CN101592664A - Hepatitis B e antibody magnetic particle chemiluminescent immunoassay kit and preparation method thereof - Google Patents
Hepatitis B e antibody magnetic particle chemiluminescent immunoassay kit and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the immuno analytical method field, relate to a kind of hepatitis B e antibody magnetic particle chemiluminescent immunoassay kit.Mainly formed by the mixed liquor of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody, hepatitis B virus e antigen, hepatitis B e antibody calibration object, chemical luminous substrate liquid, concentrated cleaning solution and reaction tube by the hepatitis B e antibody bag.The present invention can be used for content of hepatitis B e antibody in the clinical detection human serum, has advantages such as easy, quick, good reproducibility.
Description
Technical field
The present invention relates to the immunoassay field, particularly, the invention provides a kind of hepatitis B e antibody magnetic particle chemiluminescent immunoassay kit and preparation method thereof.
Background technology
Hepatitis B is a kind of serious common liver diseases, arrives the millions of people in global implication.In the present global population, surpass 2,000,000,000 people and infected hepatitis type B virus (HBV) certain period in its life, wherein, about 3.5 hundred million still are chronic infection person, become the carrier of virus.Whole world three quarters of the population is lived in the district occurred frequently of infection.The acute clinical case of annual HBV surpasses 400 ten thousand, and about 25% among the carrier, just annual 1000000 people die from chronic active hepatitis, cirrhosis or primary carcinoma of liver.
For the chronic infectious patients of the HBsAg positive, HBeAg and Anti-HBe can be used for determining the state of infection.Anti-HBe occurs behind Anti-HBc, and its appearance is relevant with the infectiousness of reduction.In the convalescence of disease, Anti-HBe will substitute HBeAg.The minimizing that HBeAg is indicating HBV DNA in the circulation to the serum conversion of antihepatitis b e antibody (Anti-HBe).HBeAg and Anti-HBe have in the patient's of the HBsAg positive state of illness monitoring and important meaning.
The immunization method that is used to detect the hepatitis B blood serum designated object at present mainly contains enzyme immunoassay (EIA) (enzymeimmunoassay, EIA), radiommunoassay (radioimmunoassay, RIA), immunofluorescence assay (fluoroimmunoassay, FIA) and chemiluminescence immune assay (chemiluminescence immunoassay, CLIA) etc.The basic theories of these ultramicron detection techniques is identical substantially, but used tracer agent and the signal that sent have nothing in common with each other.According to lot of experiment results and clinical practice data,, be followed successively by: chemiluminescence immune assay, fluoroimmunoassay, radiommunoassay and EIA enzyme immunoassay from practicality, stability, accuracy and development prospect.
Therefore radiating immuning analysis technology has certain contaminative to environment, and exists instrument cost expensive because of it uses radioelement thing that serves as a mark, and sensitivity is not high, complicated operation, measurement result instability, shortcoming such as the reagent holding time is short.Enzyme immunoassay (EIA) sensitivity is low, and influence factor is more, easily causes false negative and false positive.Chemiluminescence immunoassay is a kind of than elder generation and then effective method, can make detection sensitivity reach 10
-18Mol level, and sensing range can reach 6 orders of magnitude, because the enzyme labeling thing is stable, can uses for a long time, thereby obtain increasing concern.
Immunity magnetic particle technology is to utilize the magnetic particle of the synthetic certain particle size of macromolecular material to make carrier, and by last various immunologic active materials (antigen or antibody), making its sensitization is immune magnetic particle with method bags such as physisorption, chemical couplings.The immune magnetic particle for preparing can be suspended in the liquid, but this provides more contact area for immune response, simultaneously, the immunity magnetic particle can move in liquid, quickens the probability of antigen-antibody collision, makes reaction fast, fully, and favorable repeatability and more and more be subjected to everybody favor.
Summary of the invention
The present invention utilizes immune magnetic particle to be carrier, has developed a kind of easy, quick, special, stable hepatitis B e antibody in conjunction with chemiluminescence immunoassay technology and has measured kit.
The magnetic particle chemiluminescent immunoassay kit that the purpose of this invention is to provide a kind of hepatitis B e antibody.
