CN106257283A - HBsAg PreS chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents

HBsAg PreS chemiluminescence immune detection reagent kit and preparation method thereof Download PDF

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Publication number
CN106257283A
CN106257283A CN201610506956.8A CN201610506956A CN106257283A CN 106257283 A CN106257283 A CN 106257283A CN 201610506956 A CN201610506956 A CN 201610506956A CN 106257283 A CN106257283 A CN 106257283A
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pres
hbsag
reagent kit
detection reagent
immune detection
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钱纯亘
祝亮
王刚
夏福臻
黄湘东
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Shenzhen Yhlo Biotech Co Ltd
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Shenzhen Yhlo Biotech Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

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Abstract

The invention discloses a kind of HBsAg PreS chemiluminescence immune detection reagent kit and preparation method thereof, HBsAg PreS chemiluminescence immune detection reagent kit includes: the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody and the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody.This HBsAg PreS chemiluminescence immune detection reagent kit can complete the detection of HBsAg PreS with Full-automatic chemiluminescence immunoassay analysis meter for detection instrument.This HBsAg PreS chemiluminescence immune detection reagent kit, through experiment, the detection method sensitivity relative to traditional HBsAg PreS at least improves 10 times, and the accuracy of detection of this HBsAg PreS chemiluminescence immune detection reagent kit is higher.

Description

HBsAg PreS chemiluminescence immune detection reagent kit and preparation method thereof
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of HBsAg PreS chemiluminescence immune detection reagent kit and Its preparation method.
Background technology
Hepatitis B virus (HBV) infects the public health problem having become global, the feature that HBV is eliminated Disappear in serum for hbs antigen (HBsAg) and surface antibody (HBsAb) occurs at serum.Theoretical On, in the internal same patient that there is hbv replication, the two can not detect simultaneously, but also has partial clinical patients serum to mark In Ben, both can detect, and is more common in chronic viral hepatitis B (abbreviation hepatitis B) patient.Have research think HBsAg, The amino acid mutation of HBsAb common anode and HBVS gene main hydrophilic area α determinant and preS district, particularly pre The factors such as the Nucleic acid deletions of S2, sudden change are closely related.
The at present detection of HBsAg PreS mainly has a following several method:
One, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak point:
(1) 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate is used to be coated apparatus with anti-as antibody Answer container, 12 batches, 6 batches, 8 batches or imposite first use can only be divided in use, it is impossible to carry out independent, single The detection of person-portion；
(2) reagent type used by qualitative determination is more, and each detectable will contain with reagent bottle, and often makes Being required for changing imbibition nozzle during with a kind of reagent to be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, filling The operation of reagent is the most loaded down with trivial details；
(3) lack the corresponding mark to detection information, can only just will appreciate that by the mark checking test kit external packing box or know Know product batch number and the effect duration information of detectable, and the information known is uncontrolled during detection, has the biggest Randomness；
(4) detectable is in open space during detection, easily causes the cross-contamination between various reagent and shadow Ring the accuracy of testing result；
(5) detection process uses manual operations, and the dosage of reagent or sample is not bery accurate, and operating process is the most loaded down with trivial details and multiple Miscellaneous, it is susceptible to bust, accuracy and the precision of testing result are poor；
(6) in the quantity configuration and use of detection project reagent set, it is item number × 48/96 person-portion, if needing inspection Survey 10 projects, then configuration and the use number of reagent must be 10 × 48/96 person-portions, if the most a sample needs to detect 10 Individual different project, it is also desirable to the reagent of configuration 10 × 48/96 person-portions, the shortcoming that there is inadequate economical rationality.
Two, gene diagnosis
For many RNA viruses, RT-PCR diagnostic method is faster but also clever than traditional virus purification and diagnostic antigen method Quick.And RT-PCR can diagnose multiple virus, other diagnostic methods such as virus purification and immunofluorescence the most simultaneously Analyze and but can not.But the method also has weak point, such as PCR technology, colleges and universities' property of DNA cloning result in the dirt of denier Dye both may occur in which false positive, thus makes result distortion.In addition virus is as the invader of outer protogene, it is necessary to whole illustrating it Or during partial nucleotide sequence, just can design primer or probe, carry out making nucleic acid molecular hybridization and PCR detection.
