CN103713121A - Human vasculitis diagnostic kit and preparation method thereof - Google Patents
Human vasculitis diagnostic kit and preparation method thereof Download PDFInfo
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- CN103713121A CN103713121A CN201310737092.7A CN201310737092A CN103713121A CN 103713121 A CN103713121 A CN 103713121A CN 201310737092 A CN201310737092 A CN 201310737092A CN 103713121 A CN103713121 A CN 103713121A
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Abstract
The invention discloses a human vasculitis diagnostic kit and a preparation method thereof, and belongs to the technical field of medical laboratory diagnosis. The human vasculitis diagnostic kit comprises (1) a standard substance of a serum marker to be detected, (2) a solid phase plate enveloped with an identity marker specific antigen system, (3) an antibody linked with a marker of horseradish peroxidase, (4) a scrubbing solution, (5) a solution of a horseradish peroxidase substrate, and (6) a stop buffer. The method disclosed by the invention has the advantages of being flexible and accurate, good in repeatability, rapid in reaction and free from isotope pollution; the kit is stable in performance, simple and convenient to operate, and suitable for clinical popularization and application.
Description
Technical field
The present invention relates to a kind of people's vasculitis diagnostic kit and preparation method thereof, belong to medical experiment diagnostic techniques field.
Background technology
Systemic vasculitis (systemic vasculitis) is one group and take the disease that angionecrosis and inflammation be main pathological change, and clinical manifestation shows different because type, size, position and the pathological characteristic of involved vessels is different.Common vasculitis has vasculitis, Goodpasture syndrome and polyarteritis nodosa etc. under Wei Genashi granuloma, microscope.It often involves a plurality of systems of whole body, both can cause multisystem multiple organs dysfunction, but also can be confined to a certain internal organs.The diarrhoea that main manifestations is unknown cause clinically, and fever, stomachache, hemoproctia etc.Along with the development of the state of an illness, also there is the outer symptom of enteron aisle in various degree in some patient, as the illness of liver aspect, and arthritis, the symptom of skin and eyes aspect.Due to its complicated clinical manifestation, the state of an illness lays particular stress on, early stage diagnosis and treatment difficulty, disability rate, case fatality rate are high, poor prognosis, therefore, the early diagnosis of this disease, treat extremely important.
Distinctive anti-neutrophil leucocyte cytoplasmic antibody (anti-neutrophil cytoplasmic antibody, ANCA) be a kind of autoantibody that neutrophil leucocyte and monocyte kytoplasm composition be target antigen of take, comprised a large amount of autoantibodies, wherein Anti-proteinase 3 (PR3) antibody and anti-myeloperoxidase (MPO) antibody have clear and definite clinical diagnosis meaning, are most important ANCA antibody.Study verified anti-PR3 and anti-MPO antibody and participated in vasculitic pathogenesis, two kinds of antibody can activate human neutrophil and cause de-particle reaction, make neutrophil leucocyte discharge a large amount of harmful proteolytic enzyme and oxygen radicals, thereby cause the infringement of little vascular wall, cause vasculitic generation.
During current clinical detection systemic vasculitis, adopt ELISA method to measure anti-PR3 antibody and anti-MPO antibody, and adopt indirect immunofluorescence (IIF method) to detect ANCA.When IIF method is surveyed ANCA adopt ethanol fixedly the antigen sheet of neutrophil leucocyte carry out, easily be subject to the impact of antinuclear antibodies in sample (ANA), can on ethanol stator, there is fluorescent grain difficult and ANCA difference in ANA, especially obvious when patient's ANA is strong positive, the judgement that can produce interference experiment result.
Summary of the invention
First technical matters that the present invention will solve is to provide a kind of people's vasculitis diagnostic kit, and this kit can be got rid of ANA and disturb, and realizes the quantitative detection of anti-PR3 antibody and anti-MPO antibody.
Second technical matters that the present invention will solve is to provide the preparation method of mentioned reagent box.
