CN103698537A - Chemiluminescence immunoassay kit for quantitative detection of soluble intercellular adhesion molecule-1 and detection method - Google Patents
Chemiluminescence immunoassay kit for quantitative detection of soluble intercellular adhesion molecule-1 and detection method Download PDFInfo
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Abstract
The invention discloses a chemiluminescence immunoassay kit for quantitative detection of a soluble intercellular adhesion molecule-1 and a detection method. The kit contains a 96-well plate which is pre-coated with an anti-intercellular adhesion molecule-1 monoclonal antibody and the following bottled reagents: a phosphate wash solution, a blocking solution, a sample dilution solution, an intercellular adhesion molecule-1 standard solution, a horseradish peroxidase-labeled anti-intercellular adhesion molecule-1 polyclonal antibody IgG and a luminescent substrate, namely a luminal luminescent solution and quality control serum. The kit disclosed by the invention has the advantages of accurate and objective determination result of the soluble intercellular adhesion molecule-1 in human serum, simple and convenient method and easiness in popularization and application. The kit can reflect the autoimmune state in a patient with autoimmune thyroid disease can be used as a reference index for diagnosis of the autoimmune thyroid disease, and further has important significance for understanding the autoimmune active degree, predicting the occurrence of Graves disease and the recurrence of Graves hyperthyroidism, and determining reasonable treatment source, drug withdrawal time and other aspects.
Description
Technical field
The invention belongs to chemiluminescent immunoassay, specifically with porous ELISA Plate, do carrier and prepare an insolubilized antibody, for quantitatively detecting human serum soluble intercellular adhesion molecule-1(sICAM-1) chemiluminescence immune assay (CLIA) kit and detection method.
Background technology
AITD (autoimmune thyroid disease, AITD) be the organ specificity autoimmune disease of one group of pathogenesis complexity, comprise Graves disease (Graves ' disease, GD), Hashimoto thyroiditis (Hashimoto ' s thyroiditis, HT) etc.Human serum intercellular adhesion molecule-1 (ICAM-1) is a kind of cell surface strand glycoprotein, is divided into extracellular section, hydrophobic TMD and short intracellular region section on cell membrane.Extracellular section has immunoglobulin-like structural area, 5 extracellulars, wherein first immunoglobulin-like structural area combines with LFA-1 (lymphocyte function-associated antigen-1 LFA-1) aglucon, LFA-1 all has expression on all leucocytes, and the 3rd structure sample district combines with complement receptor 3 (claiming again Mac-1).Its molecular weight is 80-115kD, is positioned at chromosome 19.The function of ICAM-1 is by being combined and realizing with LFA-1 and Mac-1.ICAM-1 is widely distributed, as: vascular endothelial cell, thymic epithelial cells, kinds of tumor cells and mankind's thyrocytes cell etc. are all expressed, and express obviously and increase under interleukin 1 (IL-1), tumor necrosis factor α (TNF-α), interferon gamma (INF-γ) stimulate.(soluble intercellular adhesion molecule-1 sICAM-1) formed into blood by cell surface ICAM-1 comes off in soluble intercellular adhesion molecule-1.The function of ICAM-1 is to participate in antigen recognizing, complement in conjunction with, cell adherence function, and its expression is to cause one of key factor that GD occurs, and its serum levels determining and recurring significant for the generation of judgement GD, drug withdrawal opportunity.
Research in recent years shows, the expression of intercellular adhesion molecule-1 and AITD immunopathogenesis process are closely related.In GD and Hashimoto thyroiditis patient's parathyroid tissue, the ICAM-1 of lymphocyte infiltration, endothelial cell and sICAM-1 abnormal expression are active.In addition, find to increase with the GD patients serum sICAM-1 horizontal abnormality of expophthalmos, this is because of the strong expression sICAM-1 of GD patient retrobulbar tissue, so the level that serum sICAM-1 raises is many and all inflammatory infiltration degree of socket of the eye are closely related.In addition, research is discovery also, and in the therapeutic process of medicine, the improvement of thyroid function and clinical symptoms and the decline of Serum sICAM-1 are also asynchronous, and often the recovery of thyroid function to a great extent will be early than the recovery of sICAM-1.In sum, whether and whether the concentration change of soluble intercellular adhesion molecule-1 may recur etc. for the autoimmune disorder level, the drug withdrawal that judge GD significant.