From the detection method of existing HBsAg PreS, although EIA, RT-PCR diagnostic method has certain special Property and the advantage of sensitivity, but operationally need professional and technical personnel, special instrument and equipment and specific condition and time-consuming Etc. shortcoming.
Acridinium ester is compared above method as the direct chemiluminescence of label and is had detailed advantage, is mainly manifested in: anti- Should need not catalyst, as long as alkaline environment can be carried out, be swift in response, background luminescence is low, and signal to noise ratio is high, and interference factor is few, Reagent stability is good, can be with two-point calibration, and system is simple, exciting liquid low cost, and acridinium ester is easily and after protein bind, and connection Photon productivity does not reduces, and has become as the developing direction that HBsAg PreS diagnosis is new.
Summary of the invention
Based on this, it is necessary to provide the HBsAg PreS chemiluminescence immune detection reagent kit that a kind of detection sensitivity is higher And preparation method thereof.
A kind of HBsAg PreS chemiluminescence immune detection reagent kit, including: HBsAg PreS monoclonal antibody is coated Carboxylated magnetic particle and the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody.
In one embodiment, in the described coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody, described HBsAg PreS monoclonal antibody is 1:25 ~ 100 with the ratio of described carboxylated magnetic particle.
In one embodiment, in the chemiluminescent labels of described HBsAg PreS labeling of monoclonal antibody, described HBsAg PreS monoclonal antibody is 1:10 ~ 100 with the ratio of described chemiluminescent labels.
In one embodiment, the particle diameter of described carboxylated magnetic particle is 0.05 μm ~ 1 μm.
In one embodiment, described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridine Ester.
In one embodiment, also include that Chemoluminescent substrate, described Chemoluminescent substrate include A liquid and B liquid.
In one embodiment, described A liquid is H2O2Solution, described B liquid is NaOH solution.
In one embodiment, also include that HBsAg PreS calibrates product.
In one embodiment, described HBsAg PreS calibration product are the HBsAg PreS that COI is respectively 0 ~ 0.5,1 ~ 10 Solution.
The preparation method of a kind of above-mentioned HBsAg PreS chemiluminescence immune detection reagent kit, comprises the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC aqueous solution, The surface carboxyl groups of the magnetic particle of activated carboxyl, is subsequently added into HBsAg PreS monoclonal antibody, suspendible 2h ~ 10h, magnetic under room temperature Separate after removing supernatant resuspended with Tris buffer, obtain the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody； And
Take HBsAg PreS monoclonal antibody, mix after adding carbonate buffer solution, mixed after being subsequently adding chemiluminescent labels Even, under room temperature, remove impurity after lucifuge reaction 1h ~ 2h, obtains the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody.
This HBsAg PreS chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter Detection instrument, completes the detection of HBsAg PreS.This HBsAg PreS chemiluminescence immune detection reagent kit, through experiment, The detection method sensitivity relative to traditional HBsAg PreS of its detection sensitivity at least improves 10 times, this HBsAg The accuracy of detection of PreS chemiluminescence immune detection reagent kit is higher.
Accompanying drawing explanation
Fig. 1 is the flow chart of the preparation method of the HBsAg PreS chemiluminescence immune detection reagent kit of an embodiment；
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public Restriction.
The HBsAg PreS chemiluminescence immune detection reagent kit of one embodiment, including: HBsAg PreS monoclonal anti The coated carboxylated magnetic particle of body and the chemiluminescent labels of HBsAg PreS monoclonal antibody mab labelling.
Preferably, in the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody, HBsAg PreS monoclonal anti Body is 1:25 ~ 100 with the ratio of carboxylated magnetic particle.
Preferably, in the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody, HBsAg PreS monoclonal anti Body is 1:10 ~ 100 with the ratio of chemiluminescent labels.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm ~ 1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence Label is preferably acridinium ester.
