CN117031012A - Gd-IgA1 chemiluminescence quantitative determination kit - Google Patents

Gd-IgA1 chemiluminescence quantitative determination kit Download PDF

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CN117031012A
CN117031012A CN202310982989.XA CN202310982989A CN117031012A CN 117031012 A CN117031012 A CN 117031012A CN 202310982989 A CN202310982989 A CN 202310982989A CN 117031012 A CN117031012 A CN 117031012A
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iga1
concentration
solution
buffer solution
calibrator
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张乐之
吴敏华
陈学军
唐小利
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Hangzhou Kangzhi Biomedical Co ltd
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Hangzhou Kangzhi Biomedical Co ltd
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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    • G—PHYSICS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract

The invention relates to a Gd-IgA1 chemiluminescence quantitative determination kit which has high sensitivity and small error, and can specifically and quantitatively detect Gd-lgA1 content in body fluid, thereby bringing convenience to clinical diagnosis and treatment of IgAN, and comprises a magnetic particle reagent, an enzyme conjugate reagent, a calibrator, a quality control product, a substrate solution and a cleaning solution which are respectively prepared, wherein the magnetic particle reagent, the enzyme conjugate reagent, the calibrator, the quality control product, the substrate solution and the cleaning solution are independently arranged in a packaging container; the magnetic particle reagent is prepared by coupling a Gd-IgA1 capture antibody with magnetic particles, wherein the Gd-IgA1 capture antibody is coupled on the surfaces of the magnetic particles through a chemical crosslinking agent in a crosslinking buffer solution; the enzyme conjugate reagent is prepared by coupling a Gd-IgA1 detection antibody with alkaline phosphatase, and the Gd-IgA1 detection antibody is coupled with the alkaline phosphatase in a crosslinking buffer solution through a chemical crosslinking agent. The advantages are that: the Gd-IgA1 chemiluminescent quantitative determination kit has the advantages of high detection sensitivity and small detection error, and can specifically and quantitatively detect the Gd-lgA1 content in body fluid.

Description

Gd-IgA1 chemiluminescence quantitative determination kit
Technical Field
The invention belongs to the technical field of in-vitro diagnosis and immunodetection, and particularly relates to a Gd-IgA1 chemiluminescent quantitative determination kit.
Background
IgA nephropathy (IgA Nephnosis, igAN) is the most common chronic glomerulonephritis type, the global annual incidence rate of adults is higher than 2.5/10 ten thousand, asian population is more common, about 500 ten thousand people in China are plagued by IgAN, and mainly young and strong people, 30% -50% of IgAN patients can progress to uremia within 20-30 years of incidence if timely detection, diagnosis and effective intervention cannot be performed, and heavy burden is brought to individuals, families and society of patients.
The "quadruple hit" hypothesis suggests that the key initiation factor for IgAN is the deregulation of the immune response of the body after encountering causes (various infections, allergies, etc.), the intestinal mucosa produces pathogenic Galactose-deficient-lgA 1 (Gd-lgA 1) molecules (first hit); further recognizing IgG or lgA autoimmune antibody production by Gd-lgA1 (second hit); as the circulating immune complex continues to form (third hit); the immune complex eventually deposits the glomerular basement membrane area, induces an immune response, activates complement and other mediators, and causes inflammation to cause glomerular injury (fourth hit).
Currently, the definitive diagnosis and efficacy assessment of IgAN still relies on kidney aspiration biopsy, but it is an invasive examination that poses a risk of infection while causing pain to the patient. In addition, kidney puncture biopsy material limitations do not allow for an overall assessment of kidney damage; repeated renal biopsy monitoring of disease activity and evaluation of drug treatment effects is also impractical. Therefore, the development of noninvasive detection means has very important clinical value for the diagnosis and curative effect observation of IgAN.
Chinese patent (CN 114478787A) discloses an anti-Gd-lgA 1 monoclonal antibody and an ELISA kit for IgA nephropathy auxiliary diagnosis, which are used for detecting the Gd-lgA1 content in body fluid. In view of the fact that the quantitative determination method of the ELISA kit is unstable and has large error (CV is generally more than 30%), the quantitative determination kit is invented by the fact that the quantitative determination method is eliminated by national inspection science of three hospitals.
