CN105004861A - Human chromogranin A immunoassay method and determination kit - Google Patents
Human chromogranin A immunoassay method and determination kit Download PDFInfo
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- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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Abstract
The invention discloses a human chromogranin A immunoassay method. The human chromogranin A immunoassay method comprises the steps that a sample to be subjected to CGA content determination reacts with a group of antibodies, wherein at least one antibody is specifically bonded with the epitope of the 1<th>-113<th> and/or 124<th>-143<th> and/or 322<th>-364<th> and/or 373<th>-439<th> amino acids in a CGA amino acid sequence of human; at least one of the antibodies is a capture antibody, and at least one antibody is a tracer antibody. A determination kit provided by the invention mainly comprises a CGA antibody coated microporous plate, a CGA tracer antibody, an ELISA concentrated cleaning solution, an ELISA HRP matrix, an ELISA stop solution, a detection buffer solution, a CGA calibration product and a CGA quality control product. The human chromogranin A immunoassay method and the determination kit have the advantages of being high in specificity, good in fragment existence stability, high in detection accuracy and the like.
Description
Technical field
The present invention relates to immunoassay medicine technology field, specifically relate to a kind of immunoassay of people's CgA and measure kit.
Background technology
People's Chromogranin A (Chromogranin A, CgA) is a kind of Acidy soluble protein be made up of 439 amino acid, is the Major Members of chromogranin family.It is extensively present in nerve secretes in cell, and in blood plasma, the rising of CgA concentration can point out the tumour of neuroendocrine origin.Neuroendocrine tumor originates from neuroendocrine cell, typical neuroendocrine tumor is carcinoid tumor, pheochromocytoma, neuroblastoma, small-cell carcinoma of the lung, hyperparathyroidism adenoma, hypophysoma and insulinoma, and comprises MEN1 and MEN2 syndrome.This also comprises different NET syndrome, i.e. gastrinoma, insulinoma, glucagonoma of pancreas, somatostatinoma, product pancreatic polypeptide-producing tumor and non-functional NET etc.
Secrete the rising that all can occur CgA level in the diagnosis of tumour in the nerve of nearly all type, therefore, secrete in the diagnosis of tumour in nerve, CgA is valuable diagnosis marker.If using 32.7U/L as critical value, CgA is respectively 82.1% and 96.2% to the sensitivity of pancreatic neoplasm and specificity, is respectively 88.5% and 96.2% to the sensitivity of pheochromocytoma and specificity.Therefore, the quantitative detection of CgA is of great significance for the clinical diagnosis tool of cancer of pancreas and chromaffin cell cancer.
At present, the method reported the mensuration of CgA mainly contains enzyme-linked immunosorbent assay (ELISA), immunoradiometric assay (IRMA) and chemiluminometry etc.Relate to a kind of based on competitive assay in the patent of US 4758522, the CGA of usage flag and measure the method for CGA content in sample for the antibody of CGA.Measure to be combined with antibody or the unconjugated amount marking CGA as sample in the tolerance of CGA.CGA sample can derive from the tissue in incretory or adrenal gland source.The immunoassay of a kind of CGA is relate in the patent of EP1078266B1, the method comprises the sample of CgA content to be determined and one group of antibody response, and wherein the epitope specificity of the 145-234 position of at least one (monoclonal or polyclone) antibody in CGA amino acid sequence is combined.More specifically, the wherein 145-197 position of at least one (monoclonal or polyclone) antibody in CGA amino acid sequence and/or be combined with the epitope specificity of 219-234 position.Any one of these two kinds of antibody above-mentioned can be combined with the antibody of epi-position in the 250-301 bit sequence of specific binding CGA in CGA determination method.The patent No. is the immunoassay disclosing a kind of Chromogranin A in the patent of invention of CN102869681 A, the assay method of this CGA polypeptide, mainly comprise sample and the one group of monoclonal antibody reactive by preparing to measure the existence of CGA polypeptide or the amount of CGA polypeptide, the responding property of epi-position wherein in the CGA polypeptide that represents of the 236-251 amino acids sequence of at least one monoclonal antibody CGA amino acid sequence; And the responding property of epi-position wherein in the CGA polypeptide that represents of the 264-279 amino acids sequence of other monoclonal antibodies of at least one and CGA amino acid sequence.
Summary of the invention
For the deficiencies in the prior art, the object of this invention is to provide the method for immunity of people's CgA that a species specificity is high, fragment existence and stability is good, accuracy in detection is high and measure kit.
