CN101294957B - HLA-B27 magnetic ELISA reagent kit and method for producing the same - Google Patents
HLA-B27 magnetic ELISA reagent kit and method for producing the same Download PDFInfo
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- CN101294957B CN101294957B CN2007100278233A CN200710027823A CN101294957B CN 101294957 B CN101294957 B CN 101294957B CN 2007100278233 A CN2007100278233 A CN 2007100278233A CN 200710027823 A CN200710027823 A CN 200710027823A CN 101294957 B CN101294957 B CN 101294957B
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Abstract
The invention discloses an HLA-B27 magnetic enzyme-linked immunoassay kit, essentially comprising enzyme diluent, enzyme combination and a micropore plate lath coated with immunomagnetic microbeads which are covalent and coupled with an HLA-B27 anti-body; the preparing method for the immunoassay kit comprises the following steps: (1) the immunomagnetic microbeads are covalent and coupled with the HLA-B27 anti-body; (2) the preparation of the enzyme combination; (3) the micropore plate is coated; the HLA-B27 magnetic enzyme-linked immunoassay kit has the advantages of rapidness, simpleness, correctness and low cost, etc.
Description
Technical field
The invention belongs to medical instruments field, relate to a kind of HLA-B27 magnetic ELISA reagent kit and preparation method thereof specifically.
Background technology
(ankylosing spondylitis is a kind of connective tissue disease AS) to ankylosing spondylitis, and the morbidity rate in the crowd is 0.2%-0.4%, and age of onset in 30 years old, accounts for 65% mostly.Average course of disease 4-6.Cardinal symptom is waist, sacrum and joint of lower extremity pain.Characteristics such as often stiff, that night heavy, movable back is light with morning.Different with adult AS, it is less that teenager AS causes that at the initial stage of a disease the symptom of bone pathology occurs, and have children main suit's extremities joint pain of 24% or have only talagia, but the performance of tip arthropathy clearly.AS mainly shows as the inflammation and the hyperplasia of ossified, the articuli intervertebrales and the extremities joint synovial membrane of near connective tissue annulus fibrosus disci intervertebralis and the fibrous ring.The patients with terminal backbone is stiff to be tabular, limitation of activity.Late period, disability rate was high.Thereby early diagnosis, treatment are significant.
(humanleukocyte antigen HLA) is a kind of isoantigen on the leucocyte film to HLA, and the site coding according to different has the important physical function.As far back as 1973, Brewerton etc. reported HLA-B27 and ankylosing spondylitis (ankylosing spondylitis, AS) strong correlation.In AS patient, 88%-96% patient has HLA-B27 antigen, is merely 4%-8% and have B27 antigen person in the normal control group.The relative risk rate reaches 90%.
Although HLA-B27 and ankylosing spondylitis have very strong correlativity, find HLA-B27 positive and can not be diagnosed as ankylosing spondylitis, HLA-B27 is the factor of a susceptible disease of ankylosing spondylitis.
HLA-B27 detects can not be as the negative SpA of serology examining finger veins mark really, but will help diagnosis in the following aspects:
If the possibility that 1 sings and symptoms prompting joint of vertebral column pathology takes place surpasses 50%, the B27 antigen positive significantly increases the chance of correct diagnosis so.
2 backaches and tetanic patient, the strong prompting of B27 antigen negative suffers from other diseases.Without psoriasis and struvite small intestine pathology the time, the B27 antigen negative can be got rid of the AS diagnosis.
3 suffer from the teenager of struvite arthropathy, and the B27 antigen positive can be pointed out, and the negative joint of vertebral column pathology of serology may take place.
The possibility of AS takes place in 4 prediction AS patient kinsfolks.If certain member carries the haplotype relevant with AS, especially the male sex indicates that then the possibility of its morbidity is bigger, is 20%-30%, does not have this haplotype person, and the possibility of morbidity is minimum, is almost 0.
