CN1595161A - Magnetic separation enzyme-linked immune detecting method for thyroid gland peroxidase antibody - Google Patents
Magnetic separation enzyme-linked immune detecting method for thyroid gland peroxidase antibody Download PDFInfo
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Abstract
This invention provides a human hypothyroid peroxid enzyme antibody magnetic separation enzyme immune measurement method, whose steps are the following: to add sample to react with immune; to wash and to join two anti-enzyme indicate reagent and to wash again; to add monophosphatase fenolftaleina substrate solvent; to terminate color forming and to read absorbing value. The advantages in this invention are the following: it uses reformed human hypothyroid peroxid enzyme antigen fold immune magnetism micro ball as fixed phase and uses alkalescence phosphate enzyme as mark enzyme. Through the quantitative measurement of peroxid enzyme antigen of the hypothyroid patient and normal serum and other clinic symptom, it can judge whether the patient has got the self-immune hypothyroid illness.
Description
The present invention relates to the immune detection analysis field, be specifically related to a kind of human thyroid peroxidase antibody magnetic separation enzyme-linked immunosorbent assay method.
Background technology
As everybody knows, contain thyroid microsomal antigen usually among the thyroid gland autoimmune disease patients serum, this antigen is from finding over more than 20 year that its biology fundamental property is never studied clear.Along with the development of Protocols in Molecular Biology, manyly studies confirm that the principal ingredient of thyroid microsomal antigen is thyroid peroxidase (NAOKATA YOKOYAMA etc., Journal of clinical Endocrinology andMetabolism).Thyroid peroxidase is a kind of film integral protein, is positioned on the top film of follicular cells, and this enzyme is a prothetic group with the protoheme, and molecular weight is about 101KD.The traditional detection of thyroid gland autoimmune disease is usually based on thyroid microsomal antibody, based on radio-immunity and enzyme linked immunological adsorption method.Along with the molecule biological property of thyroid peroxidase and with the further investigation of thyroid microsomal antigen relation, the immunological detection method that has occurred thyroid peroxidase antibody successively, improved the accuracy that detects greatly, but still based on qualitative or half-quantitative detection.
Immune magnetic microsphere is the surface diameter that carries the mobilizing function group at the macromolecule bag of 0.1-10 mu m range by magnetic-particle.Magnetic-particle has superparamagnetism, and tool magnetic field responsiveness under magnetic field is used for the solid phase of immune detection with immune magnetic microsphere, and it is long-pending to improve reacted surface greatly, and the easier Separation of Solid and Liquid of carrying out is greatly improved the sensitivity of detection.
The about 140KD of alkaline phosphatase enzyme molecular weight from the extraction of calf intestinal mucosa, with the immunologic detection method of alkaline phosphatase as the spike enzyme, its highly sensitive horseradish peroxidase commonly used in Enzyme Linked Immunoadsorbent Assay detects, also lower (the Wu Jianmin of blank value (background), clinical chemistry robotization immunoassay, Science Press).The substrate that alkaline phosphatase is commonly used has para-nitro-pheneye phosphate (PNP), and 4-methyl umbelliferone phosphate (4-MUP) etc. also can use single phosphoric acid phenolphthalein (PPM) as substrate, develops the color after adding alkaline stop buffer.
Summary of the invention
The purpose of this invention is to provide a kind of higher stability that has, the separation enzyme-linked immunoassay analysis of human thyroid peroxidase antibody magnetic of sensitivity and accuracy.Utilize this method, use recombined human thyroid peroxidase antigen coupling magnetic micro-beads to be solid phase, it is two antienzymes mark reagent that the anti-immunoglobulin G while antibody of goat connects alkaline phosphatase, single phosphoric acid phenolphthalein solution is a substrate solution, adopt immunology principle, just can carry out the quantitative measurement of human thyroid superoxide enzyme antibody in the serum, its operation steps is as follows:
1, application of sample and immune response: in flat based tubes, add 10-30 μ l serum sample and (carry out 1 with sample dilution buffer liquid in advance: the 50-100 dilution), standard items, or quality controling serum, add 60-120 μ l 8-14mg/ml solid phase magnetic separation agent (surperficial coupling recombined human thyroid peroxidase antigen), behind the mixing, 37 ℃ incubation 15-60 minute.