Kit of the present invention comprises: the hepatitis B e antibody bag is by the mixed liquor of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody, hepatitis B virus e antigen, hepatitis B e antibody calibration object, chemical luminous substrate liquid, concentrated cleaning solution and reaction tube.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
The preparation method of kit of the present invention may further comprise the steps:
A) preparation hepatitis B e antibody bag is by the mixed liquor of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody:
Mixed liquor is to be to mix at 1: 1 by the magnetic particle of hepatitis B e antibody bag quilt and horseradish peroxidase hepatitis B e antibody with volume ratio, and is formulated with the phosphate buffer that contains 20～40% NBCS;
Described magnetic particle is 1～10 μ m particle diameter, di-iron trioxide kernel, the surperficial polymkeric substance that has reactive group that wraps up, and its working concentration is 1～10mg/mL;
Described antibody sandwich magnetic particle be by the glutaraldehyde two-step approach with the hepatitis B e antibody bag by on magnetic particle, be that 7.2 0.02mol/L phosphate buffer is the magnetic particle that quilt is wrapped in the confining liquid sealing to contain 0.5%～2.0% bovine serum albumin(BSA), pH value.
B) the preparation hepatitis B virus e antigen as in and antigen reagent: in described and antigen be hepatitis B virus e antigen for genetic engineering preparation.
C) with polyclone hepatitis B e antibody preparation calibration object: the horseradish peroxidase-labeled hepatitis B e antibody is with the preparation of improvement sodium periodate method mark.
D) preparation chemical luminous substrate liquid: described chemical luminous substrate comprises A liquid and B liquid, wherein, A liquid comprises 1 of 10mM luminol, 0.5mM, 6-two bromo-2-phenol, 0.05mM 4-iodobenzene boric acid, 0.1M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0; B liquid comprises 3.5mM urea peroxide, 0.01M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
E) preparation concentrated cleaning solution; Lavation buffer solution is the phosphate buffer that contains 0.1～0.5% Tween-20,0.1% biological preservative, and the pH value is 7.4.Use 20 times of distilled water dilutings during use.
F) above-mentioned semi-manufacture of packing and reaction tube: the reaction tube material that kit uses is transparent plastic or glass.Assembling is the finished product kit.
According to kit of the present invention, the magnetic particle of hepatitis B e antibody bag quilt earlier with in and antigen be that hepatitis B virus e antigen combines generation magnetic particle e antibody-e antigenic compound, when containing hepatitis B e antibody in the testing sample, then combine with the competition of magnetic particle compound simultaneously with the hepatitis B e antibody of enzyme labeling, when not containing hepatitis B e antibody in the sample, the then direct and magnetic particle compound reaction of the hepatitis B e antibody of enzyme labeling, after the resolvase label is removed in washing, add chemical luminous substrate liquid, by the Chemiluminescence Apparatus reading, the content of hepatitis B e antibody is inversely proportional in the signal of its generation and the sample.The present invention adopts " neutralization inhibition method " reaction pattern, has effectively utilized chemiluminescence in conjunction with magnetic particle and chemiluminescence immunoassay technology, hepatitis B e antibody in detection by quantitative human serum, the plasma sample.The magnetic particle that uses has super paramagnetic, high dispersive, characteristics that surface area is big.
The present invention's " hepatitis B e antibody magnetic particle chemiluminescent immunoassay kit " can detect the content of antihepatitis b e antibody in the sample very effectively.Have advantages such as easy, quick, good reproducibility, can be clinical hepatitis B a kind of good method that provides is provided.
Embodiment
Embodiment 1 preparation hepatitis B e antibody magnetic particle chemiluminescent immune analytic reagent kit of the present invention
One, the hepatitis B e antibody bag is by the preparation of the mixed liquor of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody
1, the hepatitis B e antibody bag is by the preparation of magnetic particle
With particle diameter is that the magnetic particle of 1～10 μ m activates with glutaraldehyde, stirring at room, and mixing is after 3 hours, add magnetic field, leave standstill 20～25min, pour out supernatant, with the pH value is that 7.4 0.01mol/L phosphate buffer cleans three times, and suspends with this solution, and concentration is 50～100mg/mL; Add hepatitis B e antibody 40～100 μ g in every milliliter of suspending liquid, after 4 ℃ stirring is spent the night down, add magnetic field, leave standstill 10～15min, pour out supernatant, sealed 3～4 hours in room temperature with the phosphate buffer (pH is 7.2) that contains 0.2%～1.0% bovine serum albumin(BSA), 0.02mol/L; At last with the pH value be 7.4, the phosphate lavation buffer solution that contains Tween-20 and biological preservative cleans 3～5 times, and is mixed with the working fluid of 5～10mg/mL with this solution.Magnetic particle is 4 ℃ of preservations.
