CN101592663A - Hepatitis B e antigen magnetic particle chemiluminescent immunoassay kit and preparation method thereof - Google Patents
Hepatitis B e antigen 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 antigen magnetic particle chemiluminescent immunoassay kit.Kit of the present invention comprises that mainly the hepatitis B e antibody bag is by the streptavidin of the mixed liquor of magnetic particle and biotin labeling hepatitis B e antibody, horseradish peroxidase-labeled, hepatitis B virus e antigen calibration object, chemical luminous substrate liquid, concentrated cleaning solution and reaction tube.The present invention utilizes the double antibody sandwich method principle, and hepatitis B virus e antigen content in the detection by quantitative serum can be used for the infection state and the hepatitis B curative effect monitoring of clinical judgment hepatitis B.
Description
Technical field
The present invention relates to a kind of hepatitis B e antigen magnetic particle chemiluminescent immunoassay kit and preparation method thereof.Kit of the present invention combines magnetic particle immunity isolation technics, biotin-avidin amplifying technique and chemiluminescence immunoassay technology.
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.
Hepatitis B virus e antigen (HBeAg) comes across hepatitis b virus infected early stage usually.The titre of HBeAg raise rapidly in the virus replication phase.The rising of the existence of HBeAg and infectious virus (Dane particle) quantity is closely related, and with serum in exist the specific archaeal dna polymerase of virus closely related.At the HBeAg positive phase, hepatitis B patient has the infectiousness of height to its contactee.The existence that HBeAg continues in the hepatitis b virus carrier is relevant with chronic active hepatitis usually.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, from practicality, stability, accuracy and development prospect, chemiluminescence immune assay is best.
Radiating immuning analysis technology uses the radioelement thing that serves as a mark, and therefore environment is produced certain radioactive contamination, and complicated operation, and 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
-15～10
-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 solid phase particle of the synthetic certain particle size size of macromolecular material to make carrier, with method bags such as physisorption, chemical coupling by on have the specificity affinity various immunologic active materials (antigen or antibody), making its sensitization is immune magnetic particle.Immune magnetic particle technology is combined the detection determinand with chemiluminescence immunoassay technology, can improve the sensitivity and the accuracy of detection greatly.This technology utilizes the micron order magnetic particle as the bag suppressed by vector, expoeridium Monoclonal Antibody immunity magnetic particle, utilize immune magnetic particle to be suspended in the liquid, can increase the contact area and the substrate light-emitting area of antigen, antibody, improve the sensitivity of reaction, and adopt rotating magnetic field to make magnetic particle play beating action and separating and combining Ag-Ab and free antibodies.
Biotin-avidin system (biotin-avidin system, BAS) be a kind of biological respinse amplifying technique of late 1970s development, because BAS affinity height, so have advantages such as high sensitivity, high specific and stability, in modern biological immunology field, be used widely, and demonstrate obvious superiority.
The combination of above-mentioned technology can provide a kind of sensitivity, method fast for hepatitis B virus e antigen, also helps lend some impetus to application and the development of chemiluminescence immunoassay technology in clinical examination.
Summary of the invention
The present invention combines the advantage of biotin-avidin immunity amplifying technique, magnetic particle immunity isolation technics and chemiluminescence immunoassay technology, but has developed a kind of kit of quick, sensitive detection by quantitative hepatitis B virus e antigen.
The object of the invention provides a kind of biotin-avidin immunity amplifying technique and the immune magnetic particle technology magnetic particle chemiluminescent immunoassay kit in conjunction with the chemiluminscence immunoassay hepatitis B virus e antigen.
Kit of the present invention comprises: the hepatitis B e antibody bag by magnetic particle and the mixed liquor of biotin labeled hepatitis B e antibody, the streptavidin of horseradish peroxidase-labeled, chemical luminous substrate liquid, concentrated cleaning solution and the reaction tube that hepatitis B virus e antigen calibration object, above-mentioned horseradish peroxidase are acted on.