2, wash: flat based tubes is placed on the magnetic separator separated 2 minutes, pour out supernatant with circular motion reversing separation vessel then, the test tube of reversing is placed on the filter paper, bounce separation vessel to remove wall built-up liquid.Add cleaning fluid 100-500 μ l in every pipe, fully behind the mixing, flat based tubes is placed on the magnetic separator separated 2 minutes, pour out supernatant with circular motion reversing separation vessel then, the test tube that reverses is placed on the filter paper, bounce separation vessel to remove wall built-up liquid.Repeat twice.
3, add two antienzymes mark reagent: in each test tube, add the anti-immunoglobulin G while antibody of the goat working fluid 30-120 μ l that alkaline phosphatase (EC3.1.3.1) connects, behind the mixing, 37 ℃ of incubations 15 minutes.
4, washing: with (2) step.
5, add single phosphoric acid phenolphthalein substrate solution: every pipe adds the single phosphoric acid phenolphthalein of 90-120 μ l substrate solution, behind the mixing, 37 ℃ incubation 15-60 minute.
6, color development stopping: every pipe adds 300-500 μ l alkalescence stop buffer, and is placed on the magnetic separator and separates more than 5 minutes.
7, read light absorption value: on the spectrophotometer or use the magnetic enzyme to exempt to read light absorption value on the analyzer I (Beijing Bei'aikang Biotechnology Co., Ltd.'s production), wavelength is 492nm/550nm.
In above-mentioned steps, being formulated as of standard items: 2g sodium chloride, 2.42 trihydroxy aminomethanes, the 5g bovine serum albumin(BSA), 300 usefulness 1000ml purified water dissolving in the general labor of 0.5ml Kelin is transferred pH to 7.6 with hydrochloric acid, 0.2 μ m filtrator filters, in 4 ℃ of preservations, as the damping fluid of standard items.With reference to WHO 1
STIRP 66/387 standard) concentration preparation standard items (the standard items scope is 0-1000IU/ml) in 4 ℃ of preservations, are used for the production standard curve.
In above-mentioned steps, being prepared as of solid phase magnetic separation agent: the magnetic microsphere of diameter range 0.1-10 μ m is activated with glutaraldehyde, and the room temperature mixing was used 0.01mol/l PBS pH7.4 buffer solution for cleaning three times after 4 hours, and suspend with this solution, concentration is 50-100mg/ml; Then, add recombined human thyroid peroxidase antigen 20-100 μ g in every milliliter of suspension, in 37 ℃ of mixing incubations 3-8 hour; Sealed 30-90 minute in 37 ℃ with isopyknic 0.01mol/l PBS 5%BSA pH7.4 damping fluid; At last, use 0.5%BSA0.02mol/l Tris-HCl pH8.0 buffer solution for cleaning three times, and be mixed with the working fluid of 8-14mg/ml with this solution.
In above-mentioned steps, two antienzymes are marked being prepared as of reagent:
(1) the anti-immunoglobulin G while antibody of goat is dissolved in the 0.01mol/l PBS pH7.4 solution concentration 5-10mg/ml; Add 20mmol/l activator N-succinimide 3-(2-pyridine dimercapto) propionic acid [SPDP] dimethyl sulfoxide solution 20-30 μ l, room temperature was placed 30-60 minute; Remove deactivator by Sephadex G25 pillar, collect protein peak; Add 500 μ l 24mg/ml dithiothreitol (DTT) (DTT) 0.01mol/l PBS pH7.4 solution in the antibody-solutions of every milliliter of activation, behind the mixing, room temperature was placed 30 minutes; Remove free dithiothreitol (DTT) by Sephadex G25 pillar, collect protein peak.
(2) alkaline phosphatase is dissolved in the 2mmol/l EDTA 20mmol/l Tris-HCl pH8.0 solution concentration 5mg/ml; Adding 0.10mg/ml Traunt reagent (sulfydryl activating reagent) room temperature placed 1 hour; Remove free activator by Sephadex G25 pillar, collect protein peak.
(3) (1) and (2) solution is mixed with alkaline phosphatase molecule mol ratio according to antibody at 1: 1, room temperature was placed 4 hours, used Superdex200 gel chromatography column separating purification then, collected first and second peaks, remove the free antibodies and the alkaline phosphatase that do not connect, connector is stored in 4 ℃.