The technical scheme that technical solution problem of the present invention adopts is: a kind of immunoassay of people's CgA, and described immunoassay comprises the sample of people's CgA content to be determined and one group of antibody response; Wherein the epitope specificity of 1st ~ 113 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 124th ~ 143 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 322nd ~ 364 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 373rd ~ 439 amino acids of at least one antibody in people's CgA amino acid sequence is combined; In the antibody that the epitope specificity of above-mentioned in people's CgA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 or 373rd ~ 439 amino acids is combined, wherein at least one is for catching antibody; In the antibody that the epitope specificity of above-mentioned in people's CgA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 or 373rd ~ 439 amino acids is combined, wherein at least one antibody is tracer antibody.
Described antibody is monoclonal antibody or polyclonal antibody.
Described seizure antibody is biotin labeled antibody or is directly fixed on Stationary liquid, and described Stationary liquid is microwell plate or magnetic bead.
Described assay method principle is double antibody sandwich method, immunoturbidimetry or Immunoturbidimetry.
The labeling method that described tracer antibody adopts is enzyme labeling method or chemiluminescent labeling.
A mensuration kit for people's CgA, contains above-mentioned tracer antibody and catches antibody in described mensuration kit.
Described mensuration kit mainly comprises following component: the microwell plate of CgA antibody bag quilt, CgA tracer antibody, ELISA concentrated cleaning solution, ELISA HRP matrix, ELISA stop buffer, detection damping fluid, CgA calibration object and CgA quality-control product; Described CgA calibration object and CgA quality-control product are the CgA antigen of freeze-dried powder state.
Further, described CgA tracer antibody is the CgA antibody of horseradish peroxidase-labeled; The principal ingredient of described ELISA concentrated cleaning solution is phosphate; The principal ingredient of described ELISA HRP matrix is 3,3 ', 5,5 '-tetramethyl benzidine; The principal ingredient of described ELISA stop buffer is the sulfuric acid of 0.5M; The principal ingredient of described detection damping fluid is phosphate and bovine serum albumin(BSA).
The assay method of said determination kit is as follows:
(1) calibration object of 15 μ L, quality-control product and sample to be tested is added in the designation hole of the microwell plate of CgA antibody bag quilt;
(2) in each hole, add 200 μ L respectively detect damping fluid;
(3) mix gently, with shrouding film sealing microwell plate, and use masking foil lucifuge, incubated at room 60 minutes under 400rpm vibration;
(4) remove masking foil and shrouding film, washing on trigger, with 350 μ L cleansing solutions, each hole is all cleaned 5 times, then blot; Remaining if any cleansing solution, pat dry gently;
(5) in each hole, add 100 μ L CgA tracer antibodies;
(6) mix gently, with shrouding film sealing microwell plate, and use masking foil lucifuge, incubated at room 60 minutes under 400rpm vibration;
(7) remove masking foil and shrouding film, washing on trigger, with 350 μ L cleansing solutions, each hole is all cleaned 5 times, then blot; Remaining if any cleansing solution, pat dry gently;
(8) in each hole, add the ELISA HRP matrix of 100 μ L;
(9) mix gently, use masking foil lucifuge, room temperature stationary incubation 20 minutes;
(10) remove masking foil, in each hole, add the ELISA stop buffer of 100 μ L, mix gently;
(11) two wavelength light density values (OD value) at 450nm/630nm place are read; According to the scope of optical density value on typical curve of sample, directly can read sample and measure concentration.
Described people's CgA measures kit and is used for neuroendocrine tumor, comprise carcinoid tumor, pheochromocytoma, neuroblastoma, small-cell carcinoma of the lung, hyperparathyroidism adenoma, hypophysoma and insulinoma, and the auxiliary diagnosis of MEN1 and MEN2 syndrome etc., efficiency evaluation and dynamic monitoring.
Said determination kit adopts " double-antibody sandwich " technology.Calibration object, quality-control product and sample to be tested are joined in the microwell plate of CgA antibody bag quilt, after hatching, cleaning microwell plate.Add CgA tracer antibody, after hatching, form " double-antibody sandwich " structure, cleaning microwell plate.Add matrix solution, after hatching, measure optical density value (OD value) by microplate reader.In microwell plate, the enzymatic activity of immunocomplex is directly proportional to the CgA concentration in sample to be tested.According to the optical density value of CgA concentration different in calibration object, according to " point-to-point " or " 4-parametric method " drawing standard curve, the CgA concentration in testing sample can directly read from typical curve.