At present, there is no domestic sICAM-1 chemiluminescence detection technology.The human serum sICAM-1 enzyme-linked immunoassay kit that abroad Jin You U.S. R & D company produces, positive rate is lower, and method is loaded down with trivial details, long flow path, and also price is very expensive, fails at home so far to promote.
summary of the invention
The present invention is the problem existing in order to overcome import reagent box, solve the demand further apply, and provide a kind of highly sensitive, specificity good, chemiluminescence immune assay detection kit and the detection method of easy and simple to handle, quantitative human serum soluble intercellular adhesion molecule-1 that once can measure a large amount of samples.
The present invention is based on immune response and chemiluminescence reaction, can detect the content of sICAM-1 in human serum, its measuring principle is by the coated porous ELISA Plate of anti-ICAM-1 monoclonal antibody, with phosphate rinsing liquid (PBST) sealing containing 1.0%-5.0% bovine serum albumin(BSA), then add respectively test serum sample and titer, wherein sICAM-1 be coated on the monoclonal antibody specific binding on solid phase carrier, binding constituents is not removed through rinsing, adding the anti-ICAM-1 polyclonal antibody IgG(of horseradish peroxidase-labeled is ELIAS secondary antibody again), carry out secondary antigen-antibody reaction, after binding constituents is removed not in rinsing, add luminous substrate luminal luminescent solution, finally on Chemiluminescence Apparatus, measure luminous value, by typical curve, calculate the sICAM-1 concentration in testing sample.
The object of the invention is to be achieved through the following technical solutions.
The chemical luminescence immune analysis reagent box of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1, in this kit, include 96 orifice plates of pre-coated Anti-intracellular Adhesion Molecule-1, and bottled following reagent: phosphate rinsing liquid, confining liquid, sample diluting liquid, intercellular adhesion molecule-1 titer, polyclonal antibody IgG in adhesion molecule-1 between the anti-cell of horseradish peroxidase-labeled, luminous substrate luminal luminescent solution and quality controlled serum.
The chemical luminescence immune analysis reagent box of described quantitative detection human serum soluble intercellular adhesion molecule-1,96 orifice plates of described pre-coated Anti-intracellular Adhesion Molecule-1, it is through the raw material of following ratio of weight and number, to make the carbonate buffer solution coated described monoclonal antibody of pH9.6,500 parts of sodium carbonate 0.78-0.80 parts, sodium bicarbonate 1.45-1.49 part, deionized water.
The chemical luminescence immune analysis reagent box of described quantitative detection human serum soluble intercellular adhesion molecule-1, its phosphate rinsing liquid is by the raw material of following ratio of weight and number, to make the liquid of pH7.4,1000 parts of potassium dihydrogen phosphate 0.3-0.5 part, sodium hydrogen phosphate 3.5-4.0 part, sodium chloride 7.8-8.2 part, potassium chloride 0.1-0.3 part, Tween-20 0.3-0.6 part and deionized waters.
The chemical luminescence immune analysis reagent box of described quantitative detection human serum soluble intercellular adhesion molecule-1, its confining liquid is the phosphate rinsing liquid containing 1.0-5.0% bovine serum albumin(BSA).
The chemical luminescence immune analysis reagent box of described quantitative detection human serum soluble intercellular adhesion molecule-1, its sample diluting liquid is to make pH7.4 liquid by the raw material of following ratio of weight and number, 1000 parts of potassium dihydrogen phosphate 0.3-0.5 parts, sodium hydrogen phosphate 3.5-4.0 part, sodium chloride 7.8-8.2 part, potassium chloride 0.1-0.3 part, Tween-20 0.3-0.6 part, bovine serum albumin(BSA) 10-15 part, deionized water.