In other examples, above-mentioned HBsAg PreS chemiluminescence immune detection reagent kit also includes at the bottom of chemiluminescence Thing liquid.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten Liquid.
In other examples, above-mentioned HBsAg PreS chemiluminescence immune detection reagent kit also includes HBsAg PreS Calibration product.
HBsAg PreS calibration product are the solution that COI is respectively the HBsAg PreS of 0 ~ 0.5 and 1 ~ 10.
Concrete, HBsAg PreS calibration product can use standard substance buffer and HBsAg PreS antigen is put into mark Quasi-product buffer becomes the solution of the HBsAg PreS that COI is respectively 0 ~ 0.5 and 1 ~ 10.
This HBsAg PreS chemiluminescence immune detection reagent kit, when HBsAg PreS detects, utilizes full-automation Learn luminescence immunoassay instrument HBsAg PreS calibration product are detected, determine Cutoff value, be built in computer software；Then survey Examination actual sample, calculates sample COI according to sample luminous value；Finally to HBsAg PreS automatic chemiluminescence immunoassay system System carries out the evaluation of performance (sensitivity, precision, interference).
This HBsAg PreS chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter Detection instrument, completes the detection this HBsAg PreS chemiluminescence immune detection reagent kit of HBsAg PreS, through experiment, its The detection sensitivity detection method sensitivity relative to traditional HBsAg PreS at least improves 10 times, this HBsAg PreS The accuracy of detection of chemiluminescence immune detection reagent kit is higher.
Additionally, this HBsAg PreS chemiluminescence immune detection reagent kit also has the advantage that
1, selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, and this luminescence system is directization Learning luminescence, compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more cost-effective；
2, select the chemiluminescence immunoassay system RLU range of readings width of acridinium ester, 200 ~ 5000000 can be reached, and traditional The range of absorbency of detection method of HBsAg PreS be 0 ~ 4.0；
3, acridinium ester chemiluminescent immunoassay system repeatability is high, and in batch and difference between batch is all within 5%, this is other chemistry Luminescence immunoassay system is unapproachable；
4, chemiluminescence immunoassay system has realized the qualitative of sample, by built-in Cutoff value to test software, only needs to survey Sample originally just can directly obtain the COI of sample；
5, chemiluminescence immunoassay system can realize the interpolation of full-automation, reagent and sample has instrument to complete entirely, operation Easier, decrease artificial error.
The preparation method of above-mentioned HBsAg PreS chemiluminescence immune detection reagent kit as shown in Figure 1, including walking as follows Rapid:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC aqueous solution, The surface carboxyl groups of the magnetic particle of activated carboxyl, is subsequently added into HBsAg PreS monoclonal antibody, suspendible 2h ~ 10h, magnetic under room temperature Separate after removing supernatant resuspended with Tris buffer, obtain the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody.
MES(2-(N-morpholine) ethyl sulfonic acid) concentration of buffer be 0.02M, pH be 5.5.
The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH.
EDC(1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides) concentration of aqueous solution is 10mg/mL ~ 20mg/ ML, EDC are 0.05:0.1 ~ 1 with the ratio of carboxylated magnetic particle.
Preferably, in the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody, HBsAg PreS monoclonal anti Body is 1:25 ~ 100 with the ratio of carboxylated magnetic particle.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm ~ 1 μm.
Take HBsAg PreS monoclonal antibody, mix after adding carbonate buffer solution, be subsequently adding chemiluminescent labels Rear mixing, under room temperature, remove impurity after lucifuge reaction 1h ~ 2h, obtains the chemiluminescent labeling of HBsAg PreS labeling of monoclonal antibody Thing.
Carbonate buffer solution concentration be 0.1M, pH be 9.0 ~ 9.5,
The operation of remove impurity is centrifugal desalting column desalination, and concrete operations are: the most respectively with pure water and TBS buffer (40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0) process centrifugal desalting column, it is eventually adding the HBsAg PreS Dan Ke obtained The chemiluminescent labels of grand antibody labeling, finally collects the liquid in centrifuge tube.
