled antibody, obtain the purifying hepatitis B surface antibody that the europium mark is crossed.
4. anti-HBs quantitative measurement diagnostic kit according to claim 1, it is characterized in that the acquisition of described hepatitis B surface antibody calibration object: demarcate the hepatitis B surface antibody sheep blood serum with the national standard product 2mIU/ml of Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 5mIU/ml, 10mIU/ml, be diluted to the calibration object that content is respectively 5mIU/ml, 10mIU/ml, 40mIU/ml, 160mIU/ml, 640mIU/ml then, get final product with the bottled amount packing of 1ml/.
5. anti-HBs quantitative measurement diagnostic kit according to claim 1, it is characterized in that the acquisition of described fluorescence enhancement solution: with anhydrous alcohol solution β-naphthoyl trifluoroacetone, trioctyl phosphine oxide, add Potassium Hydrogen Phthalate and tri-distilled water again, after 40 ℃ of dissolvings, add acetate, the ninth of the ten Heavenly Stems-(ethylene glycol) benzene Octyl Ether, transfer pH value to 3.2, and constant volume.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810043550A CN101614738A (en) | 2008-06-25 | 2008-06-25 | Diagnostic kit for quantitatively detecting hepatitis B virus surface antibody |
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810043550A CN101614738A (en) | 2008-06-25 | 2008-06-25 | Diagnostic kit for quantitatively detecting hepatitis B virus surface antibody |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102236020A (en) * | 2010-04-20 | 2011-11-09 | 上海新波生物技术有限公司 | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit |
-
2008
- 2008-06-25 CN CN200810043550A patent/CN101614738A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102236020A (en) * | 2010-04-20 | 2011-11-09 | 上海新波生物技术有限公司 | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit |
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| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Suzhou Sym-Bio Lifescience Co., Ltd. Assignor: Shanghai Xinbo Biotechnology Co., Ltd. Contract record no.: 2011320010124 Denomination of invention: Diagnostic kit for quantitatively detecting hepatitis B virus surface antibody License type: Exclusive License Open date: 20091230 Record date: 20110916 |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
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Application publication date: 20091230 |