2, the preparation of the hepatitis B e antibody of horseradish peroxidase-labeled
The horseradish peroxidase-labeled hepatitis B e antibody adopts improvement sodium periodate method, concrete labeling process is as follows: dissolving 4.4mg HRP is in 1mL distilled water, add 0.4mL sodium periodate (50mmol/L) stirring at room 20min, through the 1mmol/L sodium-acetate buffer, pH 4.4 dialysis backs add the 8mg hepatitis B e antibody, stir 2h, use 200mmol/L NaBH at last
4Reduce, after the dialysis of 0.02M PBS damping fluid, add equal-volume glycerine, preserve below-20 ℃.
3, the preparation of mixed liquor
Is 1: 1 to mix by magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody with volume ratio with the hepatitis B e antibody bag, formulated with 20～40% NBCSs.
Two, the neutralization preparation of hepatitis B virus e antigen
With pH 7.4 phosphate buffers that contain 1%BSA hepatitis B virus e antigen is diluted, select suitable concentration, in being prepared into and antigen, packing.
Three, the preparation of hepatitis B e antibody calibration object
With horse serum the high concentration hepatitis B e antibody is diluted to calibration object, concentration is respectively 0NCU/mL, 2NCU/mL, 4NCU/mL, 8NCU/mL, 16NCU/mL, 32NCU/mL, totally 6 bottles.
Four, the preparation of enzyme labeling thing
Preparation enzyme dilution contains Tris 12.12g in every 1000ml enzyme dilution earlier, BSA 5g, and glycerine 100ml, Proclin300 1ml adds distilled water to 900ml, transfers pH to 7.4 with hydrochloric acid, is settled to 1000ml with distilled water again, is made into the enzyme dilution.Adopting the square formation method to select the working concentration scope of enzyme labeling thing is 1: 1000～5000.
Five, preparation chemical luminous substrate liquid
The compound method of the chemical luminous substrate liquid of horseradish peroxidase used in the present invention (HRP):
A liquid: A liquid comprises 1 of 10mM luminol, 0.5mM, 6-two bromo-2-phenol, 0.05mM 4-iodobenzene boric acid, 0.1M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0.
B liquid: comprise 3.5mM urea peroxide, 0.01M pH7.2 phosphate buffer, its pH value is 7.0～7.6.
Using method: A, B liquid bi-component reagent mix according to the use amount equal-volume before use, or add a A liquid earlier, add a isopyknic B liquid again, mixing.
Six, preparation concentrated cleaning solution
Lavation buffer solution is the phosphate buffer that contains 0.1～0.5% Tween-20,0.1% biological preservative, and the pH value is 7.4.Use 20 times of distilled water dilutings during use.
Seven, semi-manufacture and finished product are formed
Mentioned reagent and reaction tube carry out packing through after the assay was approved, and be labelled, is semi-manufacture.Again each semi-manufacture is assembled into the finished product kit as requested.
The using method of embodiment 2 kits of the present invention
One, the preparation of sample
Adopting correct medical approaches to collect the patients serum is used for detecting.
Two, detection method
1, use this kit to experimentize before, the Avidin of the mixed liquor of taking-up antibody sandwich magnetic particle and horseradish peroxidase-labeled antibody, hepatitis B virus e antigen, calibration object/testing sample, enzyme labeling was placed 15～30 minutes in room temperature earlier, made them equilibrate to room temperature; And note constant temperature incubator or water-bath are transferred to 37 ℃; Again, be ready to suitable micro sample adding appliance and corresponding suction nozzle and check whether operate as normal of Chemiluminescence Apparatus.
2, with the reaction tube numbering, in reaction tube, add 50 μ L blood serum samples or serial calibration object solution, the every pipe of calibration object adds 0NCU/mL, 2NCU/mL, 4NCU/mL, 8NCU/mL, each 50 μ L of 16NCU/mL, 32NCU/mL.
3, every pipe adds the mixed liquor 50 μ L of antibody sandwich magnetic particle and horseradish peroxidase-labeled antibody.
4, every pipe adds each 50 μ L of hepatitis B virus e antigen, 37 ℃ of oscillating reactions 30min again.