Preferably, in the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody, HBsAg PreS monoclonal anti Body is 1:10 ~ 100 with the ratio of chemiluminescent labels.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence Label is preferably acridinium ester.
The coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody obtained and HBsAg PreS monoclonal antibody The chemiluminescent labels combination of labelling i.e. can get above-mentioned HBsAg PreS chemiluminescence immune detection reagent kit.
This HBsAg PreS chemiluminescence immune detection reagent kit is in use, in addition it is also necessary to Chemoluminescent substrate and HBsAg PreS calibrates product.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten Liquid.
Concrete, HBsAg PreS calibration product can use standard substance buffer and HBsAg PreS antigen is put into mark Quasi-product buffer becomes the solution of the HBsAg PreS that COI is respectively 0 ~ 0.5 and 1 ~ 10.
The preparation method of this HBsAg PreS chemiluminescence immune detection reagent kit is simple and convenient, the HBsAg prepared The detection sensitivity of PreS chemiluminescence immune detection reagent kit is higher, has a good application prospect.
It it is below specific embodiment.
The preparation of embodiment 1:HBsAg PreS chemiluminescence immune detection reagent kit
(1) preparation of the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody:
Take containing carboxylated magnetic particle (MagnaBind, the article No. 21353) suspension that 50mg particle diameter is 0.05 μm ~ 1 μm, Magneto separate removes supernatant, uses 0.02 M, and pH is that 5.5 MES buffer are resuspended, and the EDC adding 10mg/mL newly configured for 1mL is water-soluble Liquid, activated magnetic beads surface carboxyl groups, add 4mgHBsAg PreS monoclonal antibody (biorbyt, article No. orb48780), under room temperature Suspendible 6h, Magneto separate, remove supernatant, be resuspended to 1mg/mL with the Tris buffer that the 0.1M containing 2%BSA, pH are 8.0, obtain The coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the acridinium ester of HBsAg PreS labeling of monoclonal antibody:
Take the HBsAg PreS monoclonal antibody that 50 μ L concentration are 10mg/mL, add 150 μ L concentration be 0.1M, pH be 9.0 ~ 9.5 Carbonate buffer solution, mixing, be subsequently adding the mixing of acridinium ester solution that 1.5 μ L concentration are 5mg/mL, lucifuge reaction under room temperature, Take out after 1.5h, be centrifuged desalting column desalting processing with the zeba of 2mL, the most respectively by pure water and TBS buffering in desalination processes Liquid processes, and is eventually adding the acridinium ester solution of the HBsAg PreS labeling of monoclonal antibody obtained, and collects in centrifuge tube Liquid is in control the acridinium ester of HBsAg PreS monoclonal antibody mab labelling to preserving, and every bottle of 5mL subpackage is stored in 4 DEG C standby.
(3) preparation of HBsAg PreS calibration product:
With standard substance buffer (40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0), HBsAg PreS antigen is thrown Enter standard substance buffer being configured to COI is 0 ~ 0.5 and 1 ~ 10, and every bottle of 0.5 mL subpackage, 4 DEG C save backup.
Embodiment 2:HBsAg PreS chemical luminous immune detection method
Being detection instrument with Full-automatic chemiluminescence immunoassay analysis meter (YHLO, article No. iFlash3000), methodology pattern is double Antibody sandwich, i.e. instrument be sequentially added into the sample of 50 μ L, 50 μ L HBsAg PreS monoclonal antibody coated carboxylated Magnetic particle and the HBsAg PreS treatment fluid of 50 μ L, after reacting 20 min, then add the HBsAg PreS labelling of 100 μ L Acridinium ester, after reacting 20 min, carries out Magneto separate, and reactant mixture is sent into darkroom by instrument, is sequentially added into luminous substrate A liquid (H2O2) and B liquid (NaOH) carry out luminescence-producing reaction, finally record luminous value.