The chemical luminescence immune analysis reagent box of described a kind of quantitative detection human serum soluble intercellular adhesion molecule-1, its intercellular adhesion molecule-1 titer is the sterling intercellular adhesion molecule-1 that the U.S. R & D company of purchase produces, with sample diluting liquid dilution, form, dilute concentration is respectively 200ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2ng/ml.
The detection method of the chemiluminescence immune assay of described a kind of quantitative detection human serum soluble intercellular adhesion molecule-1, this kit detects according to the following steps:
1. take out 96 hole ELISA Plate, 10 times of sample diluting liquid dilutions for test serum, every hole adds the test serum 100 μ l after dilution; It is 200ng/ml that each hole of typical curve adds respectively concentration, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, the titer 100 μ l of 2ng/ml, 37 ℃ of incubations 1 hour;
2. get rid of reactant liquor, every hole is washed 3 times with 200-300 μ l phosphate rinsing liquid, pats dry;
3. every hole adds the anti-ICAM-1 polyclonal antibody IgG100 μ l of horseradish peroxidase-labeled, 37 ℃ of incubations 1 hour;
4. get rid of reactant liquor, every hole is washed 6 times with 200-300 μ l phosphate rinsing liquid, pats dry;
5. every hole adds luminous substrate luminal luminescent solution 100 μ l, 37 ℃ of incubations 5 minutes;
6. mix latter 30 minutes and measure luminous value on intrinsic chemical light-emitting appearance;
7. the titer that input is measured respectively in computing machine and the luminous value of testing sample, according to linear standard curve and the equation of Microsoft excel Software on Drawing, can calculate the concentration value of sICAM-1 in each testing sample automatically.
Advantage of the present invention;
1, kit of the present invention is done coated antibody with Anti-intracellular Adhesion Molecule-1, and the human serum sICAM-1 of detection is high to GD patient's positive rate.
2, kit of the present invention has been realized the quantitative measurement of human serum sICAM-1, and measurement result is accurate, objective.
3, kit of the present invention detect sensitive, stable, method is easy, to AITD, particularly whether and whether autoimmune disorder level, the drug withdrawal of GD recur etc. significant.Kit of the present invention is cheap, is easy to apply, and has solved numerous clinicians' active demand.
Accompanying drawing explanation
Fig. 1 detects human serum sICAM-1 canonical plotting;
Fig. 2 is anti-ICAM-1 monoclonal antibody and sICAM-1, VCAM-1 dosage-response curve figure.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be described in detail.
Main agents and raw material:
1. anti-ICAM-1 monoclonal antibody (voluntarily preparation)
2. carbonate buffer solution (voluntarily preparation)
3. phosphate rinsing liquid (PBST) (voluntarily preparation)
4. confining liquid (voluntarily preparation)
5.ICAM-1 standard items (production of U.S. R & D company)
6. sample diluting liquid (voluntarily preparation)
7. the anti-ICAM-1 polyclonal antibody IgG(of horseradish peroxidase (HRP) mark is ELIAS secondary antibody) (preparation voluntarily)
8. luminous substrate luminal luminescent solution (Beijing Ke Yuezhong pattern Bioisystech Co., Ltd, 250ml/ bottle)
9. quality controlled serum (voluntarily preparation)
One, the preparation of anti-ICAM-1 monoclonal antibody
1. by 10 μ g ICAM-1 albumen and adjuvant mixing lumbar injection BALB/C mouse three times, every minor tick 3 weeks, gets mouse angular vein blood and surveys immunizing potency;
2. get and tire mouse tail vein injection booster immunization high once, after three days, get mouse boosting cell, under PEG1650 (polyethylene glycol, polyglycol 1650) effect, carry out Fusion of Cells with myeloma cell.After one week, get Growth of Cells hole supernatant and survey antibody-secreting situation through ELISA, positive secretory pit is expanded and cultivates and carry out subclone.After three subclones, monoclonal porocyte is expanded and cultivated, carry out the evaluation of hybridoma and monoclonal antibody;
3. adopt the method inducing in animal body to prepare in a large number monoclonal antibody, finally with Protein G, purify ascites and obtain the monoclonal antibody of anti-ICAM-1.