(4) with 1mmol/1MgCl2 20mmol/l Tris-HCl pH8.0 solution connector is mixed with 0.5-2 μ g/ml working fluid, uses 0.2 μ m filtrator to filter.
In above-mentioned steps, the preparation of cleaning fluid is formed:
Sodium chloride (NaCl) 2g, sodium dihydrogen phosphate (NaH2PO4.12H2O) 2.9g, dipotassium hydrogen phosphate (K2HPO4) 0.2g, potassium chloride (KCl) 0.2g, triton x-100 0.5ml, polysorbas20 0.2ml, Blanc Ni Daokesi (Bronidox-L) 5g, be made into 1000ml with purified water, filter with 0.2 μ m filtrator, in room temperature preservation.
In above-mentioned steps, the preparation of substrate solution is formed:
Monoethanolamine 100g, sodium chloride (NaCl) 2g, single phosphoric acid phenolphthalein 4g is made into 1000ml with purified water, transfers pH to 8.4 with 37% hydrochloric acid, filters with 0.2 μ m filtrator, in 4 ℃ of preservations.
In above-mentioned steps, the preparation of stop buffer is formed:
Sodium chloride (NaCl) 1g, sodium carbonate (Na2CO3) 15g, EDTA-Na2 10g is made into 1000ml with purified water, filters with 0.2 μ m filtrator, in room temperature preservation.
The invention has the advantages that: utilized the antigen coated immunity magnetic micropearls of recombined human thyroid peroxidase as solid phase, and adopted the alkaline phosphatase enzyme that serves as a mark, improved the sensitivity and the accuracy that detect greatly.Thyroid disease patient and normal human serum are carried out the quantitative measurement of thyroid peroxidase antibody, just can judge in conjunction with other clinical symptoms (as thyroglobulin antibody) whether the examiner suffers from autoimmune thyroid disease.
Embodiment
Material and instrument
1, standard items 0,10,40,100,250, and 500IU/ml is (with reference to WHO 1
STIRP 66/387 standard) each 1ml.
2, magnetic separation agent (10mg/ml) 20ml.
3, two antienzymes mark reagent (1 μ g/ml) 20ml.
4, cleansing solution 100ml;
5, substrate solution 100ml.
6, stop bath 200ml.
7, sample dilution 100ml.
8, thyroid gland autoimmune disease patients serum is 100 parts, 100 parts of normal human serums.
9, the separation enzyme-linked immunoassays instrument of magnetic I type machine (Beijing Bei'aikang Biotechnology Co., Ltd.'s production).
10, water bath (being used for 37 ℃ of temperature bathes).
Reagent preparation:
The preparation of above-mentioned solution 1-6 is carried out with reference to the process for preparation described in the inventive method.
Being formulated as follows of dilution: use calf serum to add 0.2% Sodium azide, with 0.45 μ m filtrator filter operation step:
At first, serum sample is carried out 1: 100 dilution with the sample dilution.Then, carry out according to following operation steps.
1, application of sample and immune response: add 15 μ l serum samples in flat based tubes, standard items, or quality controling serum add 60 μ l 10mg/ml magnetic separation agents (surperficial coupling recombined human thyroid peroxidase antigen), behind the mixing, and 37 ℃ of incubations 30 minutes.
2, washing: flat based tubes is placed on the magnetic separator separated 2 minutes, then with one greatly and slowly circular motion reversing separation vessel pour out supernatant, the test tube that reverses is placed on the filter paper, bounce separation vessel to remove wall built-up liquid.Add cleaning fluid 150 μ l in every pipe, fully behind the mixing, flat based tubes is placed on the magnetic separator separated 2 minutes, then with one greatly and slowly circular motion reverse separation vessel and pour out supernatant, the test tube of reversing is placed on the filter paper, bounces separation vessel to remove wall built-up liquid.Repeat twice.
3, add two antienzymes mark reagent: in each test tube, add the anti-immunoglobulin G while antibody of the goat working fluid 30 μ l that alkaline phosphatase (EC3.1.3.1) connects, behind the mixing, 37 ℃ of incubations 15 minutes.
4, washing: with (2) step.
5, add single phosphoric acid phenolphthalein substrate solution: every pipe adds the single phosphoric acid phenolphthalein of 100 μ l substrate solution, behind the mixing, and 37 ℃ of incubations 15 minutes.
6, color development stopping: every pipe adds 300 μ l alkalescence stop buffer, and is placed on the magnetic separator and separated 10 minutes.
