CN113009151A - Glycosylated CD59 detection kit based on chemiluminescence method and application thereof - Google Patents

Glycosylated CD59 detection kit based on chemiluminescence method and application thereof Download PDF

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CN113009151A
CN113009151A CN202110196369.4A CN202110196369A CN113009151A CN 113009151 A CN113009151 A CN 113009151A CN 202110196369 A CN202110196369 A CN 202110196369A CN 113009151 A CN113009151 A CN 113009151A
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马万山
陈振
欧兰香
李文靖
高丽鹤
苏真真
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Shandong Big Health Precision Medical Industry Technology Research Institute
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Abstract

The invention discloses a glycosylation CD59 detection kit based on a chemiluminescence method, which comprises a luminescent plate coated with an anti-gCD 59 antibody, a gCD59 standard substance gradient solution, a sample diluent, a peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, a 20 multiplied concentrated washing liquid, a luminescent liquid sealing film, a sealing bag and an instruction book, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, the 20 multiplied concentrated washing liquid, the luminescent liquid sealing film and the sealing bag. The invention also discloses application of the kit in detecting gCD 59-containing biological samples. Experiments prove that the kit disclosed by the invention is high in stability, strong in selectivity, high in detection speed, low in cost, easy to operate, and capable of overcoming the defects of low sensitivity, long operation time and the like in gCD59 detection in the prior art, the operation time is shortened from 400 minutes to 40 minutes, the quantification limit is improved from 18.75pg/mL to 5pg/mL, and the kit has a very good clinical application prospect.

Description

Glycosylated CD59 detection kit based on chemiluminescence method and application thereof
Technical Field
The invention relates to detection of glycosylated CD59(gCD59), in particular to a glycosylated CD59 detection kit based on a chemiluminescence method and application thereof, and belongs to the technical field of clinical examination.
Background
Diabetes is a group of metabolic diseases characterized by hyperglycemia. The chronic hyperglycemia results in chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves. In 70% of diabetic patients, proliferative lesions of small blood vessels and capillaries appear in whole body, and the pathogenesis of the proliferative lesions is related to long-term blood sugar rise, increase of glycated protein end products, expression of intercellular adhesion molecules, increase of certain cytokines and thickening of blood vessel basement membrane caused by intravascular subcutaneous tissue and polysaccharide substance deposition.
Glycosylated CD59(gCD59) is a novel biomarker. CD59 is a complement regulatory protein that protects "self" cells from complement-mediated damage (Davies CS et al. Glycation of CD59 antigens regulation on extracellular cells from diabatic subjects. in diabetes, CD59 is inactivated by non-enzymatic glycosylation to form gCD59. plasma gCD59 is a soluble form of CD59 that is shed from the cell membrane. CD59 is a widely distributed membrane-bound inhibitor of the complement cytolytic Membrane Attack Complex (MAC). CD59 acts by binding to C8 and/or C9 in nascent MAC and interferes with the insertion and polymerization of C9 membrane. this protein present in all cells is anchored to the outer surface of the membrane by a lipid tail. thus, it is exposed to extracellular and extracellular fluid glucose levels. soluble CD59 shed from the cell membrane is present in circulating and urine patients, gCD59 is present in diabetic patients in elevated blood glucose levels that are therefore evident in normal blood, the detection of the gCD59 content in the human body fluid has guiding significance for clinical detection and screening of diabetics.
In the prior art, a detection method of related glycosylated CD59 is reported, but no registration character number of the gCD59 detection kit is searched for both a domestic reagent and an imported reagent when a website of the national food and drug administration is searched. The related enzyme-linked immunoassay methods reported in the literature are all as follows: the monoclonal antibody Of anti CD59 is a capture antibody, the monoclonal antibody Of anti gCD59 is a detection antibody, the secondary antibody is labeled by an enzyme And is a goat anti-rabbit IgG-Horse Radish Peroxidase (HRP) marker or SA-HRP, the sensitivity Of the kit is not high, the operation time is as long as 200 minutes (a product Of G Biosciences, which is not registered at home) to 400 minutes (Pamela Ghosh, A Specific And Sensitive Assay For Blood Levels Of Cd < Glycated 59: A Novel Biomarker For Diabetes). There is no report on a detection method using a monoclonal antibody against gCD59 as a capture antibody and a monoclonal antibody against CD59 as a labeled antibody based on a chemiluminescence method, and a detection kit prepared by a chemiluminescence immunoadsorption method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a glycosylated CD59 detection kit based on a chemiluminescence method and application thereof.