For achieving the above object, the present invention is by the following technical solutions:
People's vasculitis diagnostic kit, composition comprises:
(1) standard items of serum marker to be detected;
(2) be coated with the solid phase plate of identification marking thing specific antigen system;
(3) be connected with the antibody of the marker of horseradish peroxidase;
(4) cleansing solution;
(5) solution of horseradish peroxidase substrate;
(6) stop buffer.
The standard items of described (1) serum marker to be detected are anti-human PR3 and MPO antigen immune Lysozyme.
The solid phase plate that described (2) are coated with identification marking thing specific antigen system comprises: the solid phase plate of the solid phase plate of coated human granular leukocyte, coated anti-human PR3 antigen immune Lysozyme and the solid phase plate of coated anti-human MPO antigen immune Lysozyme;
The antibody that described (3) are connected with the marker of horseradish peroxidase is anti-non-human immunoglobulin g antibody.
Described (4) cleansing solution is phosphate buffer.
The solution of described (5) horseradish peroxidase substrate comprises A liquid and B liquid, and wherein A liquid is superoxol, and B liquid is tetramethyl biphenyl amine aqueous solution, or A liquid is tetramethyl benzidine, and B liquid is sodium acetate/citrate buffer.
Described (6) stop buffer is 2M sulfuric acid.
The preparation method of the standard items of described (1) serum marker to be detected is: in the serum of the anti-human PR3 of high-titer and MPO, prepare immunoglobulin G antiserum, purification IgG, through QAE chemical reagent, process serum separation of serum, the immunoglobulin G serum of the anti-PR3 of high-titer and MPO is mixed with, deactivation complement, add again stabilizer treatment, obtained sample is done to purity detecting with electrophoresis, calculated purity.
The preparation method of the solid phase plate of described coated human granular leukocyte is: comprise and carry out granulocyte extraction, calculate cell purity and active detection, cell envelope step, the method of its cell envelope is for diluting granulocyte with 1 ╳ Hank balanced salt solution (HBSS), make it be about 100-1000000cells/mL to final concentration, then suspension is joined in 96 porocyte culture plates, every hole 100 μ L, be statically placed in room temperature, remove supernatant, drying at room temperature, fixes and is dried with cold methanol afterwards, with capping film, by the microwell plate capping of fixed cell, place 20 ℃ and save backup; The preparation method of the solid phase plate of the solid phase plate of described coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme is: using 0.05M phosphate buffer as coated damping fluid, the concentration of envelope antigen is 0.04-1.0 microgram/ml, coated solution in the hole of microwell plate 4 ℃, 24 hours, use deionized water washed twice, 2% bovine serum albumin(BSA) room temperature sealing is after 1.5 hours, in temperature, be that 20-28 ℃ and humidity are less than or equal under 30% condition dry 12 hours, then pack sealing, be stored in 4 ℃ stand-by.
A preparation method for people's vasculitis diagnostic kit, kit comprises:
(1) standard items of serum marker to be detected;
(2) be coated with the solid phase plate of marker specific antigen system: the solid phase plate that comprises the solid phase plate of coated human granular leukocyte, the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme;
(3) be connected with the antibody of the marker of horseradish peroxidase;
(4) cleansing solution;
(5) solution of horseradish peroxidase substrate;
(6) stop buffer;
The preparation method of the standard items of described serum marker to be detected is: in the serum of the anti-human PR3 of high-titer and MPO, prepare immunoglobulin G antiserum, purification IgG, through QAE chemical reagent, process serum separation of serum, the immunoglobulin G serum of the anti-PR3 of high-titer and MPO is mixed with, deactivation complement, add again stabilizer treatment, then obtained sample is done to purity detecting with electrophoresis, calculated purity.