Two, the preparation of 96 hole ELISA Plate coated antibodies
1. (0.79 gram of sodium carbonate, 1.47 grams of sodium bicarbonates, 500 ml deionized water, pH9.6) be diluted to 100ng/100 μ l-2 μ g/100 μ l will to resist coated carbonate buffer solution for ICAM-1 monoclonal antibody;
2. every hole adds the antibody liquid 100 μ l after dilution, and 4 ℃ are spent the night coated;
3. get rid of coating buffer, by phosphate rinsing liquid (PBST) (0.3 gram of potassium dihydrogen phosphate, 3.6 grams of sodium hydrogen phosphates, 8.0 grams of sodium chloride, 0.2 gram of potassium chloride, 0.5 milliliter of Tween-20,1000 ml deionized water, pH7.4) rinsing is 3 times, each every hole rinse-added liquid 200-300 μ l, again with the PBST confining liquid sealing containing 1.0-5.0% BSA, every hole adds confining liquid 100 μ l, 25-37 ℃ of incubation 1 hour;
4. get rid of confining liquid, then use rinsing liquid rinsing 3 times, pat dry rear standby.
Three, the foundation of typical curve
The sterling intercellular adhesion molecule-1 that the U.S. R & D company buying produces forms with sample diluting liquid dilution, and the curve each point concentration that settles the standard is respectively 200ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2ng/ml.
Take ICAM-1 standard items concentration as horizontal ordinate (common coordinate), take its corresponding luminance value as ordinate (common coordinate), in linear coordinate axis, do figure Criterion curve.The results are shown in Figure shown in 1.
Four, the preparation of quality controlled serum
Get the above GD patients serum of 30 example, mix, with the repeated multiple times mensuration of chemical luminescence immune analysis reagent box of described quantitative detection soluble intercellular adhesion molecule-1, packing after definite value, freeze drying ,-30 ℃ keep in Dark Place, are quality controlled serum.
Five, the preparation of the anti-ICAM-1 polyclonal antibody IgG of HRP mark
1.HRP 5mg is dissolved in 1ml aqua sterilisa.
2.0.5ml sodium periodate adds in the HRP of 1ml, and at this moment the color of HRP water is by the blue cyan of xanthochromia, and stirring at room 25 minutes, dialyses to the sodium acetate of PH4.4.
3. get IgG 1ml dialysed overnight in the carbonate buffer solution of PH9.5.
4. get the HRP having dialysed and be placed in small beaker, with 0.2M carbonate buffer solution, adjust PH9.5-9.6, add the IgG having dialysed, 4 ℃ are stirred 2 hours.
5. get 0.4ml sodium borohydride and join in the HRP+IgG being stirred, 4 ℃ standing 2 hours, be reduced into stable enzyme labelled antibody.
6. the PBS dialysed overnight to 1L by standing good HRP and IgG.
7. go up G200 gel column, collect first peak and be the anti-ICAM-1 polyclonal antibody IgG that mark is good.
Six, with kit, detect the operation steps of human serum sICAM-1:
1. take out 96 hole ELISA Plate, phosphate sample diluting liquid for test serum (0.3 gram of potassium dihydrogen phosphate, 3.6 grams of sodium hydrogen phosphates, 8.0 grams of sodium chloride, 0.2 gram of potassium chloride, 0.5 milliliter of Tween-20,10 grams of BSA, 1000 ml deionized water, pH7.4) dilution is 10 times, and every hole adds the test serum 100 μ l after dilution; Each hole of typical curve adds respectively each concentration (200ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2ng/ml) titer 100 μ l, 37 ℃ of incubations 1 hour.
2. get rid of reactant liquor, every hole is washed 3 times with 200-300 μ l phosphate rinsing liquid, pats dry.
