CN101545911A - Chemoluminescence immunoassay quantitative measuring kit of cytokeratin 18 and preparation method thereof - Google Patents
Chemoluminescence immunoassay quantitative measuring kit of cytokeratin 18 and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chemoluminescence immunoassay quantitative measuring kit of cytokeratin 18 and a preparation method thereof in the field of immunoassay and biomedicine. The kit comprises a monoclonal antibody enzyme marker of the cytokeratin 18, a monoclonal antibody coating carrier of the cytokeratin 18 and a cytokeratin 18 calibration sample. The method for preparing the kit comprises the steps of preparing the calibration sample with pure cytokeratin 18 product and coating the carrier with the monoclonal antibody of the cytokeratin 18. The kit can fast detect a great batch of samples simultaneously, provides the main reagents in the working solution mode, has the advantages of convenient, fast, sensitive and accurate detection, and the like, and has great application value in clinic diagnosis.
Description
Technical Field
The invention relates to a quantitative determination kit for chemiluminescence immunoassay of cytokeratin 18(CyK-18) and a preparation method thereof, belonging to the fields of immunoassay and biomedicine.
Background
Among the numerous tumor markers is a relatively special class of biological macromolecules: a cytoskeletal protein. This cellular component was once thought to be a cellular scaffold, stable in nature, and without any biological function. However, a recent decade of research has led us to reconsider this important cellular component: this large family supports the maintenance of normal morphology of cells and tissues, but is also involved in differentiation and development of cellular tissues, in interactions (including signaling) between cells and cells, between cells and the extracellular matrix, and in response to external stimuli, protecting cells from mechanical, pharmaceutical, etc. damage. During the transformation process of tumor cells, the differentiation state, cell morphology and other aspects are changed, and the changes are also shown in the expression pattern, distribution position and other aspects of cytoskeletal components. Therefore, the component is widely applied to tumor diagnosis, and some components can even be used for treating tumors. Clinically, cytokeratins (cKs) in this cellular component are most widely used: the immunohistochemistry is used for judging the tissue type, the source and the differentiation degree of the tumor cells; the serology is used for early diagnosis of tumors such as lung cancer, colorectal cancer, gastric cancer, breast cancer and the like, good and malignant differentiation, clinical staging, monitoring of treatment effect, early discovery of micrometastasis and follow-up of patients.
Cytokeratins are the most complex group of proteins in the cytoskeletal component, the intermediate filament family, which comprises approximately 20 members and has a molecular weight between 40 and 6OKDa and can be divided into two types according to their isoelectric points: type I (basic keratin, CK 1-CK 8, PI: 6.1-7.8), type 11 (acidic keratin, CK 9-CK 20, PI: 4.9-5.7). CYFRA21-1, TPAcyk and TPS are clinically better immunoassay methods aiming at cytokeratin components at present, and respectively aim at cytokeratin 19 fragments; fine chest keratin 8, 18, 19: an epitope of cytokeratin 18M 3. The CYFRA21-1 is applied to lung cancer, especially squamous cell carcinoma, has a total detection rate of 61 percent on the lung cancer, has average detection rates of 52 percent, 79 percent and 54 percent on small cell lung cancer, squamous cell carcinoma and adenocarcinoma respectively, can be used as a basis for differential diagnosis of the small cell lung cancer and non-small cell lung cancer when being combined with NSE, can be used for disease monitoring after radiotherapy and chemotherapy of the lung cancer, early detection of relapse and survival time prediction, and is the most effective tumor marker of the non-small cell lung cancer at present.
TPS is a soluble fragment of a tissue antigen recognized by a cytokeratin 18 antibody, and malignant tumors and metastatic tumors of epithelial origin have high expression, and particularly TPS is highly expressed and greatly enters blood during the period of active tumor cell proliferation, so that the biological behavior of the tumors can be better reflected. Zheng et al reported that the positive rate of TPS in serum of lung cancer patients was 55.1%, while the positive rate in bronchoalveolar lavage fluid was 100%, indicating that TPS, a marker for lung cancer, is more sensitive than CEA.
Research and application of a labeling immunoassay technology have been rapidly developed in the last decade, and the labeling immunoassay technology has been widely applied to various fields of biomedical basic theory research and clinical disease diagnosis. Methods for detecting serological indicators mainly include radioisotope immunoassays, enzyme-linked immunosorbent assays, and chemiluminescent immunoassays. These methods can be used as both preliminary screening tests and confirmation tests, and are currently widely used.