The preparation method of the solid phase plate of described coated human granular leukocyte is: comprise and carry out granulocyte extraction, calculate cell purity and active detection, cell envelope step, the method of its cell envelope is for diluting granulocyte with 1 ╳ Hank balanced salt solution (HBSS), make it be about 100-1000000cells/mL to final concentration, then suspension is joined in 96 porocyte culture plates, every hole 100 μ L, be statically placed in room temperature, remove supernatant, drying at room temperature, fixes and is dried with cold methanol afterwards, with capping film, by the microwell plate capping of fixed cell, place 20 ℃ and save backup; The preparation method of the solid phase plate of the solid phase plate of described coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme is: using 0.05M phosphate buffer as coated damping fluid, the concentration of envelope antigen is 0.04-1.0 microgram/ml, coated solution in the hole of microwell plate 4 ℃, 24 hours, use deionized water washed twice, 2% bovine serum albumin(BSA) room temperature sealing is after 1.5 hours, in temperature, be that 20-28 ℃ and humidity are less than or equal under 30% condition dry 12 hours, then pack sealing, be stored in 4 ℃ stand-by;
The employing sodium iodate legal system of described horseradish peroxidase connection antibody is standby;
The solution of described horseradish peroxidase substrate comprises A liquid and B liquid, and wherein A liquid is superoxol, and B liquid is that tetramethyl biphenyl amine aqueous solution or A liquid are tetramethyl benzidine; B liquid is sodium acetate/citrate buffer;
Described stop buffer is sulfuric acid, and cleansing solution is phosphate buffer.
The invention provides a kind of detection to the immune analysis determination method of the special serum marker of vasculitis and the test kit of preparing according to the method, (1) measure uniqueness and the monopoly of content: whole markers that will detect in China at present all belong to innovative product, and the vasculitis external diagnosis reagent of developing for marker be also in current Chinese external diagnosis reagent market aspect vasculitis unique product.(2) measure highly sensitive, the high specificity, reproducible of kit, measurement result reliability is strong.Can be when seroreaction be very low or measure determinand at the commitment of disease development.Different from some other non-specific inflammation method of testing, this method of testing can detect the distinctive serum marker of vasculitis reliably.(3) measure the clinical value of kit: to the detection technique of vasculitis marker comprised to the detection of marker and in mensuration process binding immunoassay fluorescent technique can differentiate the type of target antigen.These testing results can assist a physician to vasculitic diagnosis, and these results also provide important reference value to treatment prognosis and treatment monitoring simultaneously.
The present invention compared with prior art, tool has the following advantages and beneficial effect: it is solid phase carrier that kit of the present invention adopts microwell plate, by immune response by the special serum marker of vasculitis the specific antigen system on being fixed to microwell plate be combined, then be combined with the immunoglobulin (Ig)-horseradish peroxidase of specific recognition marker, at joined enzyme, make under to the enzymatic reaction of substrate substrate present color change come quantitative measurement vasculitis special serum marker.The present invention adopts methyl alcohol fixed tablet cell detection plate, has got rid of the interference of ANA in blood samples of patients sample, and meanwhile, this kit can be realized the anti-PR3 antibody of quantitative measurement and anti-MPO antibody.This assay method is sensitive, accurately, reproducible, and fast, No Parity element pollutes in reaction; The kit stable performance forming, easy and simple to handle, be suitable for clinical application.
Below in conjunction with specification drawings and specific embodiments, describe the present invention in detail, with this, do not limit practical range of the present invention.
Accompanying drawing explanation
Fig. 1 is the typical curve of marker
Fig. 2 distributes for detecting the OD value of 99 routine vasculitides human serums
Embodiment
Embodiment 1: people's vasculitis diagnostic kit
One. kit forms:
(1) standard items of serum marker to be detected: anti-human PR3 and MPO antigen immune Lysozyme.
(2) be coated with the solid phase plate of identification marking thing specific antigen system: the solid phase plate that comprises the solid phase plate of coated human granular leukocyte, the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme;
(3) be connected with the antibody of the marker of horseradish peroxidase: the goat anti-human igg antibody (goat anti-human igg antibody, American I mmunoReagents Inc) who is connected with horseradish peroxidase
(4) cleansing solution: phosphate buffer;
(5) solution of horseradish peroxidase substrate: comprise A liquid and B liquid, wherein A liquid is superoxol, and B liquid is tetramethyl biphenyl amine aqueous solution, or A liquid is tetramethyl benzidine, and B liquid is sodium acetate/citrate buffer;
(6) stop buffer: sulfuric acid.
Two. reagent method for making:
(1) preparation of the standard items of serum marker to be detected
In the serum with the anti-human PR3 of high-titer and MPO by the detection system confirmation by the anti-human PR3 of immunofluorescence and MPO antibody target antigen-specific, prepare immunoglobulin G antiserum.