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:
1) preparation hepatitis B e antibody bag is by the mixed liquor of magnetic particle and biotin labeling hepatitis B e antibody:
Described magnetic particle is 1～10 μ m particle diameter, di-iron trioxide kernel, the surperficial polymkeric substance that has amino or carboxyl 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.Described biotin labeling hepatitis B e antibody is realized with the antibody coupling reaction under the alkalescence condition by the biotin succinimide ester.
Described mixed liquor is to be mixing in 1: 1 by the magnetic particle of hepatitis B e antibody bag quilt and biotin labeled hepatitis B e antibody with volume ratio, and is formulated with 20～40% NBCS.
2) with the horseradish peroxidase-labeled streptavidin:
Adopt improvement sodium periodate method with the horseradish peroxidase-labeled streptavidin.
3) with hepatitis B virus e antigen preparation calibration object;
With horse serum hepatitis B virus e antigen is diluted to calibration object, concentration is respectively 0NCU/mL, 0.04NCU/mL, 0.12NCU/mL, 0.5NCU/mL, 2.2NCU/mL, 9.0NCU/mL.
4) the preparation chemical luminous substrate liquid that horseradish peroxidase acted on:
Described chemical luminous substrate liquid comprises A liquid and B liquid.A liquid comprises 10mM luminol, 0.2mM pyrogallol, 0.05mM 4-iodobenzene boric acid, 0.2M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0.B liquid comprises 3.5mM urea peroxide, 0.05% ovalbumin, 0.2M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
5) concentrated cleaning solution:
Described concentrated cleaning solution is the PBST cleansing solution, carries out 20 times dilution with deionized water earlier in use.
6) the above-mentioned calibration object of packing, hepatitis B e antibody bag are by the mixed liquor of magnetic particle and biotin labeling antibody, enzyme labeling streptavidin, chemical luminous substrate liquid and reaction tube:
Wherein the reaction tube material that uses of kit is transparent plastic or glass.
7) be assembled into the finished product kit.
According to kit of the present invention, the magnetic particle and the biotin labeled hepatitis B e antibody potpourri that in reaction tube, add hepatitis B e antibody bag quilt earlier, add sample again, if contain hepatitis B virus e antigen in the sample, then form the double-antibody sandwich composite structure of " hepatitis B e antibody-hepatitis B virus e antigen that magnetic particle combines-biotinylation hepatitis B e antibody " with the former, high affinity by the biotin Avidin makes enzyme mark streptavidin combine with this compound afterwards, form " magnetic particle antibody-determined antigen-biotinylated antibody-enzyme mark Avidin " compound, after free enzyme labeling thing is removed in washing, add chemical luminous substrate and produce light signal, carry out reading at the tubular type light-emitting appearance.
The present invention's " hepatitis B e antigen magnetic particle chemiluminescent immune analytic reagent kit " can detect the content of hepatitis B virus e antigen in the sample very effectively.Have advantages such as quick, sensitive, stable, and every index all reaches the level of similar import reagent box.
Embodiment
Embodiment 1 preparation hepatitis B e antigen 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 biotin labeling hepatitis B e antibody
1, the preparation of the magnetic particle of hepatitis B e antibody bag quilt
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 sodium azide antiseptic 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 biotin labeled hepatitis B e antibody
The biotin labeling hepatitis B e antibody with the biotin succinimide ester under the alkalescence condition with monoclonal antibody generation coupling reaction, PBS is fully dialysed, the biotin labeling thing dilutes with NBCS, adds proclin300, preserves below-20 ℃.
3, the preparation of mixed liquor
Is to mix at 1: 1 the magnetic particle of hepatitis B e antibody bag quilt and biotin labeled hepatitis B e antibody with volume ratio, formulated with 20～40% NBCSs.
Two, the preparation of the streptavidin of horseradish peroxidase-labeled
Adopt improvement sodium periodate method with the horseradish peroxidase-labeled streptavidin, concrete labeling process is as follows: dissolving 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 streptavidin, 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 ℃.