The invention has the beneficial effects as follows: compared with prior art, immunoassay and the mensuration kit of people's CgA provided by the invention have the advantages such as specificity is high, fragment existence and stability is good, accuracy in detection is high; In CgA immunoassay provided by the invention, be combined by the epitope specificity of antibody in CGA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 and 373rd ~ 439, realize the quantitative measurement of CGA in sample; Mensuration kit provided by the invention can reach some performance index following: (1) lowest detectable limit: sample is the bovine serum albumin(BSA) of 5%, lowest detectable limit≤5ng/ml; (2) range of linearity: 10 ~ 670ng/ml, r >=0.990; (3) precision: CV≤12% in batch; (4) accuracy: TIANZHU XINGNAO Capsul 85% ~ 115%.
Accompanying drawing explanation
Fig. 1 is the canonical plotting of CgA in the present invention.
Fig. 2 is the fundamental diagram (double-antibody sandwich) measuring kit in the present invention.
Fig. 3 is the bound site point diagram of antibody and CgA amino acid sequence in the present invention.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
An immunoassay for people's CgA, described immunoassay comprises the sample of people's CgA content to be determined and one group of antibody response; Wherein the epitope specificity of 1st ~ 113 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 124th ~ 143 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 322nd ~ 364 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 373rd ~ 439 amino acids of at least one antibody in people's CgA amino acid sequence is combined; In the antibody that the epitope specificity of above-mentioned in people's CgA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 or 373rd ~ 439 amino acids is combined, wherein at least one is for catching antibody; In the antibody that the epitope specificity of above-mentioned in people's CgA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 or 373rd ~ 439 amino acids is combined, wherein at least one antibody is tracer antibody.
The antibody adopted in the present embodiment is monoclonal antibody.
In the present embodiment, CgA catches antibody and is fixed on Stationary liquid, and this Stationary liquid is microwell plate.
In the present embodiment, CgA tracer antibody adopts horseradish peroxidase-labeled.
In the present embodiment, the mensuration kit of employing mainly comprises following component: the microwell plate of CgA antibody bag quilt, CgA tracer antibody, ELISA concentrated cleaning solution, ELISA HRP matrix, ELISA stop buffer, detection damping fluid, CgA calibration object and CgA quality-control product; Described CgA calibration object and CgA quality-control product are the CgA antigen of freeze-dried powder state.
Wherein, CgA tracer antibody is the CgA antibody of horseradish peroxidase-labeled; The principal ingredient of ELISA concentrated cleaning solution is phosphate; The principal ingredient of ELISA HRP matrix is 3,3 ', 5,5 '-tetramethyl benzidine; The principal ingredient of ELISA stop buffer is the sulfuric acid of 0.5M; The principal ingredient detecting damping fluid is phosphate and bovine serum albumin(BSA).
In the present embodiment, the assay method measuring kit is as follows:
1, the preparation of reagent:
(1), before using, all reagent is placed in rewarming under room temperature;
(2) described ELISA concentrated cleaning solution before use by 30 × extension rate distilled water or deionized water dilute;
(3) calibration object, quality-control product often pipe add the distilled water of 0.5mL or deionized water dissolving, leave standstill 10 minutes, mixing of turning upside down, guarantee that all solids dissolve complete.
2, checking procedure:
(1) calibration object of 15 μ L, quality-control product and sample to be tested is added in the designation hole of the microwell plate of CgA antibody bag quilt;
(2) in each hole, add 200 μ L respectively detect damping fluid;
(3) mix gently, with shrouding film sealing microwell plate, and use masking foil lucifuge, incubated at room 60 minutes under 400rpm vibration;
(4) remove masking foil and shrouding film, washing on trigger, with 350 μ L cleansing solutions, each hole is all cleaned 5 times, then blot; Remaining if any cleansing solution, pat dry gently;
(5) in each hole, add 100 μ L CgA tracer antibodies;
(6) mix gently, with shrouding film sealing microwell plate, and use masking foil lucifuge, incubated at room 60 minutes under 400rpm vibration;
(7) remove masking foil and shrouding film, washing on trigger, with 350 μ L cleansing solutions, each hole is all cleaned 5 times, then blot; Remaining if any cleansing solution, pat dry gently;
(8) in each hole, add the ELISA HRP matrix of 100 μ L;
(9) mix gently, use masking foil lucifuge, room temperature stationary incubation 20 minutes;
(10) remove masking foil, in each hole, add the ELISA stop buffer of 100 μ L, mix gently;
(11) two wavelength light density values (OD value) at 450nm/630nm place are read; According to the scope of optical density value on typical curve of sample, directly can read sample and measure concentration.