Get QAE-SephadexA25 or A50 balance in acid treatment the phosphate buffer at 0.05mol/LpH7.5, by moisture pump, claim weight in wet base 1g to be added in 10ml serum, after room temperature 30min, centrifugal or remove by filter ion exchanger.For another example this processes once supernatant, obtains purer IgG.After QAE chemical reagent is processed serum separation, the immunoglobulin G serum of the anti-PR3 of high-titer and MPO is mixed with, be placed in 56 ℃ of water temperature case 30min, deactivation complement, add again ProClin300 and do stabilizer treatment, then obtained sample is done to purity detecting with electrophoresis, by IgG pillar location on running gel and mark band, contrast, its molecular size range of Analysis deterrmination, calculates the purity that it is prepared.
The typical curve that accompanying drawing 1 is positive serum that coated granulocytic solid phase plate is done.This method to positive criteria product gradient dilution after, the typical curve obtaining with expert data process software, its R value can reach 0.999, linear relationship is good.Patients serum to be checked, after this method detects and to obtain absorbance, marks bent fitting formula or matrix diagram converts by this, can obtain the content that patients serum contains antibody to be checked.
(2) be coated with the preparation of the solid phase microwell plate of marker specific antigen system
A. extract granulocyte: from healthy, extract fresh peripheral blood standby to being added with the bloodletting tube of anti-coagulants; The whole blood of collection is diluted in proportion with the HBSS that does not contain endotoxin, calcium, magnesium ion; Whole blood after dilution adds containing above Ficoll-paque plus solution layer, centrifugal after, take out peripheral blood lymphocytes, and resuspended gently at HBSS damping fluid, standby; Rapidly cell is mixed gently with glucosan, sedimentation is also collected neutrophil leucocyte.Mixed with HBBS again, 20 ℃ centrifugal.By botal blood volume certain proportion, add cytolysate, with the HBSS damping fluid of cell pyrolysis liquid volume, stop solubilizing reaction, 4 ℃ centrifugal.Supernatant discarded, obtains the granulocyte needing; Supernatant discarded, with cell washing liquid, cell precipitation is resuspended, washing, 4 ℃ are centrifugal; The cell obtaining is resuspended standby with HBSS.
Granulocyte extraction ratio and purity detecting: use pipettor obtained cell suspension, drip on microslide and be dried; With organic reagent, fix; Dyeing liquor is done cell dyeing and is observed its form on microslide; Water rinses, and dries and micro-Microscopic observation; Counting cells kind calculate granulocyte purity under microscope; Cytoactive detects: the cell suspension that takes a morsel adds trypan blue, detects under the microscope cell survival situation and calculates granulocytic cell concentration.
B. be coated with the preparation method of the solid phase plate of human granular leukocyte: the granulocyte obtaining with 1 ╳ Hank balanced salt solution (HBSS) dilution step A, make it be about 1000000cells/mL to final concentration, then suspension is joined in 96 porocyte culture plates, every hole 100 μ L, are statically placed in room temperature, remove supernatant, drying at room temperature, with cold methanol, fix and be dried afterwards, with capping film, by the microwell plate capping of fixed cell, place 20 ℃ and save backup;
C. the preparation method of the solid phase plate of the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme is: using 0.05M phosphate buffer as coated damping fluid, the concentration of envelope antigen is 1.0 micrograms/ml, coated solution in the hole of microwell plate 4 ℃, 24 hours, use deionized water washed twice, 2% bovine serum albumin(BSA) room temperature sealing is after 1.5 hours, in temperature, be that 20-28 ℃ and humidity are less than or equal under 30% condition dry 12 hours, then pack sealing, be stored in 4 ℃ stand-by.