The method of early detection HLA-B27 antigen be microlymphocytotoxicity test (microlymphocytotoxicity test, MLCT).This method wastes time and energy, and (flow cytometry FC) replaces by streaming gradually at present.But FC costs an arm and a leg, complicated operation.Recently, having developed again is the method on basis with PCR, though this method accuracy is high, is not suitable for doing the large sample rapid screening.
Enzyme-labeled immunity magnetic antibody separation detection technology (Magnetic Anti-body Immuno Assay MAIA) is a highly sensitive enzyme immune technology, and its detection sensitivity can reach the level of radioimmunology.It introduces the enzyme-linked immunoassay field with the immunity magnetic micropearls isolation technics.The monoclonal antibody that magnetic micro-beads connects is with after corresponding sample antigen combines, and under the effect of magnetic force, antigen and other separating substances of specificity combination are passed through the enzyme linked immunological chromogenic reaction again, measure its content.Up to now, also the someone arrives the HLA-B27 Detection of antigen with this technical application.
Summary of the invention
One of technical issues that need to address of the present invention provide a kind of HLA-B27 magnetic ELISA reagent kit, and this kit is based upon the magnetic immuno separation and enzyme detects on the principle, have quick, simple, accuracy advantages of higher.
The technology contents that solves the problems of the technologies described above is:
A kind of HLA-B27 magnetic ELISA reagent kit mainly comprises the enzyme dilution, and enzyme conjugates also includes:
Be coated with the microwell plate lath that joins immunity magnetic micropearls with HLA-B27 antibody covalency coupling.
Preferably, said enzyme conjugates is the HRP that coupling is associated with anti-CD45 antibody, promptly anti-CD45 enzyme conjugates.
The dilutability of HLA-B27 antibody and anti-CD45 enzyme conjugates is 1:50.
This kit also includes, and the HRP coupling joins the positive control of HLA-B27 antibody molecule.
The enzyme dilution is preferably the 0.01M PBS damping fluid that is added with 1%BSA (bSA), 0.1%Proclin300.
HLA-B27 magnetic ELISA reagent kit according to the invention is used to detect the whole blood sample of EDTA-K3 (ethylenediamine tetraacetic acid tripotassium) as anti-coagulants.
Another technical issues that need to address of the present invention provide the preparation method of above-mentioned HLA-B27 magnetic ELISA reagent kit.
A kind of preparation method of HLA-B27 magnetic ELISA reagent kit mainly may further comprise the steps:
(A) the covalency coupling joins immunity magnetic micropearls and HLA-B27 antibody: PBS damping fluid (NaCl137mmol/L, KCl2.7mmol/L, Na
2HPO
44.3mmol/L, KH
2PO
41.4mmol/L), pH6.5-7.5 washing 1ml immunity magnetic micropearls 1-5 time adds antibody 8-12mg/ml, and 1ml behind the mixing, adds difunctional coupling molecule BS altogether
3(Bis [sulfosuccinimidyl] suberate), making its final concentration is 0.8-1.2mM, incubated at room 20-40min; Add stop buffer 0.7-1.2M Tris damping fluid, pH6.5.-7.5, incubated at room 7-13min; With PBS washing 2-5 times, recover volume 1ml;
(B) preparation of enzyme conjugates;
(C) encapsulate microwell plate:
To join immunity magnetic micropearls 2-4ul/ hole with HLA-B27 antibody covalency coupling joins in the microwell plate.
The HLA-B27 antibody covalency coupling couplet immunity magnetic micropearls that joins in the microwell plate is preferably the 3ul/ hole.
Preferably, the covalency coupling joins immunity magnetic micropearls and HLA-B27 antibody comprises: PBS, pH7.0 washing 1ml immunity magnetic micropearls 3 times adds antibody 10mg/ml, altogether 1ml.Behind the mixing, add difunctional coupling molecule BS
3, making its final concentration is 1mM, incubated at room, and 30min adds stop buffer 1M Tris damping fluid, pH7.0, incubated at room 10min with PBS washing 3 times, recovers volume 1ml.Add 1%BSA then as stabilizing agent, 0.1%NaN3 is as antiseptic.