Disclosure of Invention
The invention aims to provide a Gd-IgA1 chemiluminescence quantitative determination kit, which solves the problems of the prior art, realizes high sensitivity, small error and specific quantitative determination of Gd-lgA1 content in body fluid, and thereby brings convenience to clinical diagnosis and treatment of IgAN.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention relates to a Gd-IgA1 chemiluminescent quantitative determination kit, which comprises a magnetic particle reagent, an enzyme conjugate reagent, a calibrator, a quality control product, a substrate solution and a cleaning solution which are respectively prepared, wherein the magnetic particle reagent, the enzyme conjugate reagent, the calibrator, the quality control product, the substrate solution and the cleaning solution are independently arranged in a packaging container; the magnetic particle reagent is prepared by coupling a Gd-IgA1 capture antibody with magnetic particles, wherein the Gd-IgA1 capture antibody is coupled on the surfaces of the magnetic particles through a chemical crosslinking agent in a crosslinking buffer solution; the enzyme conjugate reagent is prepared by coupling a Gd-IgA1 detection antibody with alkaline phosphatase, and the Gd-IgA1 detection antibody is coupled with the alkaline phosphatase in a crosslinking buffer solution through a chemical crosslinking agent.
Preferably, the chemical crosslinking agent is at least one or a mixture of more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide and 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester; the cross-linking buffer solution is one of MES buffer solution, PBS buffer solution, HEPES buffer solution and boric acid buffer solution which do not contain amino groups, and the pH value of the cross-linking buffer solution is 5.0-8.0.
Preferably, the magnetic particle reagent is prepared by the following method:
(1) Adding magnetic particles containing carboxyl into 50mmol-200mmol of crosslinking buffer solution, wherein the pH value of the crosslinking buffer solution is 5.0-8.0, enabling the concentration of the magnetic particles to be 0.1% -2.0%, adding EDC with the concentration of 0.02% -0.1% and NHS with the concentration of 0.05% -0.2%, stirring at room temperature for 10 min-15 min, standing in a magnetic separator for 1 min-3 min, discarding the supernatant, and resuspending with the crosslinking buffer solution, and obtaining an activated magnetic particle solution with the concentration of 0.05% -0.5% after resuspension;
(2) Adding a Gd-IgA1 capture antibody into the activated magnetic particle solution, wherein the mass ratio of the added Gd-IgA1 capture antibody to the magnetic particles is 0.5-2.0 mg/10 mg;
(3) Fully and uniformly mixing magnetic particles and Gd-IgA1 capture antibody, incubating at room temperature for 2-4 hours, adding 1-5% bovine serum albumin solution, incubating at room temperature for 1-2 hours, standing in a magnetic separator for 1-3 minutes, discarding supernatant, washing with magnetic particle preservation solution for one time, re-suspending and preserving with magnetic particle preservation solution, and diluting with diluent to the working concentration to obtain the magnetic particle reagent.
Preferably, the enzyme conjugate reagent is prepared by the following method:
(1) Dissolving alkaline phosphatase with 50-200 mmol of cross-linking buffer solution, wherein the pH value of the cross-linking buffer solution is 5.0-8.0, adding SMCC, uniformly mixing, keeping the temperature at 25 ℃ in the uniformly mixing process for 20-60 min, desalting for 2-3 times with a 30kD ultrafiltration tube, and finally re-dissolving with the cross-linking buffer solution;
(2) Dissolving Gd-IgA1 detection antibody with 50mmol-200mmol of crosslinking buffer solution, wherein the pH value of the crosslinking buffer solution is 5.0-8.0, adding Traut's Reagent, and uniformly mixing, wherein the temperature in the uniformly mixing process is kept at 25 ℃ for 20-60 min, desalting for 2-3 times by using a 100kD ultrafiltration tube, and finally re-dissolving by using the crosslinking buffer solution;
(3) Mixing 2 reaction liquids, reacting for 16-18 hours at the temperature of 2-8 ℃ after uniform mixing, adding a preservation solution, namely an enzyme conjugate stock solution, and diluting to the working concentration by using a diluent to obtain the enzyme conjugate reagent.