The invention relates to a glycosylation CD59 detection kit based on a chemiluminescence method, which is composed of a luminescent plate coated with an anti-gCD 59 antibody, a gCD59 standard substance gradient solution, a sample diluent, a peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, a 20 multiplied concentrated washing solution, a luminescent liquid, a sealing plate film, a sealing bag and an instruction book, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, the luminescent liquid, the sealing plate film, the sealing bag;
the method is characterized in that:
the luminescent plate is a transparent polystyrene 96-hole luminescent plate, and each hole of the luminescent plate is coated with a mouse monoclonal antibody Mab1 with the concentration of 5 mug/mL and resisting gCD 59;
the gCD59 standard gradient solutions were: 7 standard solutions with concentration gradients of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, wherein 0pg/mL is a standard buffer;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, SDS 0.1g, casein sodium salt 5g, TritonX-1000.1 mL, Proclin 3001 mL;
the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate is horseradish peroxidase-labeled anti-CD 59 mouse monoclonal antibody Mab 2; the enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate has a working titer of 1:10000, and the diluent is an enzyme conjugate diluent;
the formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the luminescent solution comprises solution A and solution B, and a mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L paraiodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
In the above chemiluminescence-based glycosylation CD59 detection kit, the preparation method of the luminescent plate comprises the following steps:
1.1) preparing a coating liquid: namely, preparing 0.01mol/L phosphate buffer solution and adjusting the pH value to 7.4;
Figure BDA0002946842650000021
filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
Figure BDA0002946842650000022
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
Figure BDA0002946842650000023
Figure BDA0002946842650000031
filtering for sterilization, and storing at 4 deg.C;
1.4) diluting a mouse monoclonal antibody Mab1 of anti-gCD 59 to a working concentration of 5 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking the marked enzyme label plate, using 8-pore channel gun array to spot the plate, leading the antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
1.6) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-6 hours, then adding 5 mu L of 0.01 percent CTAB aqueous solution into each hole, and coating for 12-16 hours overnight;
1.7) taking out the coated plate, balancing at room temperature for 30min, throwing off antibody liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid into each hole for each time, washing for 2 times, and beating the absorbent paper to be dry;
1.8) adding 250 mu L of sealing liquid into each hole, and sealing for 16 hours at 2-8 ℃ overnight;
1.9) taking out the coated plate, balancing the coated plate at room temperature for 30min, throwing off confining liquid, and patting the coated plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.10) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
In the above-mentioned glycosylation CD59 detection kit based on chemiluminescence method: the mouse monoclonal antibody Mab1 resisting gCD59 and the mouse monoclonal antibody Mab2 resisting CD59 are preferably products of Shandongbao Biotechnology GmbH; wherein the amino acid sequence of antibody Mab1 directed against an epitope is: DACLITKAGLQVYNKCWKFEHC, the amino acid sequence of antibody Mab2 directed against an epitope is: LQCYNCPNPTADCKTAVNCSS are provided.
In the above glycosylation CD59 detection kit based on chemiluminescence method, the preparation method of the gCD59 standard substance gradient solution is: preparing a gCD59 standard sample in simulated serum by using the purified recombinant gCD59 protein according to the proportion of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL; preparing a 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing 5 wt% of bovine serum albumin and 0.85 wt% of sodium chloride, wherein the pH value is 7.0, and obtaining a standard buffer solution; and preparing 7 standard substance solutions with standard substance concentration gradient of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL by using the standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution.