(3) prepare the marker antibody that horseradish peroxidase connects:
The preparation method that horseradish peroxidase connects antibody is sodium iodate method.Claim that horseradish peroxidase is dissolved in DDW, add freshly prepared 0.1M NaIO
4solution.Room temperature lucifuge.The dialysis of Dichlorodiphenyl Acetate sodium damping fluid, 4 ℃ are spent the night, and add carbonate buffer solution, then add the carbonate buffer solution that contains antibody (goat anti-human igg antibody, American I mmunoReagents Inc), room temperature reaction 2 hours.Add again freshly prepared NaBH, mix, reaction at 4 ℃.4 ℃ of PBS dialysis are spent the night.Under agitation dropwise add subsequently equal-volume saturated ammonium sulfate.3000rpm is centrifugal 0.5 hour afterwards, abandons supernatant, and sediment is dissolved in to PBS. then to 4 ℃ of PBS dialysis 4 hours, centrifugal 30 minutes of 10000rpm.The bond that makes enzyme, adds glycerine ,-20 ℃ of preservations.The extension rate of the antibody that enzyme connects is 1:70000.Dilution is phosphate buffer.After having configured, mix, place and detect by an unaided eye without crystallization or Precipitation after 30 minutes.
(4) preparation of cleansing solution
Cleansing solution is phosphate buffer, and composition is 135mM NaCl, 2.7mM KCl, 1.5mM KH
2pO
4, 8mMK
2hPO
4(pH7.2)
(5) solution of horseradish peroxidase substrate: A liquid is superoxol, B liquid is tetramethyl biphenyl amine aqueous solution; Purchased from ThermoFisher company
(6) stop buffer: 2M sulfuric acid.
Embodiment 2: people's vasculitis diagnostic kit
One. kit forms:
(1) standard items of serum marker to be detected: anti-human PR3 and MPO antigen immune Lysozyme.
(2) be coated with the solid phase plate of identification marking thing specific antigen system: the solid phase plate that comprises the solid phase plate of coated human granular leukocyte, the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme;
(3) be connected with the antibody of the marker of horseradish peroxidase: the goat anti-human igg antibody (goat anti-human igg antibody, American I mmunoReagents Inc) who is connected with horseradish peroxidase
(4) cleansing solution: phosphate buffer;
(5) solution of horseradish peroxidase substrate: comprise A liquid and B liquid, A liquid is tetramethyl benzidine, and B liquid is sodium acetate/citrate buffer;
(6) stop buffer: 2M sulfuric acid.
Two. reagent method for making:
(1) preparation of the standard items of serum marker to be detected
In the serum with the anti-human PR3 of high-titer and MPO by the detection system confirmation by the anti-human PR3 of immunofluorescence and MPO antibody target antigen-specific, prepare immunoglobulin G antiserum.
Get QAE-SephadexA25 or A50 balance in acid treatment the phosphate buffer at 0.05mol/LpH8.6, by moisture pump, claim weight in wet base 1g to be added in 10ml serum, after room temperature 30min, centrifugal or remove by filter ion exchanger.For another example this processes once supernatant, obtains purer IgG.After QAE chemical reagent is processed serum separation, the immunoglobulin G serum of the anti-PR3 of high-titer and MPO is mixed with, be placed in 56 ℃ of water temperature case 30min, deactivation complement, add again ProClin300 and do stabilizer treatment, then obtained sample is done to purity detecting with electrophoresis, by IgG pillar location on running gel and mark band, contrast, its molecular size range of Analysis deterrmination, calculates the purity that it is prepared.
(2) be coated with the preparation of the solid phase microwell plate of marker specific antigen system
A. extract granulocyte: from healthy, extract fresh peripheral blood standby to being added with the bloodletting tube of anti-coagulants; The whole blood of collection is diluted in proportion with the HBSS that does not contain endotoxin, calcium, magnesium ion; Whole blood after dilution adds containing above Ficoll-paque plus solution layer, centrifugal after, take out peripheral blood lymphocytes, and resuspended gently at HBSS damping fluid, standby; Rapidly cell is mixed gently with glucosan, sedimentation is also collected neutrophil leucocyte.Mixed with HBBS again, 20 ℃ centrifugal.By botal blood volume certain proportion, add cytolysate, with the HBSS damping fluid of cell pyrolysis liquid volume, stop solubilizing reaction, 4 ℃ centrifugal.Supernatant discarded, obtains the granulocyte needing; Supernatant discarded, with cell washing liquid, cell precipitation is resuspended, washing, 4 ℃ are centrifugal; The cell obtaining is resuspended standby with HBSS.