The preparation of enzyme conjugates comprises: accurately take by weighing 10mgHRP (horseradish peroxidase) and place clean container, add 0.2mol/L pH and be 5.6 acetate buffer, treat the enzyme dissolving after, add 0.06mol/LNaIO
4Solution room temperature reaction 20 minutes; Add 0.16mol/L monoethylene glycol-10% sodium chloride solution, after reacting 20 minutes under the room temperature; Above-mentioned enzyme liquid is packed in the bag filter, and using 0.001mol/LpH is 4.0 acetate buffer dialysed overnight; Add 2mol/L pH and be 9.6 carbonate buffer solution, add anti-CD45 antibody 10mg to be marked after, 4 ℃ of stirring reactions 2 hours.The NaBH that adds new preparation then
4Solution (concentration is 5mg/ml), 4 ℃ of stirring reactions 2 hours; Drip saturated (NH4)
2SO
4Solution stir to be placed 30 minutes for 4 ℃, 3500 rev/mins centrifugal 20 minutes, abandon supernatant, it is 7.4 phosphate buffer that deposition is dissolved in 0.02mol/LpH; Using 0.02mol/LpH is 7.4 phosphate buffer dialysis 24 hours, takes out dislysate, add equal-volume enzyme protection liquid after, add equal-volume glycerine again ,-20 ℃ of preservations behind the mixing.
This kit can also comprise enzyme linked immunological kit substrate solution A commonly used, substrate solution B, stop buffer, 20 times of concentrated washing lotions, negative control.
Patients blood sample (EDTA-K3 anti-freezing) all joins in the microwell plate micropore that contains HLA-B27 antibody mediated immunity magnetic micro-beads with the anticellular antibody of HRP mark; If on the leucocyte in the sample HLA-B27 antigen is arranged; Through hatching in short-term; Immunity magnetic micropearls combines with leucocyte, and this moment, the anticellular antibody of HRP mark was connected on the cell.After the washing, immunity magnetic micropearls, cell and enzyme are retained; If do not have B27 antigen on the leucocyte, through washing, cell and enzyme all are cleaned; Add the colour developing of zymolyte liquid afterwards, 450nm reads OD value.
HLA-B27 magnetic ELISA reagent kit of the present invention has advantages such as stable, that performance is good.
Carry out 310 parts of blood sample tests with HLA-B27 magnetic ELISA reagent kit according to the invention.Make streaming HLA-B27 reagent simultaneously and be used for contrast.
Table 1 and streaming contrast statistics
| Streaming is positive | Streaming is negative | ||
| Positive | 35 | 8 | 43 |
| Negative | 2 | 265 | 267 |
| 37 | 273 | 310 |
With the streaming method as goldstandard; The sensitivity of HLA-B27 magnetic ELISA reagent kit of the present invention is 35/37=94.6%; Specificity is 265/273=97.1%, and accuracy is (35+265)/310=96.8%, explains that the result of this kit and streaming meets fine.
The enzyme conjugates stability test:
Experimental technique
Enzyme conjugates and dilution that branch installs were placed seven days in 37 ℃ of biochemical incubators, tested simultaneously with the enzyme conjugates and the dilution that are kept at 4 ℃ after the taking-up balance room temperature.
Experimental result
37 ℃ of table 2 antibody-enzyme conjugates and 4 ℃ of contrasts of depositing after seven days
Can know that by table 2 after anti-CD45 enzyme conjugates was put 37 ℃ of biochemical incubators and deposited seven days, except that the OD value decreased slightly, to other index did not influence, it had good stability, and can be used for HLA-B27 magnetic EIA enzyme immunoassay fully.
The preparation method of the HLA-B27 magnetic ELISA reagent kit that preparation method of the present invention obtains also has fast, simple, accurate, low cost and other advantages.
Embodiment
A kind of HLA-B27 magnetic ELISA reagent kit; The enzyme dilution that comprises the 0.01M PBS of 1%BSA, 0.1%Proclin300; Anti-CD45 enzyme conjugates is coated with the microwell plate lath that joins immunity magnetic micropearls with HLA-B27 antibody covalency coupling, and the HRP coupling joins the positive control of HLA-B27 antibody molecule, is equipped with enzyme linked immunological kit substrate solution A, substrate solution B, stop buffer, 20 times of concentrated washing lotions commonly used when using; 0.2MPBS negative control; Wherein, during use, the dilutability of HLA-B27 antibody and CD45 enzyme conjugates is 1:50.