In the above-mentioned glycosylation CD59 detection kit based on chemiluminescence method, the formula of the enzyme conjugate diluent is: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL.
The precision and accuracy of the kit are verified by testing a median gCD59 standard product in parallel; the accuracy of the kit is verified from the aspect of sample recovery rate of the kit; the sensitivity of the kit is determined by detecting different amounts gCD59 with gCD59 concentration gradient.
The invention discloses application of a glycosylated CD59 detection kit based on a chemiluminescence method in detection of a gCD 59-containing biological sample.
Wherein the method for detecting the gCD 59-containing sample comprises the following steps:
1) sample adding of the standard: setting a standard substance hole and a sample hole, wherein the standard substance hole is sequentially added with 10 mu L of standard substances with different concentrations according to concentration gradients;
2) sample adding: respectively arranging blank holes and sample holes to be detected on an enzyme-labeled coating plate, namely a luminescent plate; adding 190 mu L of sample diluent into a sample hole to be detected, and then adding 10 mu L of sample to be detected, so that the final dilution of the sample is 20 times; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent; the blank control hole is not added with a sample and an enzyme labeling reagent, and the rest steps are operated in the same way;
3) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 20 minutes at 37 ℃;
4) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
5) adding an enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate into each hole, except blank holes;
6) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 15 minutes at 37 ℃;
7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
8) luminescence: adding 100 mu L of luminous liquid A and 100 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing at 37 ℃ in a dark place for 4 minutes;
9) and (3) determination: measuring absorbance of each hole, namely an RLU value by using a full-automatic chemiluminescence analyzer;
10) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value; multiplying by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
The glycosylation CD59 detection kit based on the chemiluminescence method is prepared based on the chemiluminescence immunoadsorption method, and tests on clinical samples show that the glycosylation CD59 detection kit (chemiluminescence method) can effectively detect the gCD59 content in a human body and is consistent with the judgment result of clinical diabetic patients, so that the glycosylation CD59 detection kit has better clinical application value. It has the obvious advantages that:
1. since gCD59 and CD59 have only a very small difference in protein structure, it is important to prepare a specific monoclonal antibody that can specifically recognize gCD59 but not CD 59. Through extensive and repeated screening and verification, the gCD59 detection antibody in the invention is a monoclonal antibody which specifically recognizes gCD59 and does not bind CD59, and therefore, the gCD59 detection antibody provides a reliable biological raw material for the specific detection gCD59 in the invention.
2. In the detection method, the gCD59 monoclonal antibody is used as the capture antibody, and the first step, namely the specificity capture gCD59 protein, is adopted in the methodology, so that the non-specific adsorption phenomenon is greatly reduced, the detection method is more direct, and the result is more accurate. This overcomes the disadvantage of non-specific binding in the first step of the reported detection method.
3. The kit provided by the invention is simple to operate, the marker is stable, the sensitivity is high, and the defects of complicated and unstable operation steps of the conventional enzyme-linked immunoassay method are completely overcome.
4. CTAB is added in the coating process, so that decontamination and impurity removal are performed, and the coating effect is improved, thereby increasing the sensitivity of the kit.
The glycosylation CD59 detection kit based on the chemiluminescence method provides a more convenient detection method based on an ELISA method, and the method has the advantages of high stability, high sensitivity, strong selectivity, high detection speed, low cost, easy operation and the like. The method overcomes the defects of low sensitivity, long operation time and the like in gCD59 detection in the prior art, shortens the operation time from 400 minutes to 40 minutes, improves the limit of quantification from 18.75pg/mL to 5pg/mL, and has excellent clinical application prospect.
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FIG. 1: the glycosylation CD59 detection kit based on the chemiluminescence method quantitatively analyzes a chart.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified.
Example 1: gCD59 preparation of protein
The published gCD59 amino acid sequence (DACLITKAGLQVYNKCWKFEHC) and CD59 amino acid sequence (LQCYNCPNPTADCKTAVNCSS) are used to obtain gCD59 and CD59 corresponding nucleotide sequences by protein expression technology known to those skilled in the biological field.