3. every hole adds the anti-ICAM-1 polyclonal antibody IgG 100 μ l of horseradish peroxidase-labeled, 37 ℃ of incubations 1 hour.
4. get rid of reactant liquor, every hole is washed 6 times with 200-300 μ l phosphate rinsing liquid, pats dry.
5. every hole adds luminous substrate luminal luminescent solution 100 μ l, 37 ℃ of incubations 5 minutes.
6. mix latter 30 minutes and measure luminous value on intrinsic chemical light-emitting appearance.
7. the titer that input is measured respectively in computing machine and the luminous value of testing sample, according to linear standard curve and the equation of Microsoft excel Software on Drawing, can calculate the concentration value of sICAM-1 in each testing sample automatically.
Seven, kit performance index
1. sensitivity
Replication 20 hole zero standard product, calculating kit detection sensitivity is 13.8ng/ml.
2. specificity
With ICAM-1 standard items and VCAM-1 (with the ICAM-1 structure proximate) standard items that decuple ICAM-1 standard lines each point concentration, make respectively line, the dosage of ICAM-1-response curve shows: with ICAM-1 concentration, increase, luminous value raises and the relation that is in line gradually, and ICAM-1 monoclonal antibody and ICAM-1 specific binding are described; The dosage of VCAM-1-response curve shows: with VCAM-1 concentration, increase, luminous value is without significant change and be background level, illustrates that ICAM-1 monoclonal antibody and VCAM-1 are without specific binding.The results are shown in Figure shown in 2.
3. precision
(1) variation within batch: once measure respectively each 6 samples of same positive quality control serum and negative quality controlled serum in experiment, calculate variation within batch (CV%) and be respectively 3.36% and 3.25%.
(2) batch variation: same positive quality control serum and negative quality controlled serum, METHOD FOR CONTINUOUS DETERMINATION 6 times, between calculating batch, CV% value is respectively 7.87% and 8.62%.
4. accuracy
The sample that 3 parts of sICAM-1 substrate concentrations are 10.28ng/ml adds respectively after 100ng/ml, 50ng/ml, 5ng/ml ICAM-1, by this kit measured concentration, be 90.3ng/ml, 49.9ng/ml, 4.84ng/ml, calculate recovery rate is respectively 90.3%, 99.8%, 96.7%, average recovery rate 95.6%.
Seven, this kit clinical detection result
This kit detection normal person sICAM-1 concentration (
± s) be 158 ± 59ng/ml, range of normal value (
± 2s) be (40 ng/ml-276ng/ml), normal person surpasses 3 examples that have of 276 ng/ml, and positive rate is 2.3% (3/132); GD patient sICAM-1 concentration (
± s) be 459 ± 142ng/ml, surpass 87 examples that have of 276 ng/ml, positive rate is 91.6% (87/95); HT patient sICAM-1 concentration (
± s) be 382 ± 110ng/ml, surpass 61 examples that have of 276 ng/ml, positive rate is 84.7% (61/72).GD patient and HT patient sICAM-1 concentration and positive rate are all significantly higher than normal person.The results are shown in Table shown in 1.
Table 1 is human serum sICAM-1 CLIA clinical detection result quantitatively
Note:
■represent and normal person's comparison
p<0.01.
Claims (7)
1. a chemical luminescence immune analysis reagent box that quantitatively detects human serum soluble intercellular adhesion molecule-1, it is characterized in that: 96 orifice plates that include pre-coated Anti-intracellular Adhesion Molecule-1 in this kit, and bottled following reagent: phosphate rinsing liquid, confining liquid, sample diluting liquid, intercellular adhesion molecule-1 titer, between the anti-cell of horseradish peroxidase-labeled, polyclonal antibody IgG in adhesion molecule-1 is second antibody, luminous substrate luminal luminescent solution and quality controlled serum.