Three, the preparation of hepatitis B virus e antigen calibration object
With horse serum hepatitis B virus e antigen is diluted to calibration object, concentration is respectively 0NCU/mL, 0.04NCU/mL, 0.12NCU/mL, 0.5NCU/mL, 2.2NCU/mL, 9.0NCU/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
The compound method of the chemical luminous substrate liquid of horseradish peroxidase used in the present invention (HRP): A liquid: contain 10mM luminol, 0.2mM pyrogallol, 0.05mM 4-iodobenzene boric acid, 0.2M pH8.7 boric acid-borate buffer solution, its pH value is 8.0～10.0.
B liquid: contain 3.5mM urea peroxide, 0.05% ovalbumin, 0.2M 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.
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 is carried out packing through after the assay was approved, and is 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
Before using this kit to experimentize, the Avidin of the mixed liquor of taking-up antibody sandwich magnetic particle and biotin labeling antibody, calibration object/testing sample, enzyme labeling was placed 15～30 minutes in room temperature earlier, made them equilibrate to room temperature; Afterwards, 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.
Use kit of the present invention as follows according to the concrete operations step that the method for embodiment 2 experimentizes:
1, with after the round bottom polystyrene test tube numbering, add 50 μ L blood serum samples or serial calibration object solution in test tube, the every pipe of calibration object adds 0NCU/mL, 0.04NCU/mL, 0.12NCU/mL, 0.5NCU/mL, each 50 μ L of 2.2NCU/mL, 9.0NCU/mL,
2, the mixed liquor 50 μ L that add antibody sandwich magnetic particle and biotin labeling antibody again, 37 ℃ of oscillating reactions 30min.
3, 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 3 times.
4, the streptavidin 100 μ L that add horseradish peroxidase-labeled, 37 ℃ of oscillating reactions 15min.
5, test tube rack is placed separate 5min on the magnetic separator, the separation vessel that reverses is then poured out supernatant, the test tube of reversing is placed on the filter paper blots, and bounces separation vessel to remove wall built-up liquid.
6, as step 3 cleansing solution repeated washing 5 times.
7, each pipe adds chemical luminous substrate 200～400 μ L, and fully mixing places in the magnetic separator, treat that magnetic particle is enriched in the bottom after, 10min is placed in the dark place.
8, then on tubular type chemiluminescence measuring instrument, measure the luminous intensity (RLU) of each pipe in regular turn.Log value with calibration object concentration is a horizontal ordinate, and the Log value of RLU is drawn typical curve (double logarithmic curve) for ordinate, finds the concentration of the HBeAg 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 5 kits.With the HBeAg calibration object of the kit measurement 0.2NCU/mL that extracted among the embodiment 1 10 times.Calculate the coefficient of variation of measuring concentration, the result shows that the coefficient of variation is between 4.3%～8.7%.
2, the kit accuracy is measured
Detect with visiting center HBeAg 2NCU/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 10 months, and the result shows that every index meets clinical requirement fully.This kit was placed 10 monthly energy by national reference material standard in 6 days and 2～8 ℃ 37 ℃ of placements, had good stability, and met clinical needs fully.
Utilize the inventive method to detect, highly sensitive, high specificity, sensing range is wide, and is simple to operate, "dead" pollution, the kit cost is low, and clinical applicability is strong, more is applicable to China's clinical detection and examination laboratory.
