CN113009151B - 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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CN113009151B
CN113009151B CN202110196369.4A CN202110196369A CN113009151B CN 113009151 B CN113009151 B CN 113009151B CN 202110196369 A CN202110196369 A CN 202110196369A CN 113009151 B CN113009151 B CN 113009151B
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gcd
plate
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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 glycosylated CD59 detection kit based on a chemiluminescence method, which consists of a luminescent plate coated with an anti-gCD 59 antibody, gCD standard substance gradient solution, sample diluent, peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, 20X concentrated washing liquid, luminescent liquid, a sealing plate film, a sealing bag and instructions, wherein the luminescent plate is arranged in the kit. The invention also discloses application of the kit in detecting a gCD 59-containing biological sample. Experiments prove that the kit has high stability, strong selectivity, high detection speed, low cost and easy operation, overcomes the defects of low sensitivity, long operation time and the like in the detection of gCD in the prior art, shortens the operation time from 400 minutes to 40 minutes, and has excellent clinical application prospect when the quantitative limit is increased from 18.75pg/mL to 5 pg/mL.

Description

Glycosylated CD59 detection kit based on chemiluminescence method and application thereof
Technical Field
The invention relates to detection of glycosylated CD59 (gCD 59), 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. Long-standing hyperglycemia leads to chronic damage, dysfunction, of various tissues, especially the eyes, kidneys, heart, blood vessels, nerves. In 70% of the diabetic population, systemic small blood vessels and microvasculature develop proliferative lesions, the pathogenesis of which is related to long-term blood glucose elevation, increased glycated protein end products, intercellular adhesion molecule expression, increased cytokines and polysaccharide deposition, and thickening of the vascular basement membrane caused by the hypoendothelium.
Glycosylated CD59 (gCD 59) is a novel biomarker. CD59 is a complement regulator protein that protects "self" cells from complement-mediated damage (Davies CS et al, glycation of CD59 impairs complement regulation on erythrocytes from diabetic subjects.) in diabetes, CD59 is inactivated by nonenzymatic glycosylation to form gCD59. Plasma gCD is a soluble form of CD59 that is shed from the cell membrane CD59 is a widely distributed membrane-bound inhibitor of 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 membranes.
Methods for detecting the related glycosylated CD59 have been reported in the prior art. The related enzyme-linked immunosorbent assay methods reported in the literature are as follows: the monoclonal antibody against CD59 is a capture antibody, the monoclonal antibody against gCD59 is a detection antibody, and the secondary antibody is a goat anti-rabbit IgG-horseradish peroxidase (HRP) marker or SA-HRP by enzyme labeling, the sensitivity of the kit is not high, and the operation time is as long as 200 minutes (G Biosciences product, which is not registered in China) to 400 minutes (PamelaGhosh, A Specific And Sensitive Assay For Blood Levels Of Glycated Cd59: ANovel Biomarker For Diabetes). The detection method using a monoclonal antibody of anti gCD59 as a capture antibody and a monoclonal antibody of anti CD59 as a labeled diabody based on a chemiluminescence method and a detection kit prepared based on a chemiluminescence immunoadsorption method have not been reported yet.