Preferably, the calibrator is prepared by the following method:
1.0ug of Gd-IgA1 protein is taken and added into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 1000ug/L, and the Gd-IgA1 protein solution is used as a calibrator S7; then, the calibrator S7 was diluted with a dilution gradient to prepare a calibrator S6 having a concentration of 500ug/L, a calibrator S5 having a concentration of 250ug/L, a calibrator S4 having a concentration of 125ug/L, a calibrator S3 having a concentration of 62.5ug/L, a calibrator S2 having a concentration of 31.25ug/L, and a calibrator S1 having a concentration of 15.625ug/L, and the dilution was used as a calibrator S0 having a concentration of 0.
Preferably, the quality control product is prepared by the following method:
adding 0.5ug of Gd-IgA1 protein into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 500ug/L as a quality control product C1; then, the quality control product C1 is diluted into a quality control product C2 with the concentration of 200ug/L and a quality control product C3 with the concentration of 50ug/L by using a diluent in a gradient way.
Preferably, the diluent is PBS buffer solution added with bovine serum albumin, tween-20 and Proclin300, the concentration of the PBS buffer solution is 10-100mM, the pH of the PBS buffer solution is 6.0-8.0, the content of the bovine serum albumin is 0.5-1.0% of the total mass of the diluent, the content of the Tween-20 is 0.02-0.1% of the total mass of the diluent, and the content of the Proclin300 is 0.02-0.1% of the total mass of the diluent.
Preferably, the substrate solution is prepared by dissolving AMPPD in a substrate buffer solution; the substrate buffer comprises the following components: purified water, tris (hydroxymethyl) aminomethane, 2-amino-2-methyl-1-propanol, magnesium sulfate and zinc chloride and sodium dodecyl sulfonate, wherein the concentration of tris (hydroxymethyl) aminomethane in the substrate buffer is 10mM-100mM, the concentration of 2-amino-2-methyl-1-propanol in the substrate buffer is 0.01M-0.5M, the concentration of magnesium sulfate in the substrate buffer is 0.02-0.2M, the concentration of zinc chloride in the substrate buffer is 0.02-0.2M, and the concentration of sodium dodecyl sulfonate in the substrate buffer is 0.05-0.5M.
Preferably, the cleaning solution is prepared by the following method:
1000g of purified water is weighed, 60.7g of tris (hydroxymethyl) aminomethane and 5.0g of sodium chloride are added, stirring and dissolving are carried out, HCl is used for regulating the pH value to 8.0, 0.5ml of Tween-20 and 0.25ml of Proclin300 are added, and stirring is carried out uniformly, thus obtaining the cleaning liquid.
Preferably, the magnetic particles are immunomagnetic particles, the surfaces of the immunomagnetic particles contain carboxyl groups, and the particle size of the immunomagnetic particles is 1um-3um.
The invention has the following beneficial effects:
1. the Gd-IgA1 chemiluminescence quantitative determination kit has the advantages of high detection sensitivity and small detection error, and can specifically and quantitatively detect the Gd-lgA1 content in body fluid, thereby bringing convenience to clinical diagnosis and treatment of IgAN, and in addition, the detection requires a small sample size.
2. A Gd-IgA1 chemiluminescent quantitative determination kit, which has a determination variation coefficient CV of 4.53-7.29% when being subjected to a precision test; when the sensitivity test is carried out, the detection limit is 2.826ug/L; when the accuracy test is carried out, the recovery rate is between 85 and 115 percent; when the stability test is carried out, the reagent can be stored and used for a long time (12 months) at the temperature of 2-8 ℃, has good stability, can be stored and used for a short time (7 days) at the temperature of 37 ℃, and has good heat stability.
3. The patent published by the invention is a Gd-IgA1 chemiluminescent quantitative determination kit, which is still blank in China at present, and a medicine supervision bureau does not have a wholesale product, namely fills the blank of the detection technology in China.