Constructing a plasmid for expressing gCD59, namely pcDNA3.1-gCD59 expression vector, transfecting CHO cells, screening positive clones, culturing and purifying to obtain gCD59 protein.
The CD59 protein can be prepared by similar method.
Example 2: preparation of anti-gCD 59 murine monoclonal antibody Mab1 and anti-CD 59 murine monoclonal antibody Mab2
The obtained gCD59 and CD59 proteins were immunized to healthy BALB/c mice (8 weeks old), respectively, and after the mice produced antibodies, anti-gCD 59 murine monoclonal antibody Mab1 and anti-CD 59 murine monoclonal antibody Mab2 were prepared by conventional myeloma fusion cell technology, respectively.
Experiments prove that the amino acid sequence of the prepared antibody Mab1 for the antigen epitope is as follows: DACLITKAGLQVYNKCWKFEHC, the amino acid sequence of antibody Mab2 directed against an epitope is: LQCYNCPNPTADCKTAVNCSS are provided.
Wherein, the obtained Mab1 was verified not to cross-react with CD59, and Mab2 was verified not to cross-react with gCD 59. The specific process adopts hybridoma antibody preparation technology commonly known by those in the biological field.
Further, the antibody Mab2 was used to prepare enzyme conjugates by the sodium periodate-labeled HRP method.
Example 3: preparation of glycosylation CD59 detection kit based on chemiluminescence method
1. Preparing the luminescent plate:
preparing a coating solution: preparing 0.01mol/L phosphate buffer solution and adjusting the pH value to 7.4;
Figure BDA0002946842650000061
filtering for sterilization, and storing at 4 deg.C;
the formula and the preparation method of the washing liquid are as follows:
Figure BDA0002946842650000062
filtering for sterilization, and storing at 4 deg.C;
preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
Figure BDA0002946842650000063
filtering for sterilization, and storing at 4 deg.C;
diluting a mouse monoclonal antibody Mab1 of gCD59 to a working concentration of 5 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
taking a marked enzyme label plate, using an 8-pore row gun to spot the plate, enabling the coated antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
placing in a refrigerator at 2-8 ℃ for coating for 4-6 hours, then adding 5 mu L of 0.01% CTAB aqueous solution into each hole, and coating for 12-16 hours overnight;
taking out the coated plate, balancing at room temperature for 30min, throwing off antibody liquid, drying absorbent paper, adding 300 μ L of washing solution into each hole, washing for 2 times, and drying absorbent paper;
adding 250 mu L of sealing liquid into each hole, sealing for 16 hours at 2-8 ℃ overnight;
taking out the coated plate, balancing at room temperature for 30min, throwing off confining liquid, and patting dry absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
vacuum packaging with aluminum foil bag, marking, and storing at 2-8 deg.C;
2. preparation of a kit solution:
preparation of the base solution
The sample diluent formula is:
Figure BDA0002946842650000071
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C;
the formula of 20 × concentrated washing solution is: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
gCD 59A standard gradient solution was prepared as follows: preparing a gCD59 standard sample in simulated serum by using the purified recombinant gCD59 protein according to the proportion of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL; preparing a 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing 5 wt% of bovine serum albumin and 0.85 wt% of sodium chloride, wherein the pH value is 7.0, and obtaining a standard buffer solution; preparing 7 standard substance solutions with standard substance concentration gradient of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL by using the standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution;
the formulation of the enzyme conjugate diluent was: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL;
the luminous liquid comprises a liquid A and a liquid B, and the formula of the luminous liquid is as follows: the solution A is a mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L iodophenol solution in equal volume ratio; the solution B is hydrogen peroxide with the concentration of 7.5 mmol/L; the solution A and solution B were mixed at a volume ratio of 1: 1.