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 glycosylated CD59 detection kit based on a chemiluminescence method, which consists of a luminescent plate coated with an anti-gCD 59 antibody, gCD standard substance gradient solution, sample diluent, peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, 20X concentrated washing liquid, luminescent liquid, a sealing plate film, a sealing bag and a specification, wherein the luminescent plate is arranged in the kit;
the method is characterized in that:
the luminous plate is a transparent polystyrene 96-hole luminous plate, and each hole of the luminous plate is coated with a murine monoclonal antibody Mab1 of a antigen gCD59 with the concentration of 5 mug/mL;
the gCD standard gradient solutions are respectively: 7 standard solutions with concentration gradients of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000 pg/mL, wherein 0pg/mL is a standard buffer;
the formula of the sample diluent is as follows: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, SDS 0.1g, casein sodium salt 5g, tritonX-1000.1mL and Proclin300 mL;
the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate is horseradish peroxidase-labeled anti-CD 59 mouse monoclonal antibody Mab2; the working titer of the enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate is 1:10000, and the diluent is the diluent of the enzyme conjugate;
the formula of the 20 x concentrated washing liquid is as follows: the sterilized 50mL double distilled water contains 8.0g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 2.9g of O and 0.5mL of Tween 20;
the luminous solution comprises a solution A and a solution B, wherein the mixed solution of luminol solution with the concentration of 3.0mmol/L and p-iodophenol solution with the equal volume ratio is named as the solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; when in use, the solution A and the solution B are mixed according to the volume ratio of 1:1.
In the glycosylated CD59 detection kit based on the chemiluminescence method, 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;
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
the volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.3 Preparing a sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 ·2H 2 O 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
sucrose 30g
BSA 10g
Casein sodium salt 10g
Tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.4 Diluting the murine monoclonal antibody Mab1 of the antigen gCD59 to the working concentration of 5 mug/mL by using a coating solution, uniformly mixing and standing for 15 minutes;
1.5 Taking a marked ELISA plate, using an 8-channel gun-discharging spot plate to enable the antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
1.6 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;
1.7 Taking out the coating plate, balancing at room temperature for 30min, throwing away the anti-body fluid, beating to dry by using water-absorbing paper, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating to dry by using the water-absorbing paper;
1.8 250 μl of blocking solution was added to each well and blocked for 16 hours at 2deg.C-8deg.C overnight;
1.9 Taking out the coating plate, balancing at room temperature for 30min, throwing off the sealing liquid, and drying by beating with water-absorbing paper; drying in a silica gel dryer, and maintaining humidity below 30% at room temperature for 5 hr;
1.10 Vacuum packaging the coated plate with aluminum foil bag, marking, and storing at 2-8deg.C for use.
In the above-described chemiluminescence-based glycosylated CD59 detection kit: the anti-gCD 59 murine monoclonal antibody Mab1 and the anti-CD 59 murine monoclonal antibody Mab2 are preferably manufactured by Shandong Leibo biotechnology Co., ltd; wherein the amino acid sequence of antibody Mab1 against the epitope is: DACLITKAGLQVYNKCWKFEHC the amino acid sequence of antibody Mab2 against the epitope is: LQCYNCPNPTADCKTAVNCSS.
In the glycosylated CD59 detection kit based on the chemiluminescence method, the preparation method of the gCD standard substance gradient solution comprises the following steps: gCD59 standard in simulated serum was prepared with purified recombinant gCD protein at a ratio of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000 pg/mL; preparing a buffer solution containing 5wt% of bovine serum albumin and 0.85wt% of sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate to obtain a standard buffer solution; and 7 standard solution with standard concentration gradients of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL are prepared by using the standard buffer, wherein 0pg/mL is the standard buffer.
In the above-mentioned chemiluminescence method-based glycosylated CD59 detection kit, the enzyme conjugate diluent comprises the following components: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, surfactant S9 3g, casein sodium salt 5g, BSA 10g and Proclin300 mL.
The precision and accuracy of the kit are verified by parallel testing of a median gCD59 standard, and the precision of the kit is verified; the accuracy of the kit is verified in terms of the sample recovery rate of the kit; the assay for the sensitivity of the kit was to determine the sensitivity of the kit reaction by detecting different amounts gCD59 with a gCD concentration gradient.
The invention relates to an application of a glycosylated CD59 detection kit based on a chemiluminescence method in detecting a biological sample containing gCD59.