Drawings
FIG. 1 is a calibration curve of a calibrator in a Gd-IgA1 chemiluminescent quantification assay kit.
Detailed Description
Example 1. A Gd-IgA1 chemiluminescent quantitative determination kit comprises a magnetic particle reagent, an enzyme conjugate reagent, a calibrator, a quality control product, a substrate solution and a cleaning solution which are respectively prepared, wherein the magnetic particle reagent, the enzyme conjugate reagent, the calibrator, the quality control product, the substrate solution and the cleaning solution are independently arranged in a packaging container; the magnetic particle reagent is prepared by coupling a Gd-IgA1 capture antibody with magnetic particles, wherein the Gd-IgA1 capture antibody is coupled on the surfaces of the magnetic particles through a chemical crosslinking agent in a crosslinking buffer solution (namely, the magnetic particle reagent couples the Gd-IgA1 capture antibody on the surfaces of the magnetic particles through a chemical crosslinking method, and the chemical crosslinking method is carried out in the crosslinking buffer solution through the chemical crosslinking agent); the enzyme conjugate reagent is prepared by coupling a Gd-IgA1 detection antibody with alkaline phosphatase, and the Gd-IgA1 detection antibody is coupled with the alkaline phosphatase in a crosslinking buffer solution through a chemical crosslinking agent. (i.e., the enzyme conjugate reagent is a Gd-IgA1 detection antibody conjugated to alkaline phosphatase (AKP) by chemical cross-linking in a cross-linking buffer by a chemical cross-linking agent).
The chemical cross-linking agent is at least one or more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide and 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester; the cross-linking buffer solution is one of MES buffer solution, PBS buffer solution, HEPES buffer solution and boric acid buffer solution which do not contain amino groups, and the pH value of the cross-linking buffer solution is 5.0-8.0.
The magnetic particle reagent is prepared by the following method:
(1) Adding magnetic particles containing carboxyl into 50mmol-200mmol of crosslinking buffer solution, wherein the pH value of the crosslinking buffer solution is 5.0-8.0, enabling the concentration of the magnetic particles to be 0.1% -2.0%, adding EDC with the concentration of 0.02% -0.1% and NHS with the concentration of 0.05% -0.2%, stirring at room temperature for 10 min-15 min, standing in a magnetic separator for 1 min-3 min, discarding the supernatant, and resuspending with the crosslinking buffer solution, and obtaining an activated magnetic particle solution with the concentration of 0.05% -0.5% after resuspension;
(2) Adding Gd-IgA1 capture antibody into activated magnetic particle solution, wherein the mass ratio of the added Gd-IgA1 capture antibody to the magnetic particles is 0.5-2.0mgGd-IgA1 capture antibodies)∶10mg(Magnetic particles)；
(3) Fully and uniformly mixing magnetic particles and Gd-IgA1 capture antibody, incubating at room temperature for 2-4 hours, adding 1-5% bovine serum albumin solution, incubating at room temperature for 1-2 hours, standing in a magnetic separator for 1-3 minutes, discarding supernatant, washing with magnetic particle preservation solution for one time, re-suspending and preserving with magnetic particle preservation solution, and diluting with diluent to the working concentration to obtain the magnetic particle reagent.
The enzyme conjugate reagent is prepared by the following method:
(1) Dissolving alkaline phosphatase with 50-200 mmol of cross-linking buffer solution, wherein the pH value of the cross-linking buffer solution is 5.0-8.0, adding SMCC, uniformly mixing, keeping the temperature at 25 ℃ in the uniformly mixing process for 20-60 min, desalting for 2-3 times with a 30kD ultrafiltration tube, and finally re-dissolving with the cross-linking buffer solution;
(2) Dissolving Gd-IgA1 detection antibody with 50mmol-200mmol of crosslinking buffer solution, wherein the pH value of the crosslinking buffer solution is 5.0-8.0, adding Traut's Reagent, and uniformly mixing, wherein the temperature in the uniformly mixing process is kept at 25 ℃ for 20-60 min, desalting for 2-3 times by using a 100kD ultrafiltration tube, and finally re-dissolving by using the crosslinking buffer solution;
(3) Mixing 2 reaction liquids, reacting for 16-18 hours at the temperature of 2-8 ℃ after uniform mixing, adding a preservation solution, namely an enzyme conjugate stock solution, and diluting to the working concentration by using a diluent to obtain the enzyme conjugate reagent.