Embodiment 4 kits of the present invention are with the clinical blood sample measured value comparison of external kit
Import HBeAg chemical luminescence reagent kit with kit of the present invention and external renowned company detects simultaneously to 1024 portions of normal human serums and 176 parts of hepatitis B great three positive patients serums respectively, and its testing result sees Table 1
The clinical blood examination result of table 1
Contrast agents susceptibility is 100%, and specificity is 99.9%, and reagent susceptibility of the present invention is 100%, and specificity is 99.9%, and coincidence rate is 100% between the two.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 (10)
1, a kind of hepatitis B e antigen magnetic particle chemiluminescent immunoassay kit; it is characterized in that, described kit by: hepatitis B e antibody bag by magnetic particle and the mixed liquor of biotin labeled hepatitis B e antibody, the streptavidin of horseradish peroxidase-labeled, chemical luminous substrate liquid, concentrated cleaning solution and the reaction tube that hepatitis B virus e antigen calibration object, above-mentioned horseradish peroxidase are acted on form.
2, kit as claimed in claim 1 is characterized in that, the magnetic particle of described hepatitis B e antibody bag quilt and the blending ratio of biotin labeled hepatitis B e antibody are 1: 1.
3, kit as claimed in claim 1 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
A liquid comprises 10mM luminol, 0.2mM pyrogallol, 0.05mM 4-iodobenzene boric acid, 0.2M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0;
B liquid comprises 3.5mM urea peroxide, 0.05% ovalbumin, 0.2M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
4, a kind of method for preparing the described kit of claim 1 is characterized in that may further comprise the steps: 1) preparation hepatitis B e antibody bag is by the mixed liquor of magnetic particle and biotin labeling hepatitis B e antibody; 2) with the horseradish peroxidase-labeled streptavidin; 3) with genetic engineering hepatitis B virus e antigen preparation calibration object; 4) the preparation chemical luminous substrate liquid that horseradish peroxidase acted on; 5) preparation concentrated cleaning solution; 6) above-mentioned semi-manufacture of packing and reaction tube; And 7) be assembled into the finished product kit.
5, method as claimed in claim 4 is characterized in that, in the step 1) of the mixed liquor of described preparation antibody sandwich magnetic particle and biotin labeling antibody:
Described mixed liquor is to be to mix at 1: 1 by the magnetic particle of hepatitis B e antibody bag quilt and biotin labeled 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 amino or carboxyl that wraps up, and its working concentration is 1～10mg/mL;
6, method as claimed in claim 5, it is characterized in that, 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 biotin labeling hepatitis B e antibody is realized with antibody lysine residue generation coupling reaction under the alkalescence condition by the biotin succinimide ester.
8, method as claimed in claim 7 is characterized in that, described reactive group is amino or carboxyl.
9, method as claimed in claim 4 is characterized in that, in described step 2 with the horseradish peroxidase-labeled streptavidin) in, adopt improvement sodium periodate method with the horseradish peroxidase-labeled streptavidin.
10, method as claimed in claim 4 is characterized in that, described chemical luminous substrate liquid comprises A liquid and B liquid, wherein,
A liquid comprises 10mM luminol, 0.2mM pyrogallol, 0.05mM 4-iodobenzene boric acid, 0.2M pH8.7 boric acid-borate buffer solution, and its pH value is 8.0～10.0;
B liquid comprises 3.5mM urea peroxide, 0.05% ovalbumin, 0.2M pH7.2 phosphate buffer, and its pH value is 7.0～7.6.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048454A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nanometer magnetic particle chemiluminescence detection kit for hepatitis B virus e antigen as well as preparation method thereof and detecting method thereof |
| CN103616513A (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 virus e-antigen measurement kit and detection method thereof |
-
2008
- 2008-05-30 CN CN 200810113811 patent/CN101592663A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048454A (en) * | 2012-12-18 | 2013-04-17 | 苏州浩欧博生物医药有限公司 | Nanometer magnetic particle chemiluminescence detection kit for hepatitis B virus e antigen as well as preparation method thereof and detecting method thereof |
| CN103048454B (en) * | 2012-12-18 | 2015-08-26 | 苏州浩欧博生物医药有限公司 | Nano magnetic particulate chemistry luminescent assay kit of a kind of hepatitis B virus e antigen and preparation method thereof |
| CN103616513A (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 virus e-antigen measurement kit and detection method thereof |
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