Wherein, the method for detecting the sample containing gCD59 comprises the following steps:
1) Sample addition of standard substance: setting a standard substance hole and a sample hole, wherein 10 mu L of standard substances with different concentrations are sequentially added into the standard substance hole according to concentration gradients;
2) Sample adding: respectively arranging blank holes and sample holes to be tested on an enzyme-labeled coated plate, namely a light-emitting 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 to enable the final dilution of the sample to be 20 times; when in sample adding, a sample is added to the bottom of a hole of the plate, the hole wall is not touched as much as possible, and the mixture is gently shaken and uniformly mixed; the blank control hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same;
3) Incubation: incubating for 20 minutes at 37 ℃ after membrane sealing by a sealing plate;
4) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the steps for 5 times, and beating;
5) Adding enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate, except blank holes;
6) Incubation: incubating for 15 minutes at 37 ℃ after membrane sealing by a sealing plate;
7) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the steps for 5 times, and beating;
8) And (3) emitting light: adding 100 mu L of luminous solution A and 100 mu L of luminous solution B into each hole, gently shaking and mixing uniformly, and developing for 4 minutes at 37 ℃ in a dark place;
9) And (3) measuring: measuring the absorbance of each well, namely the RLU value, by using a full-automatic chemiluminescence analyzer;
10 Drawing a standard curve on a coordinate paper by taking each concentration of the standard substance as an abscissa and 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 the sample by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of the 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 the concentration by the dilution multiple to obtain the actual concentration of the sample.
The glycosylated CD59 detection kit based on the chemiluminescence method is a detection kit prepared based on a chemiluminescence immunoadsorption method, and clinical samples are tested to show that the glycosylated CD59 detection kit (chemiluminescence method) can effectively detect the content of gCD in a human body and is consistent with the judgment result of a clinical diabetic patient, so that the glycosylated CD59 detection kit has a good clinical application value. The method has the remarkable advantages that:
1. because gCD differs only slightly in protein structure from CD59, it is particularly important to prepare specific monoclonal antibodies that specifically recognize gCD59 but not CD59. Through extensive and repeated screening and validation, the gCD detection antibody of the present invention is a monoclonal antibody that specifically recognizes gCD59 without binding to CD59, which provides a reliable biological feedstock for the specific detection gCD59 of the present invention.
2. In the detection method, the gCD59 monoclonal antibody is used as a capture antibody, the gCD59 protein is specifically captured in the first step from the methodology, the non-specific adsorption phenomenon is greatly reduced, the detection method is more direct, and the result is more accurate. This overcomes the reported disadvantage of the first step of non-specific binding in the detection method.
3. The kit disclosed by the invention is simple to operate, stable in marker and high in sensitivity, and the defects of complicated and unstable operation steps of the conventional ELISA detection method are completely overcome.
4. CTAB is added in the coating process, so that decontamination and impurity removal are realized, and the coating effect is improved, so that the sensitivity of the kit is increased.
The invention provides a glycosylated CD59 detection kit based on a chemiluminescence method, and provides a more convenient detection method based on an ELISA method. Overcomes the defects of low sensitivity, long operation time and the like in the detection of gCD in the prior art, shortens the operation time from 400 minutes to 40 minutes, improves the quantitative limit from 18.75pg/mL to 5pg/mL, and has excellent clinical application prospect.
Drawings
Fig. 1: quantitative linear analysis chart of glycosylated CD59 detection kit based on chemiluminescence method.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
In the following examples, materials, reagents and the like used, unless otherwise specified, were obtained commercially.
Example 1: preparation of gCD protein 59
The corresponding nucleotide sequences for gCD and CD59 were obtained using published gCD amino acid sequence (DACLITKAGLQVYNKCWKFEHC), CD59 amino acid sequence (LQCYNCPNPTADCKTAVNCSS), respectively, using protein expression techniques well known to those skilled in the art of biology.
Constructing a plasmid for expressing gCD59, namely a pcDNA3.1-gCD59 expression vector, transfecting CHO cells, screening positive clones, culturing and purifying to obtain gCD59 protein.
A similar procedure also produced CD59 protein.