The calibrator is prepared by the following steps:
1.0ug of Gd-IgA1 protein is taken and added into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 1000ug/L, and the Gd-IgA1 protein solution is used as a calibrator S7; then, the calibrator S7 was diluted with a dilution gradient to prepare a calibrator S6 having a concentration of 500ug/L, a calibrator S5 having a concentration of 250ug/L, a calibrator S4 having a concentration of 125ug/L, a calibrator S3 having a concentration of 62.5ug/L, a calibrator S2 having a concentration of 31.25ug/L, and a calibrator S1 having a concentration of 15.625ug/L, and the dilution was used as a calibrator S0 having a concentration of 0.
The quality control product is prepared by the following method:
adding 0.5ug of Gd-IgA1 protein into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 500ug/L as a quality control product C1; then, the quality control product C1 is diluted into a quality control product C2 with the concentration of 200ug/L and a quality control product C3 with the concentration of 50ug/L by using a diluent in a gradient way.
The dilution liquid is PBS buffer solution added with bovine serum albumin, tween-20 and Proclin300, the concentration of the PBS buffer solution is 10-100mM, the pH value of the PBS buffer solution is 6.0-8.0, the content of the bovine serum albumin is 0.5-1.0% of the total mass of the dilution liquid, the content of the Tween-20 is 0.02-0.1% of the total mass of the dilution liquid, and the content of the Proclin300 is 0.02-0.1% of the total mass of the dilution liquid.
The substrate solution is prepared by dissolving AMPPD in a substrate buffer solution; the substrate buffer comprises the following components: purified water, tris (hydroxymethyl) aminomethane, 2-amino-2-methyl-1-propanol, magnesium sulfate and zinc chloride and sodium dodecyl sulfonate, the concentration of tris (hydroxymethyl) aminomethane in the substrate buffer being 10mM-100mM (mM ismmol/L) The concentration of the 2-amino-2-methyl-1-propanol in the substrate buffer solution is 0.01M-0.5M%M is mol/L) The concentration of magnesium sulfate in the substrate buffer solution is 0.02-0.2M, the concentration of zinc chloride in the substrate buffer solution is 0.02-0.2M, and the concentration of sodium dodecyl sulfate in the substrate buffer solution is 0.05-0.5M.
The substrate solution is prepared by the following method:
1000g of purified water is weighed, 6.07g of tris (hydroxymethyl) aminomethane is added, after dissolution, 17.83g of 2-amino-2-methyl-1-propanol, 6.02g of magnesium sulfate, 6.82g of zinc chloride and 13.62g of sodium dodecyl sulfate are added, the pH value is adjusted to 9.5, then 0.382mg of AMPPD and 50mg of sodium fluorescein are added, and the substrate solution is obtained after stirring and dissolution.
The cleaning solution is prepared by the following steps:
1000g of purified water is weighed, 60.7g of tris (hydroxymethyl) aminomethane and 5.0g of sodium chloride are added, stirring and dissolving are carried out, HCl is used for regulating the pH value to 8.0, 0.5ml of Tween-20 and 0.25ml of Proclin300 are added, and stirring is carried out uniformly, thus obtaining the cleaning liquid.
The magnetic particles are immunomagnetic particles, the surfaces of the immunomagnetic particles contain carboxyl groups, and the particle size of the immunomagnetic particles is 1um-3um.