The preparation method of above-mentioned HLA-B27 magnetic ELISA reagent kit mainly may further comprise the steps:
(A) the covalency coupling joins immunity magnetic micropearls and HLA-B27 antibody:
PBS, pH7.0 washing 1ml immunity magnetic micropearls 3 times adds antibody 10mg/ml, and 1ml behind the mixing, adds difunctional coupling molecule BS altogether
3(Bis [sulfosuccinimidyl] suberate), making its final concentration is 1mM, incubated at room 30min adds stop buffer 1M Tris damping fluid, Ph7, incubated at room 10min with PBS washing 3 times, recovers volume 1ml.Add 1%BSA (bSA) then as stabilizing agent, 0.1%NaN
3As antiseptic.
(B) preparation of enzyme conjugates:
Accurately take by weighing 10mgHRP (horseradish peroxidase) and place a clean container, add 0.2mol/L pH and be 5.6 acetate buffer 1ml, treat the HRP dissolving after, add 0.06mol/LNaIO
4Solution 1ml, room temperature reaction 20 minutes; Add 0.16mol/L monoethylene glycol-10% sodium chloride solution 1ml, after reacting 20 minutes under the room temperature; Above-mentioned enzyme liquid is packed in the bag filter, and using 0.001mol/LpH is 4.0 acetate buffer dialysed overnight; Add 2mol/L pH and be 9.6 carbonate buffer solution, add anti-CD45 antibody 10mg to be marked after, 4 ℃ of stirring reactions 2 hours.The NaBH that adds new preparation then
4Solution (concentration is 5mg/ml) 2ml, 4 ℃ of stirring reactions 2 hours; Drip saturated (NH4)
2SO
4Solution 5ml stir to place 30 minutes for 4 ℃, 3500 rev/mins centrifugal 20 minutes, abandon supernatant, it is 7.4 phosphate buffer 5ml that deposition is dissolved in 0.02mol/LpH; Using 0.02mol/LpH is 7.4 phosphate buffer dialysis 24 hours, takes out dislysate, add equal-volume enzyme protection liquid after, add equal-volume glycerine again ,-20 ℃ of preservations behind the mixing.
(C) encapsulate microwell plate
To join immunity magnetic micropearls with HLA-B27 antibody covalency coupling by the 3ul/ hole and join in the microwell plate Vacuum Package.
The used said substrate solution A of present embodiment, substrate solution B, stop buffer, 20 times of concentrated washing lotions also can prepare by following method available from Anqun Bioengineering Co., Ltd., Shenzhen:
The preparation of substrate solution A:
In mixer, add production 0.2MPBS5%;
Press 5g/1000ml in the mixer and add H
2O
2. be stirred to fully evenly.
Add pure water to producing cumulative volume, stir; Packing.
The preparation of substrate solution B:
In mixer, add production 0.2MPBS5%;
Adding TMB. by 3g/1000ml in the mixer is stirred to fully evenly.
Add pure water to producing cumulative volume, stir; Packing.
The preparation of 20 times of concentrates:
In mixer, add production 0.2MPBS85%;
Press 0.5ml/1000ml in the mixer and add polysorbas20, be stirred to fully evenly.
Add 0.2MPBS to producing cumulative volume, stir; Packing
The preparation of stop buffer:
In mixer, add production pure water 60%;
Add the concentrated sulphuric acid by 125ml in the mixer. stir;
Add pure water to producing cumulative volume, stir; Packing.
Because people's cell is difficult to preserve, so HLA-B27 molecule coupling is joined upward HRP, as the positive control of kit of the present invention, its coupling linked method is identical with antileukocyte antibody (anti-CD45 antibody) coupling couplet HRP.
More than all reagent all should be in the storage of 4 ℃ of lucifuges.