5, every pipe adds cleansing solution 500 μ L, and fully mixing places and separates 5min on the magnetic separator, pours out supernatant, the test tube that reverses is placed on the filter paper blots, and bounces separation vessel to remove wall built-up liquid, repeats 5 times.
6, every pipe adds chemical luminous substrate A liquid 100 μ L, adds B liquid 100 μ L again, fully mixing, place in the magnetic separator, after treating that magnetic particle is enriched in the bottom, 10min is placed in the dark place, then measures the luminous intensity (RLU) of each pipe on tubular type chemiluminescence measuring instrument in regular turn.
7, the Log value with calibration object concentration is a horizontal ordinate, and the Logit value of RLU is drawn typical curve (double logarithmic curve) for ordinate, finds the concentration of the hepatitis B e antibody of this serum on typical curve with each test serum RLU value.
The methodology calibrating of embodiment 3 kits of the present invention
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is examined and determine, the result is as follows:
1, kit precision is measured
(1) calibration object precision experiment
The kit of preparation among the embodiment 1 is got three batches respectively carry out the precision experiment, every batch is extracted 10 kits.With the HBeAb calibration object of the kit measurement 4NCU/mL that extracted among the embodiment 15 times.Calculate its coefficient of variation, the result shows that the coefficient of variation is between 4.6%～7.7%.
2, the kit accuracy is measured
Detect with visiting center hepatitis B e antibody 4NCU/ml quality controlled serum, the measured value deviation is less than 10%.
3, kit specificity, sensitivity experiment
Do not see cross reaction with diseases such as HAV, HCV, rheumatoid disease, systemic loupus erythematosuses.Detect with national reference material, wherein 15 parts of national reference materials of feminine gender all detect negative match-rate 100% (15/15), 10 parts of national reference materials of the positive all detect the positive, coincidence rate 100% (10/10), and three serial dilution sensitivity reference serums all detect the positive for 9 parts.
4, kit stability experiment
After the kit of embodiment 1 carried out 37 ℃ of 6 days accelerated tests, place the high, medium and low value quality-control product of the parallel detection of kit with 4 ℃, the result shows that 37 ℃ of quality controlled serum measured value deviations after 6 days are all less than 15%, embodiment 1 kit is carried out 2～8 ℃ of tracking tests of 8 months, and the result shows that every index meets clinical requirement fully.This kit was placed 8 monthly energy by national reference material standard in 6 days and 2～8 ℃ 37 ℃ of placements, had good stability, and met clinical needs fully.
Embodiment 4 kits of the present invention are with the clinical blood sample measured value comparison of external kit
Respectively to 1056 examples blood sample at random, its testing result sees Table 1 with the import HBeAb chemical luminescence reagent kit of kit of the present invention and external renowned company
Table 1 clinical examination result
Contrast agents HBeAb positive rate is 18.84%, and the clinical positive rate of reagent of the present invention is 18.94%; Total coincidence rate between the two is 99.72%.Show by above result, reagent sensitivity height of the present invention, specificity is good, and clinical accordance is good, and pollution-free, has excellent popularization for clinical detection and is worth.
Claims (9)
1, a kind of hepatitis B e antibody magnetic particle chemiluminescent immunoassay kit; it is characterized in that described kit comprises chemical luminous substrate liquid, concentrated cleaning solution and the reaction tube that the hepatitis B e antibody bag is acted on by the mixed liquor of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody, hepatitis B virus e antigen, hepatitis B e antibody calibration object, above-mentioned horseradish peroxidase.
2, kit as claimed in claim 1 is characterized in that, described hepatitis B e antibody bag is 1: 1 by the blending ratio of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody.
3, kit as claimed in claim 1 is characterized in that, described hepatitis B virus e antigen be in and antigen, adopt the genetic engineering hepatitis B virus e antigen.
4, kit as claimed in claim 1 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
A liquid comprises 1 of 10mM luminol, 0.5mM, 6-two bromo-2-phenol, 0.05mM 4-iodobenzene boric acid, 0.1M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0;
B liquid comprises 3.5mM urea peroxide, 0.01M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
5, a kind of method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
Preparation hepatitis B e antibody bag is by the mixed liquor of magnetic particle and horseradish peroxidase-labeled hepatitis B e antibody; Prepare in the hepatitis B virus e antigen conduct and antigen reagent; With polyclone hepatitis B e antibody preparation calibration object; The preparation chemical luminous substrate liquid that horseradish peroxidase acted on; The preparation concentrated cleaning solution; Above-mentioned semi-manufacture of packing and reaction tube.