Example 2: on the basis of example 1. Preparation of Gd-IgA1 chemiluminescent quantitative determination kit
(1) Preparation of magnetic microparticle reagents
10mg of magnetic particles containing carboxyl are taken, added into 1.0ml of 50mmol cross-linking buffer solution with pH of 8.0, vortex mixed uniformly, then 50ug of EDC and 100ug of NHS are added, vortex mixed uniformly, activated for 15 minutes at room temperature, then the mixture is placed in a magnetic separator for 1-3 minutes, the supernatant is discarded, and 1.0ml of cross-linking buffer solution is used for resuspension, thus obtaining activated magnetic particle solution after resuspension;
adding 0.5mg of Gd-IgA1 capture antibody into the activated magnetic particle solution, vortex mixing uniformly, vibrating at room temperature for incubation for 2 hours, adding 0.5ml of 2% bovine serum albumin solution after incubation is finished, incubating at room temperature for 1 hour, standing in a magnetic separator for 1-3 minutes, discarding the supernatant, washing once with 1.0ml of magnetic particle preservation solution, and re-suspending and preserving with 1.0ml of magnetic particle preservation solution; and then diluting to the working concentration by using 10ml of diluent, namely the magnetic particle reagent.
(2) Enzyme conjugate reagent preparation
10mg of alkaline phosphatase was dissolved in 1ml of PBS buffer (0.1M, pH 7.2), and 4ul of SMCC was added and mixed at 25℃for 20 minutes. Desalting for 2-3 times with 30kD ultrafiltration tube, and redissolving with 1ml buffer solution;
dissolving 5mg of Gd-IgA1 detection antibody in 1ml of PBS buffer solution (0.1M, pH 8.0), adding 40ul of Traut's Reagent, uniformly mixing at 25 ℃ for 60min, desalting for 2-3 times by using a 100kD ultrafiltration tube, and finally re-dissolving by using 1ml of buffer solution;
and (3) mixing the 2 reaction solutions, uniformly mixing, reacting for 16-18 hours at the temperature of 2-8 ℃, adding a preservation solution, namely an enzyme conjugate stock solution, and diluting to the working concentration by using a diluent to obtain the enzyme conjugate reagent.
(3) Preparation of a calibrator
1.0ug of Gd-IgA1 protein is taken and added into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 1000ug/L, and the Gd-IgA1 protein solution is used as a calibrator S7. Then the mixture is diluted with the diluent in a gradient manner to obtain the calibration material S6-S1 with different gradient concentrations of 500ug/L,250ug/L,125ug/L,62.5ug/L,31.25ug/L and 15.625ug/L, and the diluent is used as the calibration material S0 with 0 concentration.
(4) Preparation of quality control product
Adding 0.5ug of Gd-IgA1 protein into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 500ug/L as a quality control product C1; then the mixture is diluted into two different concentrations of 200ug/L and 50ug/L by a dilution liquid gradient, and the two different concentrations are respectively used as quality control products C2 and C3.
(5) Preparation of the substrate solution
1000g of purified water is weighed, 6.07g of tris (hydroxymethyl) aminomethane is added, after dissolution, 17.83g of 2-amino-2-methyl-1-propanol, 6.02g of magnesium sulfate, 6.82g of zinc chloride and 13.62g of sodium dodecyl sulfate are added, the pH value is adjusted to pH=9.5, then 0.382mg of AMPPD and 50mg of sodium fluorescein are added, and the substrate solution is obtained after stirring and dissolution.
(6) Preparation of cleaning liquid
1000g of purified water is weighed, 60.7g of tris (hydroxymethyl) aminomethane and 5.0g of sodium chloride are added, stirring and dissolving are carried out, HCl is used for regulating the pH value to 8.0, 0.5ml of Tween-20 and 0.25ml of Proclin300 are added, and stirring is carried out uniformly, thus obtaining the cleaning liquid.