Embodiment 2
People's CgA measures the typical curve of kit
Provide one group of 5 calibration object in the present embodiment, its value is respectively calibration object 1 for 0ng/mL, and calibration object 2 is 17.6ng/mL, and calibration object 3 is 64ng/mL, and calibration object 4 is 186ng/mL, and calibration object 5 is 674ng/mL.
Calibration object in kit is CgA antigen.By the OD of 5 calibration objects to the mapping of ng/mL value, build calibration curve.The result table 1 that calibration object is measured and Fig. 1.
Table 1: the result that calibration object is measured
Embodiment 3
People's CgA measures the lowest detectable limit of kit
Detect as sample with 5% bovine serum albumin(BSA), replication 20 times, calculate the mean value (M) and standard deviation (SD) that measure optical density value (OD value), draw the OD value corresponding to M+2SD, carry out 2 regression fits according to the concentration-OD value result between zero-dose calibration object and adjacent calibration object and draw linear function, OD value corresponding to M+2SD is brought in above-mentioned equation, obtains corresponding concentration value, be lowest detectable limit.
Embodiment 4
People's CgA measures the withinrun precision of kit
Detect as sample with 2 quality-control products, replication 10 times, calculate the mean value (M) and standard deviation (SD) that measure concentration, calculate the coefficient of variation (CV) by formula (1).
In formula: SD---the standard deviation of measured value;
The mean value of M---measured value.
Embodiment 5
People's CgA measures the accuracy of kit
People's CgA sample (A) of known high concentration is joined in people's CgA sample (B), add sample A volume should be no more than 10% of cumulative volume (A+B), replication 3 times, calculate the mean value measuring concentration, calculate recovery R according to formula (2).
In formula: R---the recovery;
V---the volume of sample A liquid;
V0---the volume of sample B liquid;
C---sample B liquid adds the detectable concentration after sample A liquid;
C0---the detectable concentration of sample B liquid;
Cs---the detectable concentration of sample A liquid;
Prepared by sample: sample A, B recommend by 1: 10 volume mixture.
Note: A1, A2 are people's CgA sample of high concentration, A1, A2 volume ratio joined in sample B is 1: 10.
Above embodiment is only for illustration of the present invention; and be not limitation of the present invention; the those of ordinary skill of relevant technical field; without departing from the spirit and scope of the present invention; can also make a variety of changes and modification; therefore all equivalent technical schemes also belong to category of the present invention, and scope of patent protection of the present invention should be defined by the claims.
Claims (10)
1. an immunoassay for people's CgA, is characterized in that: described immunoassay comprises the sample of people's CgA content to be determined and one group of antibody response; Wherein the epitope specificity of 1st ~ 113 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 124th ~ 143 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 322nd ~ 364 amino acids of at least one antibody in people's CgA amino acid sequence is combined; Wherein the epitope specificity of 373rd ~ 439 amino acids of at least one antibody in people's CgA amino acid sequence is combined; In the antibody that the epitope specificity of above-mentioned in people's CgA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 or 373rd ~ 439 amino acids is combined, wherein at least one is for catching antibody; In the antibody that the epitope specificity of above-mentioned in people's CgA amino acid sequence 1st ~ 113,124th ~ 143,322nd ~ 364 or 373rd ~ 439 amino acids is combined, wherein at least one antibody is tracer antibody.
2. the immunoassay of a kind of people's CgA according to claim 1, is characterized in that: described antibody is monoclonal antibody or polyclonal antibody.
3. the immunoassay of a kind of people's CgA according to claim 1, is characterized in that: described Method And Principle is double antibody sandwich method, immunoturbidimetry or Immunoturbidimetry.
4. the immunoassay of a kind of people's CgA according to claim 1, it is characterized in that: described seizure antibody is biotin labeled antibody or is directly fixed on Stationary liquid, described Stationary liquid is microwell plate or magnetic bead.
5. the immunoassay of a kind of people's CgA according to claim 1, is characterized in that: the labeling method that described tracer antibody adopts is enzyme labeling method or chemiluminescent labeling.
6. a mensuration kit for people's CgA, is characterized in that: contain the tracer antibody in the claims 1 to 5 described in any one in described mensuration kit and catch antibody.