7, read light absorption value: use the magnetic enzyme to exempt to read light absorption value on the analyzer I (Beijing Bei'aikang Biotechnology Co., Ltd.'s production), wavelength is 492nm/550nm, calculates the concentration of test sample book according to the typical curve of match.
Testing result:
| Sample | Quantity | Measured value (IU/ml) | |
| Thyroid gland autoimmunity patient | n＝100 | ?436±308 | Wherein, 96%＞40IU/ml |
| The normal person | N＝100 | ?4.07±2.52 |
Zero standard is carried out 20 repeated tests on schedule, get the mean value that zero standard measures on schedule and add 2 times standard deviation, be its sensitivity.The sensitivity of this method is＜5IU/ml.
Patients serum to 7 variable concentrations uses two batches of reagent to carry out 20 repeated tests respectively, and patients serum's concentration range is 5-1000IU/ml, calculates its variation within batch.Variation within batch (CV) average out to 4.5% as a result.
Patients serum to 7 variable concentrations uses two batches of reagent to carry out 20 repeated tests respectively with different operating personnel, and patients serum's concentration range is 5-1000IU/ml, calculates its batch variation.Batch variation (CV) average out to 8.9% as a result.
5 parts of patient's sample are carried out doubling dilution, and extension rate was tested according to standard method from 1: 2 to 1: 32, calculated its dilution recovery.The result is as follows:
| Concentration of specimens (IU/ml) | Alluvial (IU/ml) | Overall recovery (%) |
| ?110.5 | ?124.8 | ?113 |
| ?250.4 | ?275.0 | ?110 |
| ??425.3 | ??446.5 | ??105 |
| ??684.0 | ??636.2 | ??93 |
| ??901.2 | ??856.1 | ??95 |
| Average recovery rate is: 103.2 ± 8.9 | ||
5 parts of patient's sample are added recovery test, and this added the high concentration standard items according to 50: 1 every increment, tested according to standard method, calculated it and added the recovery.The result is as follows:
| Concentration of specimens (IU/ml) | Interpolation value (IU/ml) | Test value (IU/ml) | Overall recovery (%) |
| ??45.2 | ??345.2 | ??400.2 | ??103 |
| ??85.9 | ??345.2 | ??429.8 | ??100 |
| ??110.5 | ??345.2 | ??484.1 | ??108 |
| ??250.4 | ??345.2 | ??570.6 | ??93 |
| ??425.3 | ??345.2 | ??766.7 | ??99 |
| Average recovery rate is: 100.6 ± 5.5 | |||
Claims (7)
1, a kind of human thyroid peroxidase antibody magnetic separation enzyme-linked immunosorbent assay method is characterized in that: detecting step is:
A, application of sample and immune response: in flat based tubes, add 10-30 μ l serum sample standard items or quality controling serum, add 60-120 μ l 8-14mg/ml solid phase magnetic separation agent, behind the mixing, 37 ℃ incubation 15-60 minute;
B, washing: flat based tubes is placed on the magnetic separator separated 2 minutes, pour out supernatant with circular motion reversing separation vessel then, the test tube of reversing is placed on the filter paper, bounce separation vessel to remove wall built-up liquid, add cleaning fluid 100-500 μ l in every pipe, fully behind the mixing, flat based tubes is placed on the magnetic separator separated 2 minutes, pour out supernatant with circular motion reversing separation vessel then, the test tube of reversing is placed on the filter paper, bounce separation vessel to remove wall built-up liquid; Repeat twice;
C, add two antienzymes mark reagent: in each test tube, add the anti-immunoglobulin G while antibody of the goat working fluid 30-120 μ l that alkaline phosphatase EC3.1.3.1 connects, behind the mixing, 37 ℃ of incubations 15 minutes;
D, washing: with the b step;
E, add single phosphoric acid phenolphthalein substrate solution: every pipe adds the single phosphoric acid phenolphthalein of 90-120 μ l substrate solution, behind the mixing, 37 ℃ incubation 15-60 minute;
F, color development stopping: every pipe adds 300-500 μ l alkalescence stop buffer, and is placed on the magnetic separator and separates more than 5 minutes;
G, read light absorption value: on the spectrophotometer or use the magnetic enzyme to exempt to read light absorption value on the analyzer I, wavelength is 492nm/550nm.