Embodiment 3:HBsAg PreS chemiluminescence immune detection reagent kit performance evaluation
Use the method in embodiment 2 that HBsAg PreS calibration product are detected, obtain Cutoff value.
Then test actual sample, calculate sample COI according to sample luminous value.
Precision measures:
Taking COI is 5 and 50 two HBsAg PreS samples, each concentration of each sample respectively do 3 parallel, enter with three batches of test kits Row detection, calculates test kit and criticizes interior and difference between batch, and result shows that this test kit is criticized interior and difference between batch and is respectively less than 5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, glycerol Ester, adding proportion is carried out according to 1:20, the measured value of pooled serum after measuring pooled serum respectively and with the addition of various chaff interference, Calculate deviation therebetween, with ± 10% as tolerance interval.Result shows, interference all reaches the files-designated of NCCLS Standard, can be used for the accurate evaluation of clinical laboratory's HBsAg PreS situation.
Embodiment 4, the contrast experiment of HBsAg PreS chemiluminescence immune detection reagent kit
By chemical luminescence detection method and traditional enzyme linked immunosorbent assay, one group of gradient dilution sample is detected respectively, two kinds Method detection sensitivity is compared, and data are as shown in the table:
Testing time Chemiluminescence detection (COI) Enzyme linked immunosorbent assay detection (COI)
Former times of sample 1122.76 17.78
1:10 diluted sample 794.19 15.67
1:20 diluted sample 547.48 9.17
1:40 diluted sample 386.34 5.61
1:80 diluted sample 220.19 3.22
1:160 diluted sample 113.83 1.72
1:320 diluted sample 52.32 0.94
1:640 diluted sample 23.59 0.67
1:1280 diluted sample 11.50 0.44
Diluent 0.22 0.22
As can be seen from the above table, the sensitivity of chemical luminescence detection method relatively enzyme linked immunosorbent assay improves about 10 times.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but can not Therefore the restriction to the scope of the claims of the present invention it is interpreted as.It should be pointed out that, for the person of ordinary skill of the art, Without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the protection model of the present invention Enclose.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (10)

1. a HBsAg PreS chemiluminescence immune detection reagent kit, it is characterised in that including: HBsAg PreS monoclonal anti The coated carboxylated magnetic particle of body and the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described In the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody, described HBsAg PreS monoclonal antibody and described carboxyl The ratio of the magnetic particle changed is 1:25 ~ 100.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described In the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody, described HBsAg PreS monoclonal antibody and described chemistry The ratio of luminous marker is 1:10 ~ 100.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described carboxyl The particle diameter of the magnetic particle changed is 0.05 μm ~ 1 μm.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described chemistry Luminous marker is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that also including Learning luminous substrate liquid, described Chemoluminescent substrate includes A liquid and B liquid.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 6, it is characterised in that described A liquid For H2O2Solution, described B liquid is NaOH solution.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that also include HBsAg PreS calibrates product.
HBsAg PreS chemiluminescence immune detection reagent kit the most according to claim 8, it is characterised in that described HBsAg PreS calibration product are the HBsAg PreS solution of COI 1 ~ 10.
10. the system according to the HBsAg PreS chemiluminescence immune detection reagent kit according to any one of claim 1 ~ 9 Preparation Method, it is characterised in that comprise the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC aqueous solution, The surface carboxyl groups of the magnetic particle of activated carboxyl, is subsequently added into HBsAg PreS monoclonal antibody, suspendible 2h ~ 10h, magnetic under room temperature Separate after removing supernatant resuspended with Tris buffer, obtain the coated carboxylated magnetic particle of HBsAg PreS monoclonal antibody； And take HBsAg PreS monoclonal antibody, and mix after adding carbonate buffer solution, mixed after being subsequently adding chemiluminescent labels Even, under room temperature, remove impurity after lucifuge reaction 1h ~ 2h, obtains the chemiluminescent labels of HBsAg PreS labeling of monoclonal antibody.
CN201610506956.8A 2016-06-30 2016-06-30 HBsAg PreS chemiluminescence immune detection reagent kit and preparation method thereof Pending CN106257283A (en)

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