Through the repeatedly influence of the anti-coagulants of comparative determination repeatedly of experiment, in multiple anti-coagulants (for example sodium citrate, heparin and EDTA-K3), select to use the anti-coagulants of EDTA-K3 as the blood sample of the mensuration of kit according to the invention.
During use, can operate according to the following steps:
1. take out all reagent and blood sample, balance is to room temperature;
2. the patient's sample numbering is arranged into patient's sample, positive control, negative control in the micropore;
3. take out required micropore lath, all the other are put back in the sealing bag and seal, and put into refrigerator;
4. with the 50 times of dilutions of anticellular antibody of enzyme dilution with the HRP mark;
| The enzyme dilution | 50uL X (sample number+2, positive and negative contrast) |
| The anticellular antibody of HRP mark | 1uL X (sample number+2, positive and negative contrast) |
5. behind the abundant mixing, add 50ul in each micropore, pat grillage, immunity magnetic micropearls is disperseed;
6. anticoagulant tube (EDTA-K3) is put upside down mixing for several times, the every hole of arrangement of pressing in the record adds the 50ul blood sample.With pipettor piping and druming 5-6 time, make the abundant mixing of immunity magnetic micropearls and whole blood sample; Respectively add the 50uL positive, negative control in corresponding micropore; Immunity magnetic micropearls and whole blood sample must mix evenly;
7 reactions 10 minutes under room temperature, during pat frame edge for several times, make reagent and sample keep even the mixing;
Prepare washing lotion, substrate solution during 8 reactions.Washing lotion is by every hole 1500ul, with 20 times of dilutions of deionized water;
9 every holes add the 250ul washing lotion.Utilization adds fashionable impulsive force disperses immunity magnetic micropearls, as finding the group of gathering is arranged, and dispels with pipettor.Grillage is put on the magnetic sheet 2 minutes;
10 usefulness, eight road pipettors are abandoned supernatant in the micropore sidewall suction away from magnet, stay about 10uL, do not blot, and take off and pat grillage, and immunity magnetic micropearls is disperseed;
11 repeat 9,10 steps totally 5-6 times;
12 every holes add the mixed liquor 100ul of substrate A liquid, B liquid, room temperature, lucifuge, 15 minutes, observations;
13 as needing to measure the OD value, and every hole adds the 50ul stop buffer;
14 are put in grillage on the magnetic sheet, and immunity magnetic micropearls is inhaled in the lath sidewall;
15 take off grillage gently, under wavelength 450nm, read absorbance (OD).
Claims (7)
1. a HLA-B27 magnetic ELISA reagent kit mainly comprises the enzyme dilution, and enzyme conjugates is characterized in that, also includes: be coated with the microwell plate lath that joins immunity magnetic micropearls with HLA-B27 antibody covalency coupling,
The preparation of said microwell plate lath is following: the covalency coupling joins immunity magnetic micropearls and HLA-B27 antibody: PBS, and pH6.5-7.5 washing 1ml immunity magnetic micropearls 1-5 time adds antibody 8-12mg/ml, and 1ml behind the mixing, adds difunctional coupling molecule BS altogether
3, making its final concentration is 0.8-1.2mM, incubated at room 20-40min adds stop buffer 0.7-1.2M Tris damping fluid, and pH6.5-7.5, incubated at room 7-13min with PBS washing 2-5 time, recovers volume 1ml; Encapsulate microwell plate: will join immunity magnetic micropearls 2-4ul/ hole with the HLA-B27 antibody covalency coupling of step (A) preparation and join in the microwell plate;
The 0.01M PBS that said enzyme dilution is 1%BSA, 0.1%Proclin300;
Said enzyme conjugates is the HRP that joins with anti-CD45 antibody coupling, and the dilutability of HLA-B27 antibody and enzyme conjugates is 1: 50.
2. HLA-B27 magnetic ELISA reagent kit according to claim 1 is characterized in that, also includes the positive control that the HRP coupling joins the HLA-B27 antibody molecule.