Granulocyte extraction ratio and purity detecting: use pipettor obtained cell suspension, drip on microslide and be dried; With organic reagent, fix; Dyeing liquor is done cell dyeing and is observed its form on microslide; Water rinses, and dries and micro-Microscopic observation; Counting cells kind calculate granulocyte purity under microscope; Cytoactive detects: the cell suspension that takes a morsel adds trypan blue, detects under the microscope cell survival situation and calculates granulocytic cell concentration.
B. be coated with the preparation method of the solid phase plate of human granular leukocyte: the granulocyte obtaining with 1 ╳ Hank balanced salt solution (HBSS) dilution step A, make it be about 1000000cells/mL to final concentration, then suspension is joined in 96 porocyte culture plates, every hole 100 μ L, are statically placed in room temperature, remove supernatant, drying at room temperature, with cold methanol, fix and be dried afterwards, with capping film, by the microwell plate capping of fixed cell, place 20 ℃ and save backup;
C. the preparation method of the solid phase plate of the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme is: using 0.05M phosphate buffer as coated damping fluid, the concentration of envelope antigen is 0.04 microgram/ml, coated solution in the hole of microwell plate 4 ℃, 24 hours, use deionized water washed twice, 2% bovine serum albumin(BSA) room temperature sealing is after 1.5 hours, in temperature, be that 20-28 ℃ and humidity are less than or equal under 30% condition dry 12 hours, then pack sealing, be stored in 4 ℃ stand-by.
(3) prepare the marker antibody that horseradish peroxidase connects:
The preparation method that horseradish peroxidase connects antibody is sodium iodate method.Claim that horseradish peroxidase is dissolved in DDW, add freshly prepared 0.1M NaIO
4solution.Room temperature lucifuge.The dialysis of Dichlorodiphenyl Acetate sodium damping fluid, 4 ℃ are spent the night, and add carbonate buffer solution, then add the carbonate buffer solution that contains antibody (goat anti-human igg antibody, American I mmunoReagents Inc), room temperature reaction 2 hours.Add again freshly prepared NaBH, mix, reaction at 4 ℃.4 ℃ of PBS dialysis are spent the night.Under agitation dropwise add subsequently equal-volume saturated ammonium sulfate.3000rpm is centrifugal 0.5 hour afterwards, abandons supernatant, and sediment is dissolved in to PBS. then to 4 ℃ of PBS dialysis 4 hours, centrifugal 30 minutes of 10000rpm.The bond that makes enzyme, adds glycerine ,-20 ℃ of preservations.The extension rate of the antibody that enzyme connects is 1:70000.Dilution is phosphate buffer.After having configured, mix, place and detect by an unaided eye without crystallization or Precipitation after 30 minutes.
(4) preparation of cleansing solution
Cleansing solution is phosphate buffer, and composition is 135mM NaCl, 2.7mM KCl, 1.5mM KH
2pO
4, 8mMK
2hPO
4(pH7.2)
(5) solution of horseradish peroxidase substrate: A liquid is superoxol, B liquid is tetramethyl biphenyl amine aqueous solution; Purchased from ThermoFisher company
(6) stop buffer: 2M sulfuric acid.
Embodiment 3: clinical detection test
One. experiment material
1. the kit of embodiment 1
2.99 routine vasculitis patients serum's samples
Two. detection method:
1. adopt conventional medical technology to collect whole blood sample, putting blood, after 30 minutes, to draw serum stand-by.Test serum, as used in 24 hours, can be preserved in 2-8 ℃ of condition, if need long-term storage, should be stored in below-20 ℃, and avoid multigelation.
2. to being coated with in the micropore of solid phase plate of marker specific antigen system, add blood serum sample to be checked, standingly make its antigen-antibody reaction combination.
(1) with PBST, test serum is diluted in proportion, every hole adds 100uL, room temperature oscillation incubation;
(2) washing: remove solution in hole, add PBST, standing, remove PBST, and repeat 2 times;
(3) ELIAS secondary antibody: will be connected with the antibody dilution (1:500-of the marker of horseradish peroxidase with enzyme dilution
1:2,000,000), every hole 100uL, room temperature oscillation incubation.