The cytokeratin 18 determination kit (chemiluminescence method) developed by the inventor adopts a double-antibody sandwich one-step reaction mode, and a cytokeratin 18 monoclonal antibody is adopted to prepare a solid phase, so that the specific monoclonal antibody has high purity, no infectivity and small nonspecific binding reaction, and can be produced in large batch without a special laboratory.
The kit can be applied to an open chemiluminescence measuring instrument, is low in use cost and is easier to popularize and apply.
Disclosure of Invention
The invention aims to provide a quantitative determination kit for chemiluminescence immunoassay of cytokeratin 18 (CyK-18).
Another object of the present invention is to provide a method for preparing the above kit.
The kit according to the invention comprises: 1) an enzyme-labeled monoclonal antibody to cytokeratin 18; 2) a carrier coated with a monoclonal antibody to cytokeratin 18; 3) a cytokeratin 18 calibrator; and 4) a chemiluminescent substrate acted on by the enzyme.
Further, the method for preparing the above kit according to the present invention comprises the steps of:
1) monoclonal antibodies to cytokeratin 18 labeled with an enzyme; 2) coating the vector with a monoclonal antibody to cytokeratin 18; 3) preparing a cytokeratin 18 calibrator from a pure cytokeratin 18 product; 4) subpackaging the cytokeratin 18 calibrator, the enzyme-labeled monoclonal antibody of cytokeratin 18 and the chemiluminescent substrate acted by the enzyme; and 5) assembling to obtain a finished product.
In the above-mentioned kit and the preparation method thereof according to the present invention, the carrier may be a solid phase carrier, preferably a microplate, plastic beads, plastic tubes or magnetic particles. The enzyme may be horseradish peroxidase or alkaline phosphatase. The chemiluminescent substrate may be luminol or isoluminol, a 1, 2-dioxetane derivative including (adamantane) -1, 2-dioxethane, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxethane (AMPPD), CSPD or CDP-Star.
In the method according to the present invention, wherein the step 2) of coating the carrier comprises the following processes:
mixing 1000mL of coating solution containing 7.30g of trisodium citrate and 4.45g of citric acid, coating solution with the pH value of 4.5-4.8 and CyK-18 monoclonal antibody, loading the mixture on a solid phase carrier, and washing with physiological saline; blocking was performed with phosphate buffer. The confining liquid comprises 0.2gNaH based on 1000mL of the confining liquid2PO4·2H2O，2.9gNaH2PO4·12H2O, 10g BSA and 1mL Proclin300, and the pH value is 7.0-7.5.
The kit can specifically comprise CyK-18 calibrator, antibody coated plate, enzyme marker and chemiluminescence substrate solution, 20-time concentrated washing solution and the like. The CyK-18 calibrator is standard grade, the purity is not lower than 90%, an antibody coating plate is a microporous plate strip with 48 or 96 holes, an enzyme label CyK-18 monoclonal antibody is coupled alkaline phosphatase or horseradish peroxidase, chemiluminescence substrate liquid is AMPPD or luminol, and concentrated washing liquid is Tris-HCl.
The quantitative determination kit for chemoluminescence immunoassay of cytokeratin 18 can accurately and quantitatively detect the content of CyK-18 in a patient, and has important significance for monitoring the disease condition of tumors of epithelial sources such as digestive tract, mammary gland, genitourinary tract, respiratory tract and the like, evaluating the curative effect, and finding early relapse and judging prognosis according to the content of CyK-18. The method has the advantages of high specificity, high sensitivity, high accuracy, simplicity, convenience, quickness and the like. All indexes of the CyK-18 determination kit (chemiluminescence method) reach enzyme-linked immunoassay. In addition, the detection system is open-type operation, is simple, convenient and quick, does not need an expensive full-automatic chemiluminescence measuring instrument, is particularly suitable for popularization and use in vast middle and small hospitals, and provides a very valuable detection means for clinical diagnosis and scientific research work. According to the kit, the enzyme-labeled CyK-18 monoclonal antibody, CyK-18 monoclonal antibody coated on a carrier and CyK-18 antigen of a detected sample form a double-antibody sandwich structure, so that the reaction mode of a double-antibody sandwich one-step method adopted by the invention not only effectively utilizes the technical principle of chemiluminescence, but also ensures the detection sensitivity.