Example 2: preparation of anti-gCD 59 murine monoclonal antibody Mab1 and anti-CD 59 murine monoclonal antibody Mab2
The obtained gCD and CD59 proteins were immunized into healthy BALB/c mice (8 weeks old), and after the mice developed antibodies, anti-gCD 59 mouse monoclonal antibody Mab1 and anti-CD 59 mouse monoclonal antibody Mab2 were prepared by conventional myeloma fusion cell technology, respectively.
Experiments prove that the amino acid sequence of the prepared antibody Mab1 aiming at the antigen epitope is as follows: DACLITKAGLQVYNKCWKFEHC the amino acid sequence of antibody Mab2 against the epitope is: LQCYNCPNPTADCKTAVNCSS.
Wherein, mab1 was verified to be non-cross-reactive with CD59 and Mab2 was verified to be non-cross-reactive with gCD. The specific process adopts hybridoma antibody preparation technology commonly known to the person skilled in the biological field.
Further, the antibody Mab2 was used to prepare an enzyme conjugate using the sodium periodate-labeled HRP method.
Example 3: the invention relates to preparation of a glycosylated CD59 detection kit based on a chemiluminescence method
1. Preparation of a light-emitting plate:
preparing a coating liquid: preparing a phosphate buffer solution with the concentration of 0.01mol/L, and adjusting the pH value to 7.4;
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
the volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
preparing a sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 ·2H 2 O 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
sucrose 30g
BSA 10g
Casein sodium salt 10g
Tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
diluting the monoclonal antibody mAb1 of the antigen gCD59 to the working concentration of 5 mug/mL by using a coating liquid, uniformly mixing and standing for 15 minutes;
taking a marked ELISA plate, using an 8-channel gun-discharging spot plate to enable the coating antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
placing in a refrigerator at 2-8deg.C, coating for 4-6 hr, adding 5 μl of 0.01% CTAB aqueous solution into each well, and coating overnight for 12-16 hr;
taking out the coating plate, balancing at room temperature for 30min, throwing away the anti-body fluid, beating dry by using water-absorbing paper, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating dry by using the water-absorbing paper;
250 mu L of blocking solution is added into each hole, and the mixture is blocked for 16 hours at the temperature of 2-8 ℃ overnight;
taking out the coating plate, balancing at room temperature for 30min, throwing off the sealing liquid, and drying by beating with water-absorbing paper; drying in a silica gel dryer, and maintaining humidity below 30% at room temperature for 5 hr;
vacuum packaging the coated plate with aluminum foil bag, marking, and storing at 2-8deg.C;
2. preparing a kit solution:
preparation of the base solution
The sample diluent formula is:
NaCL 8.0g
NaH 2 PO 4 ·2H 2 O 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
casein sodium salt 5g
SDS 0.1g
Triton X-100 0.1mL
Proclin300 1mL
The volume of the purified water is fixed to 1000mL
Adjusting pH to 7.4, filtering, sterilizing, and preserving at 4deg.C;
the formula of the 20 x concentrated washing solution is: the sterilized 50mL double distilled water contains 8.0g NaCl and NaH 2 PO 4· 2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 2.9g of O and 0.5mL of Tween 20;
gCD59 standard gradient solution is prepared as follows: gCD59 standard in simulated serum was prepared with purified recombinant gCD protein at a ratio of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000 pg/mL; preparing a buffer solution containing 5wt% of bovine serum albumin and 0.85wt% of sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate to obtain a standard buffer solution; preparing 7 standard substance solutions with standard substance concentration gradients of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL by using a standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution;
the formulation of the enzyme conjugate dilutions was: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, surfactant S9 3g, casein sodium salt 5g, BSA 10g and Proclin300 mL;
the luminous liquid comprises liquid A and liquid B, and the formula of the luminous liquid comprises: the solution A is a mixed solution of luminol solution with the concentration of 3.0mmol/L and p-iodophenol solution with the equal volume ratio of 0.3 mmol/L; the solution B is hydrogen peroxide with the concentration of 7.5 mmol/L; when in use, the solution A and the solution B are mixed according to the volume ratio of 1:1.