(4) washing: same step (2).
(5) colour developing: add the solution of horseradish peroxidase substrate, room temperature vibration colour developing 30min, under the effect of enzyme, zymolyte converts substance that show color to.Like this, by the mensuration of the color to changed, obtain the amount of marker to be measured in sample.Ratio according to the concentration of each known standard items in typical curve with colour developing, can calculate by formula the concentration value of marker to be measured.
(6) cessation reaction: every hole adds stop buffer, then surveys OD450.
(7) according to the fixed normal value of clinical data, determine whether the marker concentration in this Blood of Patients is in normal range or in abnormal ranges subsequently.
Kit Plays product can synchronously obtain typical curve, serum gained OD value to be checked, in substitution typical curve, try to achieve the content of anti-PR3 and MPO antibody in patients serum, its antibody content surpasses the patients serum of Cut off value, again according to clinical symptoms, can make a definite diagnosis the anti-PR3 and the MPO antibody that in patient body, produce high concentration, point out it to suffer from vasculitis.
Accompanying drawing 2 is for passing through this experimental technique, detect the confirmed cases serum of clinical collection, the loose point of its absorbance distributes, as shown in Figure 2, this method can be accomplished obvious discrimination to antibody test in patient body, can obviously distinguish and have or not illness and illness degree (passing through examined antibody content is judged), give clinician certain believable reference data.
The present invention carries out examination by the method for immunofluorescence to serum and judges ANCA, can effectively get rid of the impact of ANA.
For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. people's vasculitis diagnostic kit, is characterized in that comprising:
(1) standard items of serum marker to be detected;
(2) be coated with the solid phase plate of identification marking thing specific antigen system;
(3) be connected with the antibody of the marker of horseradish peroxidase;
(4) cleansing solution;
(5) solution of horseradish peroxidase substrate;
(6) stop buffer.
2. people's vasculitis diagnostic kit according to claim 1, is characterized in that: the standard items of described (1) serum marker to be detected are anti-human PR3 and MPO antigen immune Lysozyme.
3. people's vasculitis diagnostic kit according to claim 1, is characterized in that: described (2) are coated with solid phase plate, the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and the solid phase plate of coated anti-human MPO antigen immune Lysozyme that the solid phase plate of identification marking thing specific antigen system is coated human granular leukocyte.
4. people's vasculitis diagnostic kit according to claim 1, is characterized in that: the antibody that described (3) are connected with the marker of horseradish peroxidase is anti-non-human immunoglobulin g antibody.
5. people's vasculitis diagnostic kit according to claim 1, is characterized in that: described (4) cleansing solution is phosphate buffer.
6. people's vasculitis diagnostic kit according to claim 1, it is characterized in that: the solution of described (5) horseradish peroxidase substrate comprises A liquid and B liquid, wherein A liquid is superoxol, B liquid is tetramethyl biphenyl amine aqueous solution, or A liquid is tetramethyl benzidine, B liquid is sodium acetate/citrate buffer.
7. people's vasculitis diagnostic kit according to claim 1, is characterized in that: described (6) stop buffer is 2M sulfuric acid.
8. people's vasculitis diagnostic kit according to claim 1, it is characterized in that: the preparation method of the standard items of described (1) serum marker to be detected is: in the serum of the anti-human PR3 of high-titer and MPO, prepare immunoglobulin G antiserum, purification IgG, through QAE chemical reagent, process serum separation of serum, the immunoglobulin G serum of the anti-PR3 of high-titer and MPO is mixed with, deactivation complement, then add stabilizer treatment, obtained sample is done to purity detecting with electrophoresis, calculated purity.