The kit of the invention uses an enzyme-catalyzed luminescent substrate, and a light signal generated by detecting the luminescent substrate replaces a chromogenic substrate in enzyme-linked immunosorbent assay, so that the kit has the same specificity as the enzyme-linked immunosorbent assay, has greatly improved sensitivity which is improved by about 10 times compared with the sensitivity of the prior enzyme-linked immunosorbent assay, and can provide more specific, rapid and reliable basis for monitoring the disease condition of tumors of epithelial sources such as digestive tract, mammary gland, genitourinary tract, respiratory tract and the like, evaluating the curative effect, and finding early relapse and judging prognosis.
In the course of the research of the present invention, the inventors of the present invention first performed screening experiments and quality identification on the raw materials used, including the activities of the labeled antibody and the coated antibody, the adsorption performance and variation size of the carrier (such as opaque white microplate), the activity of the enzyme, the luminescence intensity and the luminescence duration of the chemiluminescent substrate, etc. Then, the coating method is researched, different coating buffer solutions and protection solutions are used for carrying out experiments, the most suitable coating buffer solution and protection solution are selected, and the optimal concentration condition is found through experiments of different coating concentrations of antibodies. Different methods can be used for labeling the ALP, a simple labeling method with high yield, low cost and reliable quality is finally found through repeated exploration and comparison experiments, and different enzyme diluents are subjected to long-term investigation experiments to prepare diluents capable of keeping the activity of the enzyme label stable for a long time.
The inventor of the invention also carries out experimental research on the reaction mode and the reaction conditions of the kit, finally determines the reaction mode of the double-antibody sandwich one-step method, carries out experiments on the influence of different reaction time on the experimental result, and determines the most suitable reaction time.
Drawings
Fig. 1 is a calibration graph of the kit, in which a standard curve is plotted with the concentration of the calibrator as the abscissa and the RLU value as the ordinate, wherein the linear equation is log (y) ═ 3.7578+1.3117log (x), and r ═ 0.9972.
Detailed Description
EXAMPLE 1 Cyk-18 preparation of alkaline phosphatase-labeled monoclonal antibody
CyK-18 monoclonal antibody is coupled with alkaline phosphatase by glutaraldehyde method, dialyzed against PBS, added with glycerol of the same volume, and stored at-20 deg.C or below.
Example 2 Cyk-18 preparation of Horseradish peroxidase-labeled monoclonal antibody
Cyk-18 monoclonal antibody was labeled by sodium periodate oxidation. The labeling process was as follows: 5mg of HRP was weighed and dissolved in 1ml of double distilled water. 0.2ml of newly prepared 0.1M NaIO 4 solution was added to the supernatant, and the mixture was stirred at room temperature for 20 minutes in the dark. The above solution was filled into a dialysis bag, dialyzed against 1mM sodium acetate buffer pH4.4 at 4 ℃ overnight. Mu.l of 0.2M carbonate buffer pH9.5 was added to raise the pH of the above-described hydroformylation HRP to 9.0 to 9.5, and then 10mg of the antibody was immediately added to 1ml of 0.01M carbonate buffer, followed by gentle stirring at room temperature for 2 hours in the dark. Adding 0.1ml of newly prepared 4mg/ml NaBH 4 solution, uniformly mixing, and standing at 4 ℃ for 2 hours. The above solution was put into a dialysis bag, dialyzed against 0.15M PBS pH7.4C, and left overnight at 4 ℃. An equal volume of saturated ammonium sulfate was added dropwise with stirring and left at 4 ℃ for 1 hour. Centrifuge at 3000rpm for half an hour, and discard the supernatant. The precipitate was dissolved in a small amount of 0.15M PBS pH 7.4. Putting the solution into a dialysis bag, dialyzing 0.15M PB buffer saline with pH7.4, removing ammonium ions, centrifuging at 10,000rpm for 30 minutes to remove precipitates, collecting supernatant as enzyme conjugate, adding equal amount of high-quality glycerol, packaging, and storing at-20 deg.C.
EXAMPLE 3 preparation of CyK-18 quantitative determination kit of the invention
Preparation of alkaline phosphatase-labeled antibody
Preparing and weighing 12.12g of Tris, 5g of BSA and 1mL of Proclin300 by using the enzyme-labeled monoclonal antibody diluent, weighing the reagents, putting the reagents into a clean container, adding double-distilled water to 1L, dissolving and uniformly mixing.
Diluting the enzyme-labeled antibody by using an enzyme-labeled monoclonal antibody diluent in different proportions, and selecting the enzyme-labeled antibody with the working concentration of more than 1: 4000.
preparation of CyK-18 calibrator
Prepared by CyK-18 pure products, and subpackaged with 6 bottles of 0, 25, 50, 100, 200 and 400U/L.