6, method as claimed in claim 5 is characterized in that, in the step of the mixed liquor of described preparation antibody sandwich magnetic particle and horseradish peroxidase-labeled antibody:
Described mixed liquor is to be to mix at 1: 1 by the magnetic particle of hepatitis B e antibody bag quilt and horseradish peroxidase hepatitis B e antibody with volume ratio, and is formulated with the phosphate buffer that contains 20～40% NBCS;
Described magnetic particle is 1～10 μ m particle diameter, di-iron trioxide kernel, the surperficial polymkeric substance that has reactive group that wraps up, and its working concentration is 1～10mg/mL;
Described antibody sandwich magnetic particle be by the glutaraldehyde two-step approach with the hepatitis B e antibody bag by on magnetic particle, be that 7.2 0.02mol/L phosphate buffer is the magnetic particle that quilt is wrapped in the confining liquid sealing to contain 0.5%～2.0% bovine serum albumin(BSA), pH value.
7, method as claimed in claim 5 is characterized in that, described horseradish peroxidase-labeled hepatitis B e antibody is realized by improvement sodium periodate method mark.
8, method as claimed in claim 5 is characterized in that, described in and antigen be hepatitis B virus e antigen for genetic engineering preparation.
9, method as claimed in claim 5 is characterized in that, described chemical luminous substrate liquid comprises A liquid and B liquid, wherein,
A liquid comprises 1 of 10mM luminol, 0.5mM, 6-two bromo-2-phenol, 0.05mM 4-iodobenzene boric acid, 0.1M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0;
B liquid comprises 3.5mM urea peroxide, 0.01M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103616372A (en) * | 2013-12-06 | 2014-03-05 | 苏州长光华医生物医学工程有限公司 | Hepatitis B e-antibody measurement kit and detection method thereof |
| CN104597255A (en) * | 2015-02-04 | 2015-05-06 | 华中农业大学 | Test paper card for detecting pseudorabies virus gE protein antibody in porcine serum as well as preparation method and application thereof |
| CN105277695A (en) * | 2015-09-15 | 2016-01-27 | 安徽博睿生物科技有限公司 | Immune magnetic particle chemiluminescence method HCMVpp65 antigen detection kit |
| CN109541204A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection antihepatitis b e antibody kit and preparation method thereof |
| CN109541232A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection anti-HAV kit and preparation method thereof |
| CN110297091A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | A kind of multiple tube-type chemical luminescence detection kit of I type antibody of aftosa O, A, Asia |
| CN112630430A (en) * | 2020-11-16 | 2021-04-09 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
| CN113252901A (en) * | 2021-06-02 | 2021-08-13 | 中山生物工程有限公司 | Hepatitis B virus e antibody detection kit |
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2008
- 2008-05-30 CN CN 200810113812 patent/CN101592664A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103616372A (en) * | 2013-12-06 | 2014-03-05 | 苏州长光华医生物医学工程有限公司 | Hepatitis B e-antibody measurement kit and detection method thereof |
| CN104597255A (en) * | 2015-02-04 | 2015-05-06 | 华中农业大学 | Test paper card for detecting pseudorabies virus gE protein antibody in porcine serum as well as preparation method and application thereof |
| CN105277695A (en) * | 2015-09-15 | 2016-01-27 | 安徽博睿生物科技有限公司 | Immune magnetic particle chemiluminescence method HCMVpp65 antigen detection kit |
| CN109541204A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection antihepatitis b e antibody kit and preparation method thereof |
| CN109541232A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection anti-HAV kit and preparation method thereof |
| CN110297091A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | A kind of multiple tube-type chemical luminescence detection kit of I type antibody of aftosa O, A, Asia |
| CN110297091B (en) * | 2019-07-17 | 2020-12-01 | 中国农业科学院兰州兽医研究所 | Multi-tube chemiluminescence detection kit for foot-and-mouth disease O, A, Asia I-type antibody |
| CN112630430A (en) * | 2020-11-16 | 2021-04-09 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
| CN112630430B (en) * | 2020-11-16 | 2021-08-27 | 北京美联泰科生物技术有限公司 | Kit for quantitatively detecting UCHL-1 and application thereof |
| CN113252901A (en) * | 2021-06-02 | 2021-08-13 | 中山生物工程有限公司 | Hepatitis B virus e antibody detection kit |
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Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20111018 |
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