9. people's vasculitis diagnostic kit according to claim 3, it is characterized in that: the preparation method of the solid phase plate of described coated human granular leukocyte is: comprise and carry out granulocyte extraction, calculate cell purity and active detection, cell envelope step, the method of its cell envelope is for diluting granulocyte with 1 ╳ Hank balanced salt solution (HBSS), make it be about 100-1000000cells/mL to final concentration, then suspension is joined in 96 porocyte culture plates, every hole 100 μ L, be statically placed in room temperature, remove supernatant, drying at room temperature, with cold methanol, fix and be dried afterwards, with capping film by the microwell plate capping of fixed cell, placing 20 ℃ saves backup, the preparation method of the solid phase plate of the solid phase plate of described coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme is: using 0.05M phosphate buffer as coated damping fluid, the concentration of envelope antigen is 0.04-1.0 microgram/ml, coated solution in the hole of microwell plate 4 ℃, 24 hours, use deionized water washed twice, 2% bovine serum albumin(BSA) room temperature sealing is after 1.5 hours, in temperature, be that 20-28 ℃ and humidity are less than or equal under 30% condition dry 12 hours, then pack sealing, be stored in 4 ℃ stand-by.
10. a preparation method for people's vasculitis diagnostic kit, is characterized in that:
Described kit comprises:
(1) standard items of serum marker to be detected;
(2) be coated with the solid phase plate of marker specific antigen system: the solid phase plate that comprises the solid phase plate of coated human granular leukocyte, the solid phase plate of coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme;
(3) be connected with the antibody of the marker of horseradish peroxidase;
(4) cleansing solution;
(5) solution of horseradish peroxidase substrate;
(6) stop buffer;
The preparation method of the standard items of described serum marker to be detected is: in the serum of the anti-human PR3 of high-titer and MPO, prepare immunoglobulin G antiserum, purification IgG, through QAE chemical reagent, process serum separation of serum, the immunoglobulin G serum of the anti-PR3 of high-titer and MPO is mixed with, deactivation complement, add again stabilizer treatment, then obtained sample is done to purity detecting with electrophoresis, calculated purity;
The preparation method of the solid phase plate of described coated human granular leukocyte is: comprise and carry out granulocyte extraction, calculate cell purity and active detection, cell envelope step, the method of its cell envelope is for diluting granulocyte with 1 ╳ Hank balanced salt solution (HBSS), make it be about 100-1000000cells/mL to final concentration, then suspension is joined in 96 porocyte culture plates, every hole 100 μ L, be statically placed in room temperature, remove supernatant, drying at room temperature, fixes and is dried with cold methanol afterwards, with capping film, by the microwell plate capping of fixed cell, place 20 ℃ and save backup; The preparation method of the solid phase plate of the solid phase plate of described coated anti-human PR3 antigen immune Lysozyme and coated anti-human MPO antigen immune Lysozyme is: using 0.05M phosphate buffer as coated damping fluid, the concentration of envelope antigen is 0.04-1.0 microgram/ml, coated solution in the hole of microwell plate 4 ℃, 24 hours, use deionized water washed twice, 2% bovine serum albumin(BSA) room temperature sealing is after 1.5 hours, in temperature, be that 20-28 ℃ and humidity are less than or equal under 30% condition dry 12 hours, then pack sealing, be stored in 4 ℃ stand-by;
The employing sodium iodate legal system of described horseradish peroxidase connection antibody is standby;
The solution of described horseradish peroxidase substrate comprises A liquid and B liquid, and wherein A liquid is superoxol, and B liquid is that tetramethyl biphenyl amine aqueous solution or A liquid are tetramethyl benzidine; B liquid is sodium acetate/citrate buffer;
Described stop buffer is 2M sulfuric acid, and cleansing solution is phosphate buffer.
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| CN201310737092.7A CN103713121A (en) | 2013-12-23 | 2013-12-23 | Human vasculitis diagnostic kit and preparation method thereof |
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Address after: 300457 Tianjin international biological medicine Joint Research Institute, No. 220 Dongting Road, Tianjin economic and Technological Development Zone, S606 Applicant after: Tianjin Ruihua Biological Technology Co., Ltd. Address before: 300457 Tianjin international biological medicine Joint Research Institute, Tianjin 220, Dongting Road, Tianjin economic and Technological Development Zone, Binhai New Area, S1404 Applicant before: Tianjin Ruihua Biological Technology Co., Ltd. |
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