2. the chemical luminescence immune analysis reagent box of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1 as claimed in claim 1, it is characterized in that: 96 orifice plates of described pre-coated Anti-intracellular Adhesion Molecule-1, it is through the raw material of following ratio of weight and number, to make the carbonate buffer solution coated described monoclonal antibody of pH9.6,500 parts of sodium carbonate 0.78-0.80 parts, sodium bicarbonate 1.45-1.49 part, deionized water.
3. the chemical luminescence immune analysis reagent box of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1 as claimed in claim 1, it is characterized in that: described phosphate rinsing liquid is by the raw material of following ratio of weight and number, to make the liquid of pH7.4,1000 parts of potassium dihydrogen phosphate 0.3-0.5 part, sodium hydrogen phosphate 3.5-4.0 part, sodium chloride 7.8-8.2 part, potassium chloride 0.1-0.3 part, Tween-20 0.3-0.6 part and deionized waters are mixed with.
4. the chemical luminescence immune analysis reagent box of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1 as described in claim 1 or 3, is characterized in that: confining liquid is the phosphate rinsing liquid containing 1.0-5.0% bovine serum albumin(BSA).
5. the chemical luminescence immune analysis reagent box of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1 as claimed in claim 1, it is characterized in that: sample diluting liquid is to make pH7.4 liquid by the raw material of following ratio of weight and number 1000 parts of potassium dihydrogen phosphate 0.3-0.5 parts, sodium hydrogen phosphate 3.5-4.0 part, sodium chloride 7.8-8.2 part, potassium chloride 0.1-0.3 part, Tween-20 0.3-0.6 part, bovine serum albumin(BSA) 10-15 part, deionized water.
6. the chemical luminescence immune analysis reagent box of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1 as described in claim 1 or 5, it is characterized in that: intercellular adhesion molecule-1 titer is that sterling intercellular adhesion molecule-1 forms with above-mentioned sample diluting liquid dilution, dilute concentration is respectively 200ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2ng/ml.
7. the detection method of the chemiluminescence immune assay of a kind of quantitative detection human serum soluble intercellular adhesion molecule-1 as claimed in claim 1, is characterized in that: the method is carried out according to the following steps,
1. take out 96 orifice plates of pre-coated Anti-intracellular Adhesion Molecule-1,10 times of sample diluting liquid dilutions for test serum, every hole adds the test serum 100 μ l after dilution; It is 200ng/ml that each typical curve hole adds respectively concentration, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, the titer 100 μ l of 2ng/ml, 37 ℃ of incubations 1 hour;
2. get rid of reactant liquor, every hole is washed 3 times with 200-300 μ l phosphate rinsing liquid, pats dry;
3. every hole adds polyclonal antibody IgG 100 μ l in adhesion molecule-1 between the anti-cell of horseradish peroxidase-labeled, 37 ℃ of incubations 1 hour;
4. get rid of reactant liquor, every hole is washed 6 times with 200-300 μ l phosphate rinsing liquid, pats dry;
5. every hole adds luminous substrate luminal luminescent solution 100 μ l, 37 ℃ of incubations 5 minutes;
6. mix latter 30 minutes and measure luminous value on intrinsic chemical light-emitting appearance;
7. the titer that input is measured respectively in computing machine and the luminous value of testing sample, according to linear standard curve and the equation of Microsoft excel Software on Drawing, can calculate the concentration value of soluble intercellular adhesion molecule-1 in each testing sample automatically.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106568968A (en) * | 2016-10-12 | 2017-04-19 | 南京健安医疗科技有限公司 | Human soluble intercellular adhesion molecule-1 quantitative chemiluminescent enzyme-linked immunosorbent assay kit and preparation method thereof |
| CN109696550A (en) * | 2017-10-20 | 2019-04-30 | 成都蓝瑙生物技术有限公司 | Luminous ELISA humoral sample stabilisation aqueous solution for headstroke |
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| CN109696550A (en) * | 2017-10-20 | 2019-04-30 | 成都蓝瑙生物技术有限公司 | Luminous ELISA humoral sample stabilisation aqueous solution for headstroke |
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