Preparation of solid phase coated plate
(1) Coating and weighing 7.30g of trisodium citrate and 4.45g of citric acid in a 1L clean container, adding 1L of double distilled water, dissolving and uniformly mixing, adding an appropriate amount of CyK-18 monoclonal antibody, uniformly mixing, then adding into each hole of a microporous plate, wherein the pH value is 4.8, the volume of each hole is 130 mu L, and the temperature is 4 ℃ for 24 hours.
(2) Washing: washed three times with physiological saline.
(3) Closed weighing 0.2g of NaH2PO4·2H2O, 2.9g of NaH2PO4·12H2O, 10g BSA and 1mL Proclin300, the reagents are weighed and put into a clean container, double distilled water is added to 1L, the mixture is dissolved and mixed evenly, and the pH value is measured to be 7.0.
Add 300. mu.L of blocking solution to each well, and then let stand at room temperature for 3 hours. The confining liquid is thrown off and patted dry on absorbent paper. And dehumidifying and drying for 24 hours at room temperature. The bag was immediately sealed. And (5) after labeling, preserving at 2-8 ℃.
Four, chemiluminescence substrate liquid
24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane and 1mL Proclin300 were weighed, placed in a clean container, double distilled water was added to 1L, dissolved and mixed well.
Five-and 20-times washing liquid
Weighing 24g Tris, 160g NaCl, 4g KCl and 15ml HCl in a clean container, adding double distilled water to 1L, dissolving and mixing uniformly, adjusting pH to 7.4
Sixthly, semi-finished products and finished product composition
And subpackaging the products obtained in the steps to obtain semi-finished products. Three parts are extracted and qualified through specificity, precision, sensitivity and stability detection to be assembled into the cytokeratin 18 quantitative determination kit (chemiluminescence method). The kit can be delivered after being assembled and qualified by sampling inspection.
EXAMPLE 4 preparation of CyK-18 quantitative determination kit of the invention
Except that horseradish peroxidase labeled CyK-18 monoclonal antibody is used as an enzyme labeled antibody, luminol is used as a luminescent substrate, the chemiluminescent substrate comprises a solution A and a solution B, wherein,
based on 1000mL of the chemiluminescent substrate A solution, comprising 1.7716g of luminol, 0.051g of 4-hydroxybiphenyl, 0.012g of 4-iodophenylboronic acid, 11.4g of boric acid and 4.9g of borax, wherein the pH value is 8.0-10.0;
based on 1000mL of the chemiluminescent substrate B solution, the chemiluminescent substrate B solution comprises 0.329g of carbamide peroxide, 1mL of Tween20 and 51.58g of Na2HPO4·12H2O、8.74g NaH2PO4·2H2O, the pH value is 7.0-7.6. Solution A is Tris-HCl buffer solution with pH of 8.5 and 0.1M prepared by adding Tris and concentrated HCl into double distilled water, and comprises 4.0mg/mL Luminol and 0.3mg/mL p-iodophenol.
Solution B is prepared by adding trisodium citrate and citric acid into double distilled water to obtain 0.1M citric acid buffer solution with pH of 4.6, which contains 200mg/mL hydrogen peroxide solution. The quantitative determination kit CyK-18 was prepared in the same manner as in example 1 except that the contents of the kit were changed.
EXAMPLES 5 to 6 preparation of CyK-18 quantitative determination kit of the present invention
CyK-18 quantitative determination kits were prepared in the same manner as in examples 1 and 4, except that plastic beads and plastic tubes were used as carriers, respectively, and the pH of the blocking solution was 7.5.
EXAMPLE 7 preparation of CyK-18 quantitative determination kit of the invention
CyK-18 quantitative assay kit was prepared in the same manner as in examples 1 and 4, except that magnetic particles were used as carriers and CyK-18 coated monoclonal antibodies were coupled to the magnetic particles using glutaraldehyde.
EXAMPLE 8 method of Using the kit of the present invention
The specific procedure for the CyK-18 quantitative determination kit prepared in example 1 above is as follows:
1) the kit was removed from the 4 ℃ freezer and allowed to equilibrate for 15 minutes at room temperature.
2) The coating batten is taken out and inserted into the plate frame.
Table 1 Cyk-18 sample application measurement procedure table units: mu.L
And (3) drawing a standard curve by taking the concentration of the calibrator as an abscissa and the RLU value as an ordinate, and checking out the concentration of CyK-18 of the serum on the standard curve by using the RLU value of each serum to be detected.