7. the mensuration kit of a kind of people's CgA according to claim 6, is characterized in that: described mensuration kit mainly comprises following component: the microwell plate of CgA antibody bag quilt, CgA tracer antibody, ELISA concentrated cleaning solution, ELISA HRP matrix, ELISA stop buffer, detection damping fluid, CgA calibration object and CgA quality-control product; Described CgA calibration object and CgA quality-control product are the CgA antigen of freeze-dried powder state.
8. the mensuration kit of a kind of people's CgA according to claim 7, is characterized in that: described CgA tracer antibody is the CgA antibody of horseradish peroxidase-labeled; The principal ingredient of described ELISA concentrated cleaning solution is phosphate; The principal ingredient of described ELISA HRP matrix is 3,3 ', 5,5 '-tetramethyl benzidine; The principal ingredient of described ELISA stop buffer is the sulfuric acid of 0.5M; The principal ingredient of described detection damping fluid is phosphate and bovine serum albumin(BSA).
9. the mensuration kit of a kind of people's CgA according to claim 7, is characterized in that: the assay method of described mensuration kit is as follows:
(1) calibration object of 15 μ L, quality-control product and sample to be tested is added in the designation hole of the microwell plate of CgA antibody bag quilt;
(2) in each hole, add 200 μ L respectively detect damping fluid;
(3) mix gently, with shrouding film sealing microwell plate, and use masking foil lucifuge, incubated at room 60 minutes under 400rpm vibration;
(4) remove masking foil and shrouding film, washing on trigger, with 350 μ L cleansing solutions, each hole is all cleaned 5 times, then blot; Remaining if any cleansing solution, pat dry gently;
(5) in each hole, add 100 μ L CgA tracer antibodies;
(6) mix gently, with shrouding film sealing microwell plate, and use masking foil lucifuge, incubated at room 60 minutes under 400rpm vibration;
(7) remove masking foil and shrouding film, washing on trigger, with 350 μ L cleansing solutions, each hole is all cleaned 5 times, then blot; Remaining if any cleansing solution, pat dry gently;
(8) in each hole, add the ELISA HRP matrix of 100 μ L;
(9) mix gently, use masking foil lucifuge, room temperature stationary incubation 20 minutes;
(10) remove masking foil, in each hole, add the ELISA stop buffer of 100 μ L, mix gently;
(11) two wavelength light density values (OD value) at 450nm/630nm place are read; According to the scope of optical density value on typical curve of sample, directly can read sample and measure concentration.
10. the mensuration kit of a kind of people's CgA according to claim 6, it is characterized in that: described people's CgA measures kit and is used for neuroendocrine tumor, comprise carcinoid tumor, pheochromocytoma, neuroblastoma, small-cell carcinoma of the lung, hyperparathyroidism adenoma, hypophysoma and insulinoma, and the auxiliary diagnosis of MEN1 and MEN2 syndrome etc., efficiency evaluation and dynamic monitoring.
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| CN201510232661.1A CN105004861A (en) | 2015-05-06 | 2015-05-06 | Human chromogranin A immunoassay method and determination kit |
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| CN201510232661.1A CN105004861A (en) | 2015-05-06 | 2015-05-06 | Human chromogranin A immunoassay method and determination kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834447A (en) * | 2017-01-09 | 2017-06-13 | 浙江大学 | A kind of primer sets and detection method for detecting neuroblastoma |
| CN107090439A (en) * | 2017-06-02 | 2017-08-25 | 福州迈新生物技术开发有限公司 | The hybridoma cell strain of one plant of anti-Chromogranin A monoclonal antibody of secretion and its application |
| CN109507432A (en) * | 2018-11-14 | 2019-03-22 | 嘉兴行健生物科技有限公司 | A kind of CGA detection reagent and detection reference interval and detection method |
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2015
- 2015-05-06 CN CN201510232661.1A patent/CN105004861A/en active Pending
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834447A (en) * | 2017-01-09 | 2017-06-13 | 浙江大学 | A kind of primer sets and detection method for detecting neuroblastoma |
| CN107090439A (en) * | 2017-06-02 | 2017-08-25 | 福州迈新生物技术开发有限公司 | The hybridoma cell strain of one plant of anti-Chromogranin A monoclonal antibody of secretion and its application |
| CN107090439B (en) * | 2017-06-02 | 2020-05-05 | 福州迈新生物技术开发有限公司 | Hybridoma cell strain secreting anti-chromaffin A monoclonal antibody and application thereof |
| CN109507432A (en) * | 2018-11-14 | 2019-03-22 | 嘉兴行健生物科技有限公司 | A kind of CGA detection reagent and detection reference interval and detection method |
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Application publication date: 20151028 |