Example 4: method for detecting gCD 59-containing sample by using kit
1) Sample addition of standard substance: setting a standard substance hole and a sample hole, wherein 10 mu L of standard substances with different concentrations are sequentially added into the standard substance hole according to concentration gradients;
2) Sample adding: respectively arranging blank holes and sample holes to be tested on an enzyme-labeled coated plate, namely a light-emitting 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 to enable the final dilution of the sample to be 20 times; when in sample adding, a sample is added to the bottom of a hole of the plate, the hole wall is not touched as much as possible, and the mixture is gently shaken and uniformly mixed; the blank control hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same;
3) Incubation: incubating for 20 minutes at 37 ℃ after membrane sealing by a sealing plate;
4) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the steps for 5 times, and beating;
5) Adding enzyme: adding 200 mu L of enzyme-labeled reagent, namely enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate, except blank holes;
6) Incubation: incubating for 15 minutes at 37 ℃ after membrane sealing by a sealing plate;
7) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the steps for 5 times, and beating;
8) And (3) emitting light: adding 100 mu L of luminous solution A and 100 mu L of luminous solution B into each hole, gently shaking and mixing uniformly, and developing for 4 minutes at 37 ℃ in a dark place;
9) And (3) measuring: measuring the absorbance of each well, namely the RLU value, by using a full-automatic chemiluminescence analyzer;
10 Drawing a standard curve on a coordinate paper by taking each concentration of the standard substance as an abscissa and 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 the sample by the dilution times to obtain the actual concentration of the sample; or calculating a linear regression equation of the 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 the concentration by the dilution multiple to obtain the actual concentration of the sample.
Example 5: the invention relates to a detection method for linearity, accuracy, recovery rate and precision of a glycosylated CD59 detection kit based on a chemiluminescence method
1) Linearity of the kit
The standard solution is adopted to have 7 concentration gradients, which are respectively: the RLU values of the standard solutions were read using the standard procedure of the invention at 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000 pg/mL, and the linear analytical curves of the kits of the invention were plotted according to the recorded test values, see fig. 1.
As can be seen from FIG. 1, the chemiluminescence method-based glycosylated CD59 detection kit of the present invention has good linearity.
2) The accuracy of the kit was verified at sample recovery.
gCD59 was added to the standard buffer, respectively, so that the final concentrations of gCD59 were 20ng/mL, 210ng/mL, 1050ng/mL, each sample was tested 3 times in parallel, and the calculated recovery statistics are shown in Table 1.
Table 1: recovery rate
The results show that the recovery rate of the gCD 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 total recovery rate is 98.9 percent.
3) Precision test:
the results are shown in Table 2, and are tested 10 times in parallel with 100ng/mL standard.
Table 2: precision test results
Precision: CV% = 5.4%
Example 6: the invention relates to an application of a glycosylated CD59 detection kit based on a chemiluminescence method in screening clinical samples of diabetes
gCD59 is tested using a kit and the index is used to screen for diabetics.
By cooperation with the cooperating hospitals, hospitals collected 100 cases of clinical serum samples for diabetes and 200 cases of clinical serum samples for non-diabetes. The test of gCD in serum samples by using the kit provided by the invention shows that gCD is significantly higher in diabetic individuals than in non-diabetic individuals, and the test is independently related to glycosylated hemoglobin, so that the diabetic patients have high specificity and sensitivity. See table 3.