2, according to the described detection method of claim 1, it is characterized in that: being formulated as of described standard items: 2g sodium chloride, 2.42 trihydroxy aminomethane, the 5g bovine serum albumin(BSA), 0.5ml 300 usefulness 1000ml purified water dissolving in general labor Kelin is transferred pH to 7.6 with hydrochloric acid, 0.2 μ m filtrator filters, in 4 ℃ of preservations, as the damping fluid of standard items; With reference to WHO l
STIRP 66/387 normal concentration preparation standard items, its standard items scope is 0-1000IU/ml, in 4 ℃ of preservations, is used for the production standard curve.
3, according to claim 1 or 2 described detection methods, it is characterized in that: being prepared as of described solid phase magnetic separation agent: the magnetic microsphere of diameter range 0.1-10 μ m is activated with glutaraldehyde, behind the room temperature mixing 4 hours, with 0.01mol/l PBS pH7.4 buffer solution for cleaning three times, and suspend with this solution, concentration is 50-100mg/ml; Then, add recombined human thyroid peroxidase antigen 20-100 μ g in every milliliter of suspension, in 37 ℃ of mixing incubations 3-8 hour; Sealed 30-90 minute in 37 ℃ with isopyknic 0.01mol/l PBS 5%BSA pH7.4 damping fluid; At last, use 0.5%BSA 0.02mol/l Tris-HCl pH8.0 buffer solution for cleaning three times, and be mixed with the working fluid of 8-14mg/ml with this solution.
4, according to the described detection method of claim 1, it is characterized in that: above-mentioned two antienzymes are marked being prepared as of reagent:
A, the anti-immunoglobulin G while antibody of goat is dissolved in the 0.01mol/l PBS pH7.4 solution concentration 5-10mg/ml; Add 20mmol/l activator N-succinimide 3-(2-pyridine dimercapto) propionic acid [SPDP] dimethyl sulfoxide solution 20-30 μ l, room temperature was placed 30-60 minute; Remove deactivator by Sephadex G25 pillar, collect protein peak; Add 500 μ l 24mg/ml dithiothreitol (DTT) (DTT) 0.01mol/l PBS pH7.4 solution in the antibody-solutions of every milliliter of activation, behind the mixing, room temperature was placed 30 minutes; Remove free dithiothreitol (DTT) by Sephadex G25 pillar, collect protein peak;
B, alkaline phosphatase is dissolved in the 2mmol/l EDTA 20 mmol/l Tris-HCl pH8.0 solution concentration 5mg/ml; Adding 0.10mg/ml Traunt reagent (sulfydryl activating reagent) room temperature placed 1 hour; Remove free activator by Sephadex G25 pillar, collect protein peak;
C, a and b solution are mixed with alkaline phosphatase molecule mol ratio according to antibody at 1: 1, room temperature was placed 4 hours, used Superdex200 gel chromatography column separating purification then, collected first and second peaks, remove the free antibodies and the alkaline phosphatase that do not connect, connector is stored in 4 ℃;
D, connector is mixed with 0.5-2 μ g/ml working fluid, uses 0.2 μ m filtrator to filter with 1mmol/lMgCl2 20mmol/l Tris-HCl pH8.0 solution.
5, according to the described detection method of claim 1, it is characterized in that: the preparation of institute's cleaning fluid is formed: sodium chloride 2g, sodium dihydrogen phosphate 2.9g, dipotassium hydrogen phosphate 0.2g, potassium chloride 0.2g, triton x-100 0.5ml, polysorbas20 0.2ml, Blanc Ni Daokesi 5g is made into 1000ml with purified water, filter with 0.2 μ m filtrator, in room temperature preservation.
6, according to the described detection method of claim 1, it is characterized in that: the preparation of the substrate solution that goes on foot is formed: monoethanolamine 100g, sodium chloride 2g, single phosphoric acid phenolphthalein 4g is made into 1000ml with purified water, transfers pH to 8.4 with 37% hydrochloric acid, filter with 0.2 μ m filtrator, in 4 ℃ of preservations.
7, according to the described detection method of claim 1, it is characterized in that: the preparation of described stop buffer is formed:
Sodium chloride 1g, sodium carbonate 15g, EDTA-Na2 10g is made into 1000ml with purified water, filters with 0.2 μ m filtrator, in room temperature preservation.
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