3. according to the arbitrary described HLA-B27 magnetic ELISA reagent kit of claim 1-2, it is characterized in that the whole blood sample that this kit is used to detect is as anti-coagulants with EDTA-K3.
4. a method for preparing the said HLA-B27 magnetic ELISA reagent kit of claim 1 is characterized in that, mainly may further comprise the steps:
(A) the covalency coupling joins immunity magnetic micropearls and HLA-B27 antibody: PBS, and pH6.5-7.5 washing 1ml immunity magnetic micropearls 1-5 time adds antibody 8-12mg/ml, and 1ml behind the mixing, adds difunctional coupling molecule BS altogether
3, making its final concentration is 0.8-1.2mM, incubated at room 20-40min adds stop buffer 0.7-1.2M Tris damping fluid, and pH6.5-7.5, incubated at room 7-13min with PBS washing 2-5 time, recovers volume 1ml;
(B) preparation of enzyme conjugates;
(C) encapsulate microwell plate:
To join immunity magnetic micropearls 2-4ul/ hole with the HLA-B27 antibody covalency coupling of step (A) preparation joins in the microwell plate.
5. the preparation method of above-mentioned HLA-B27 magnetic ELISA reagent kit according to claim 4; It is characterized in that: being prepared as of said enzyme conjugates: take by weighing 10mgHRP and place clean container; Adding 0.2mol/L pH is 5.6 acetate buffer; After treating the enzyme dissolving, add 0.06mol/LNaIO
4Solution, room temperature reaction 10-30 minute; Add 0.16mol/L monoethylene glycol-10% sodium chloride solution, during bag filter was packed in reaction into after 10-30 minute under room temperature, use 0.001mol/LpH was 4.0 acetate buffer dialysed overnight; Add 2mol/L pH and be 9.6 carbonate buffer solution, adds anti-CD45 antibody 9-11mg to be marked after, 4 ℃ of stirring reactions 2 hours, adding the concentration of newly preparing then is the NaBH of 5mg/ml
4Solution, 4 ℃ stirring reaction 1-3 hour; Drip saturated (NH4)
2SO
4Solution stir to be placed 20-40 minute for 4 ℃, and centrifugal 10-30 minute, abandon supernatant, it is 7.4 phosphate buffer that deposition is dissolved in 0.02mol/LpH; Using 0.02mol/LpH is 7.4 phosphate buffer dialysis 18-30 hour, takes out dislysate, add equal-volume enzyme protection liquid after, add equal-volume glycerine again, mixing.
6. the preparation method of above-mentioned HLA-B27 magnetic ELISA reagent kit according to claim 4 is characterized in that: the HLA-B27 antibody covalency coupling couplet immunity magnetic micropearls that step (C) joins in the microwell plate is the 3ul/ hole.
7. according to the preparation method of the arbitrary described above-mentioned HLA-B27 magnetic ELISA reagent kit of claim 4-6; It is characterized in that: the covalency coupling joins immunity magnetic micropearls and HLA-B27 antibody is: PBS; PH7.0 washing 1ml immunity magnetic micropearls 3 times adds antibody 10mg/ml, altogether 1ml; Behind the mixing, add difunctional coupling molecule BS
3, making its final concentration is 1mM, incubated at room, and 30min adds stop buffer 1M Tris damping fluid, pH7.0, incubated at room 10min with PBS washing 3 times, recovers volume 1ml, adds 1%BSA then as stabilizing agent, 0.1%NaN
3As antiseptic.
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Non-Patent Citations (3)
| Title |
|---|
| Chung-Tei Chou等.The detection of the HLA-B27 antigen by immunomagnetic separation and enzyme-linked immunosorbent assay—comparison with a flow cytometric procedure.《Journal of Immunological Methods》.2001,第255卷第15-22页. * |
| HLA B 27检测在强直性脊柱炎临床诊断中的意义.黄翠珍等.《实用医学杂志》.2005,第21卷(第4期),第390-391页. * |
| 郑佐娅等.磁珠分离酶联免疫测定法在检测人血清肌红蛋白中的应用.《检验医学》.2005,第20卷(第1期),第17-19页. * |
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