Table 3: screening of diabetics

Claims (1)

1. The glycosylated CD59 detection kit based on the chemiluminescence method comprises a light-emitting plate coated with an anti-gCD antibody, gCD standard substance gradient solution, sample diluent, peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate, 20X concentrated washing liquid, a light-emitting liquid, a sealing plate film, a sealing bag and instructions, wherein the light-emitting plate is arranged in the kit; wherein glycosylated CD59 is abbreviated gCD59;
the method is characterized in that:
the luminous plate is a transparent polystyrene 96-hole luminous plate, and each hole of the luminous plate is coated with a murine monoclonal antibody Mab1 of a antigen gCD59 with the concentration of 5 mug/mL;
the gCD standard gradient solutions are respectively: 7 standard solutions with concentration gradients of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000 pg/mL, wherein 0pg/mL is a standard buffer;
the formula of the sample diluent is as follows: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, SDS 0.1g, casein sodium salt 5g, triton X-100.1 mL and Proclin300 mL;
the peroxidase-labeled anti-CD 59 monoclonal antibody enzyme conjugate is horseradish peroxidase-labeled anti-CD 59 mouse monoclonal antibody Mab2; the working titer of the enzyme-labeled anti-CD 59 mouse monoclonal antibody Mab2 enzyme conjugate is 1:10000, and the diluent is the diluent of the enzyme conjugate;
the formula of the 20 x concentrated washing liquid is as follows: the sterilized 50mL double distilled water contains 8.0g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 2.9g of O and 0.5mL of Tween 20;
the luminous solution comprises a solution A and a solution B, wherein the mixed solution of luminol solution with the concentration of 3.0mmol/L and p-iodophenol solution with the equal volume ratio is named as the solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; when in use, the solution A and the solution B are mixed according to the volume ratio of 1:1;
the preparation method of the light-emitting 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;
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
the volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.3 Preparing a sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 ·2H 2 O 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
sucrose 30g
BSA 10g
Casein sodium salt 10g
Tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.4 Diluting the murine monoclonal antibody Mab1 of the antigen gCD59 to the working concentration of 5 mug/mL by using a coating solution, uniformly mixing and standing for 15 minutes;
1.5 Taking a marked ELISA plate, using an 8-channel gun-discharging spot plate to enable the antibody liquid to reach 200 mu L/hole, and covering a cover plate film;
1.6 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;
1.7 Taking out the coating plate, balancing at room temperature for 30min, throwing away the anti-body fluid, beating to dry by using water-absorbing paper, adding 300 mu L of washing liquid into each hole, washing for 2 times, and beating to dry by using the water-absorbing paper;
1.8 250 μl of blocking solution was added to each well and blocked for 16 hours at 2deg.C-8deg.C overnight;
1.9 Taking out the coating plate, balancing at room temperature for 30min, throwing off the sealing liquid, and drying by beating with water-absorbing paper; drying in a silica gel dryer, and maintaining humidity below 30% at room temperature for 5 hr;
1.10 Vacuum packaging the coated plate with aluminum foil bag, marking, and storing at 2-8deg.C;
the anti-gCD 59 murine monoclonal antibody Mab1 and the anti-CD 59 murine monoclonal antibody Mab2 are selected from Shandong Leibo biotechnology limited company; wherein the amino acid sequence of antibody Mab1 against the epitope is: DACLITKAGLQVYNKCWKFEHC the amino acid sequence of antibody Mab2 against the epitope is: LQCYNCPNPTADCKTAVNCSS;
the preparation method of the gCD59 standard substance gradient solution comprises the following steps: gCD59 standard in simulated serum was prepared with purified recombinant gCD protein at a ratio of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 2000 pg/mL; preparing a buffer solution containing 5wt% of bovine serum albumin and 0.85wt% of sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate to obtain a standard buffer solution; preparing 7 standard substance solutions with standard substance concentration gradients of 0pg/mL, 10 pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL by using a standard substance buffer solution, wherein 0pg/mL is the standard substance buffer solution;
the formulation of the enzyme conjugate diluent is: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, surfactant S9 3g, casein sodium salt 5g, BSA 10g and Proclin300 mL.
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