Example 9 methodological identification of the kits of the invention
The kit prepared in example 1 was assayed according to manufacturing and assay protocols conventional in the art,
(1) sensitivity test of kit
The concentration value obtained by adding two times of standard deviation to the curve equation of the average value of 10-well repeated measurement by using the S0 calibrator is the sensitivity of the kit, and the sensitivity is 10.6U/L.
(2) Kit specificity test
The cross reaction rate of the compound and the analogue is less than 0.01 percent.
(3) Precision test of kit
Inter-batch variation
The low, medium and high quality control serum samples are respectively subjected to 10-hole parallel experiments, and the average value (x) and the standard deviation(s) of the measured values are calculated. The coefficient of variation was calculated according to the formula CV ═ s/x × 100%, and the intra-batch coefficient of variation CVs were 6.3%, 4.9%, and 4.1%, respectively.
Inter-batch variation
5 serum samples with different concentrations are selected to perform 3 times of repeated measurement on each serum, and the inter-batch variation coefficient (CV%) is calculated, and the inter-batch variation CV is 7.21-9.5%.
(4) Accuracy test of kit
Diluting the high-concentration calibrator raw material with normal human serum into four different concentration values, performing a 5-well parallel experiment on each concentration, and respectively calculating the recovery rate to be within the range of 91-108%.
(5) Stability test of kit
The storage temperature of the kit is 2-80 ℃, all indexes of the kit after 15 months of determination meet requirements, the influence on the kit in the transportation and use processes is considered, an accelerated experiment of 370 ℃ for 7 days is carried out, and the experiment result shows that all indexes of the kit completely meet the requirements.
The sensitivity, specificity, precision, accuracy and stability of the quantitative determination kit for the chemiluminescence immunoassay of the cytokeratin 18 are completely qualified.
Claims (7)
1. A quantitative cytokeratin 18(CyK-18) assay kit, characterized in that said kit comprises an enzyme-labeled CyK-18 monoclonal antibody; a vector coated with a monoclonal antibody of CyK-18; CyK-18 calibrant and wash solution; and the chemiluminescent substrate acted by the enzyme is a 1, 2-dioxyethane derivative.
2. The kit of claim 1, wherein the enzyme is alkaline phosphatase or horseradish peroxidase.
3. The kit of claim 1, wherein the carrier is a microplate, plastic beads, plastic tubing, or magnetic particles.
4. The kit of claim 1, wherein the chemiluminescent substrate is a 1, 2-dioxeine derivative, luminol or isoluminol.
5. A method for preparing the kit of claim 1, comprising the steps of:
1) an enzyme-labeled CyK-18 monoclonal antibody; 2) coating the vector with a monoclonal antibody of CyK-18; 3) preparing CyK-18 calibration products from CyK-18 pure products; 4) subpackaging the CyK-18 calibrator, the enzyme-labeled CyK-18 monoclonal antibody and a chemiluminescent substrate acted by the enzyme, namely 1, 2-dioxane derivative, luminol or isoluminol and washing liquid; and 5) assembling to obtain a finished product.
6. The method of claim 5, wherein the step 2) of coating the carrier comprises the process of: mixing a citric acid coating solution with the pH value of 4.5-4.8 and 0.046M with the monoclonal antibody CyK-18, loading the mixture on a solid phase carrier, and washing the carrier with normal saline; sealing with phosphate with pH value of 7.0-7.5.
7. The kit of claim 5, wherein the chemiluminescent substrate is formulated in 24g Tris, 160g NaCl, 4g KCl, 15mL HCl, 200mL 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxyethane, 1mL Proclin300, 1000mL double distilled water.
Or,
the chemiluminescent substrate comprises a solution A and a solution B, wherein,
based on 1000mL of the chemiluminescent substrate A solution, comprising 1.7716g of luminol, 0.051g of 4-hydroxybiphenyl, 0.012g of 4-iodophenylboronic acid, 11.4g of boric acid and 4.9g of borax, wherein the pH value is 8.0-10.0;
based on 1000mL of the chemiluminescence substrate B solution, the chemiluminescence substrate B solution comprises 0.329g of carbamide peroxide, 1mL of Tween20, 51.58g of Na2HPO 4.12H 2O and 8.74g of NaH2PO 4.2H 2O, and the pH value of the chemiluminescence substrate B solution is 7.0-7.6.
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