Example 4: method for detecting gCD 59-containing sample by using kit of the invention
1) Sample adding of the standard: setting a standard substance hole and a sample hole, wherein the standard substance hole is sequentially added with 10 mu L of standard substances with different concentrations according to concentration gradients;
2) sample adding: respectively arranging blank holes and sample holes to be detected on an enzyme-labeled coating plate, namely a luminescent plate; adding 190 mu L of sample diluent into a sample hole to be detected, and then adding 10 mu L of sample to be detected, so that the final dilution of the sample is 20 times; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent; the blank control hole is not added with a sample and an enzyme labeling reagent, and the rest steps are operated in the same way;
3) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 20 minutes at 37 ℃;
4) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
5) adding an enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate into each hole, except blank holes;
6) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 15 minutes at 37 ℃;
7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
8) luminescence: adding 100 mu L of luminous liquid A and 100 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing at 37 ℃ in a dark place for 4 minutes;
9) and (3) determination: measuring absorbance of each hole, namely an RLU value by using a full-automatic chemiluminescence analyzer;
10) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value; multiplying by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
Example 5: the glycosylation CD59 detection kit based on the chemiluminescence method has the advantages of detection of linearity, accuracy, recovery rate and precision
1) Linearity of the kit
The standard solution is adopted to have 7 concentration gradients which are respectively as follows: 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, RLU value readings of standard solutions were performed using the standard protocol of the present invention, and the linear assay curves of the kits of the present invention were plotted against the recorded test values, see FIG. 1.
As can be seen from FIG. 1, the glycosylation CD59 detection kit based on the chemiluminescence method has good linearity.
2) The accuracy of the kit was verified by sample recovery.
gCD59 was added to the standard buffer to give gCD59 final concentrations of 20ng/mL, 210ng/mL, 1050ng/mL, respectively, and each sample was tested in3 replicates and the recovery statistics calculated are shown in Table 1.
Table 1: recovery rate
Figure BDA0002946842650000081
The result shows that the recovery rate of gCD59 samples with high, medium and low concentrations is between 85.8 and 108.9 percent, the average recovery rate is between 88.6 and 106 percent, and the overall recovery rate is 98.9 percent.
3) Precision test:
the standard substance with 100ng/mL is tested 10 times in parallel, and the statistics of the results are shown in Table 2.
Table 2: results of precision test
Figure BDA0002946842650000091
Precision: CV% ═ 5.4%
Example 6: application of glycosylation CD59 detection kit based on chemiluminescence method in diabetes clinical sample screening
gCD59 is detected by using the kit, and the index is simultaneously applied to screening in the diabetic patients.
Through cooperation with a cooperative hospital, the hospital collects 100 diabetic clinical serum samples and 200 non-diabetic clinical serum samples. The result of using the kit to detect gCD59 in the serum sample shows that gCD59 is obviously higher in individuals with diabetes than in individuals without diabetes, and is independently related to glycosylated hemoglobin, so that the diabetic patients are determined to have high specificity and sensitivity. See table 3.
Table 3: diabetic patient screening
Figure BDA0002946842650000092

Claims (7)

1. A glycosylation CD59 detection kit based on chemiluminescence method comprises a luminescent plate coated with anti-gCD 59 antibody, gCD59 standard substance gradient solution, sample diluent, peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, 20 Xconcentrated washing solution, luminescent solution, a sealing plate film, a sealing bag and an instruction book, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, the 20 Xconcentrated washing solution, the luminescent solution, the sealing plate film, the sealing bag and the instruction book are arranged;
the method is characterized in that:
the luminescent plate is a transparent polystyrene 96-hole luminescent plate, and each hole of the luminescent plate is coated with a mouse monoclonal antibody Mab1 with the concentration of 5 mug/mL and resisting gCD 59;
the gCD59 standard gradient solutions were: 7 standard solutions with concentration gradients of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, wherein 0pg/mL is a standard buffer;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, SDS 0.1g, casein sodium salt 5g, TritonX-1000.1 mL, Proclin 3001 mL;
the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate is horseradish peroxidase-labeled anti-CD 59 mouse monoclonal antibody Mab 2; the enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate has a working titer of 1:10000, and the diluent is an enzyme conjugate diluent;
the formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double steamIn water, NaCl 8.0g and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the luminescent solution comprises solution A and solution B, and a mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L paraiodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
2. The chemiluminescence-based glycosylation CD59 assay kit according to claim 1, wherein the luminescent plate is prepared by:
1.1) preparing a coating liquid: namely, preparing 0.01mol/L phosphate buffer solution and adjusting the pH value to 7.4;
Figure FDA0002946842640000011
filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
Figure FDA0002946842640000012
Figure FDA0002946842640000021
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
Figure FDA0002946842640000022
filtering for sterilization, and storing at 4 deg.C;
1.4) diluting a mouse monoclonal antibody Mab1 of anti-gCD 59 to a working concentration of 5 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking the marked enzyme label plate, using 8-pore channel gun array to spot the plate, leading the antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
1.6) placing in a refrigerator with the temperature of 2-8 ℃ for coating for 4-6 hours, then adding 5 mu L of 0.01 percent CTAB aqueous solution into each hole, and coating for 12-16 hours overnight;
1.7) taking out the coated plate, balancing at room temperature for 30min, throwing off antibody liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid into each hole for each time, washing for 2 times, and beating the absorbent paper to be dry;
1.8) adding 250 mu L of sealing liquid into each hole, and sealing for 16 hours at 2-8 ℃ overnight;
1.9) taking out the coated plate, balancing the coated plate at room temperature for 30min, throwing off confining liquid, and patting the coated plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.10) packaging the coated board in an aluminum foil bag in vacuum, marking, and storing at 2-8 ℃ for later use.
3. The chemiluminescence-based glycosylated CD59 detection kit according to claim 1, wherein: the mouse monoclonal antibody Mab1 resisting gCD59 and the mouse monoclonal antibody Mab2 resisting CD59 are products of Torilebo Biotech, Inc., Shandong; wherein the amino acid sequence of antibody Mab1 directed against an epitope is: DACLITKAGLQVYNKCWKFEHC, the amino acid sequence of antibody Mab2 directed against an epitope is: LQCYNCPNPTADCKTAVNCSS are provided.
4. The chemiluminescence-based glycosylated CD59 detection kit according to claim 1, wherein: the preparation method of the gCD59 standard substance gradient solution comprises the following steps: preparing a gCD59 standard sample in simulated serum by using the purified recombinant gCD59 protein according to the proportion of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL; preparing a 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing 5 wt% of bovine serum albumin and 0.85 wt% of sodium chloride, wherein the pH value is 7.0, and obtaining a standard buffer solution; and preparing 7 standard substance solutions with standard substance concentration gradient of 0pg/mL, 10pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000pg/mL by using the standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution.
5. The glycosylated CD59 enzyme-linked immunoassay kit according to claim 1, wherein the enzyme conjugate diluent is formulated as: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL.
6. Use of the chemiluminescence-based glycosylated CD59 detection kit of claim 1 for detecting gCD 59-containing biological samples.
7. The use of claim 6, wherein the gCD 59-containing sample is detected by:
1) sample adding of the standard: setting a standard substance hole and a sample hole, wherein the standard substance hole is sequentially added with 10 mu L of standard substances with different concentrations according to concentration gradients;
2) sample adding: respectively arranging blank holes and sample holes to be detected on an enzyme-labeled coating plate, namely a luminescent plate; adding 190 mu L of sample diluent into a sample hole to be detected, and then adding 10 mu L of sample to be detected, so that the final dilution of the sample is 20 times; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent; the blank control hole is not added with a sample and an enzyme labeling reagent, and the rest steps are operated in the same way;
3) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 20 minutes at 37 ℃;
4) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
5) adding an enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate into each hole, except blank holes;
6) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 15 minutes at 37 ℃;
7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;
8) luminescence: adding 100 mu L of luminous liquid A and 100 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing at 37 ℃ in a dark place for 4 minutes;
9) and (3) determination: measuring absorbance of each hole, namely an RLU value by using a full-automatic chemiluminescence analyzer;
10) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, and finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value; multiplying by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
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