Example 3: preparation of a Standard Curve
Taking 2ul of each concentration of calibrator solution in example 2 and 100ul of magnetic particle reagent, adding into a blank microplate, carrying out shaking reaction for 30 minutes at 37 ℃, washing for 5 times by using a magnetic separation plate washer, adding 100ul of enzyme conjugate reagent, carrying out shaking reaction for 30 minutes at 37 ℃, washing for 5 times by using the magnetic separation plate washer, adding 100ul of substrate solution, reading by using a full-automatic chemiluminescence reader, and fitting the signal value and the corresponding concentration value by using a Logistic curve (four parameters), wherein the fitting result is shown in table 1:
calibrator (ug/L) Signal value
0 2697
15.63 4659
31.25 11332
62.5 21448
125 35915
250 71523
500 125130
1000 208738
TABLE 1
The Logistic curve fit (four parameters) is as follows:
logistic curve fitting (four parameter) equation
y＝(A-D)/[1+(x/C)^B]+D
A＝593294.47034
B＝-1.02077
C＝1837.10577
D＝1870.27133
r^2＝0.99971
A calibration curve of the fitted calibration material in the Gd-IgA1 chemiluminescent quantitative determination kit is shown in a figure 1, wherein an X-axis value is a concentration value of the calibration material, and a Y-axis value is a signal value.
Example 4: precision test
The quality control product in example 2 was taken as a sample for testing, and the testing procedure was the same as in example 3. The test was repeated 10 times for each concentration of the sample, the mean (M) and Standard Deviation (SD) of the 10 test results were calculated, and the coefficient of variation (CV%) thereof was calculated.
The coefficient of variation calculation formula:
the results of the measurements are shown in Table 2 below:
# C1：50ug/L C2：200ug/L C3：500ug/L
1 51.82 210.51 525.90
2 45.10 189.81 514.27
3 48.90 214.45 494.06
4 43.09 209.71 483.95
5 52.11 210.03 523.12
6 51.86 190.13 526.06
7 45.10 189.62 483.91
8 49.90 219.93 525.09
9 51.85 210.49 464.18
10 53.10 190.01 524.94
mean M 49.282 203.469 506.548
Standard deviation SD 3.591 12.064 22.963
Coefficient of variation CV 7.29％ 5.93％ 4.53％
TABLE 2
Example 5:sensitivity test
The test was performed using a blank (calibrator dilution) as a sample, and the test procedure was the same as in example 3. The test was repeated 20 times, the mean value (M) and Standard Deviation (SD) of the blank response (signal value) were calculated, and (m+2sd) was substituted into the fitted standard curve to calculate the concentration result. The measurement results are shown in Table 3 below:
# signal value # Signal value
1 2501 11 1520
2 1750 12 2156
3 2137 13 2032
4 2094 14 2100
5 1687 15 1836
6 2082 16 2073
7 2634 17 1975
8 1961 18 2167
9 2006 19 2746
10 2046 20 2121
Mean M 2081.2 Standard deviation SD 291.75
Coefficient of variation CV 14.02％ M+2SD 2664.69
Detection limit (ug/L) 2.826
TABLE 3 Table 3
Example 6: accuracy test
Taking 0.5ug of a calibrator raw material Gd-IgA1 protein, adding the calibrator raw material Gd-IgA1 protein into 1.0ml of diluent to prepare a high-concentration sample (500 ug/L), and diluting the high-concentration sample with 100X diluted normal human serum to prepare 4 recovered samples with different concentration values, wherein the preparation mode is shown in Table 4:
TABLE 4 Table 4
High concentration samples, 100X diluted serum and recovered samples were tested separately, and the test procedure was the same as in example 3. Each sample was tested 3 times repeatedly and its recovery was calculated to be between 85-115% and the results are shown in table 5.
TABLE 5
Example 7: stability test
The reagents of example 2 were stored at 2-8deg.C and assayed with quality controls at 0 month, 3 months, 6 months, and 12 months, respectively. The test procedure was the same as in example 3. The measurement results are shown in Table 6 below. The results show that the kit provided by the invention has good stability.
TABLE 6
The reagent of example 2 was stored in an oven at 37℃and assayed with quality control on days 1, 3, 5 and 7, respectively. The test procedure was the same as in example 3. The measurement results are shown in Table 7 below.
The results show that the kit provided by the invention has good thermal stability.
# C1：50ug/L C2：200ug/L C3：500ug/L
0 52.22 207.39 525.32
37 ℃ for 1 day 49.10 197.81 514.27
37 ℃ for 3 days 48.00 189.45 493.06
37 ℃ for 5 days 47.38 183.37 482.72
37 ℃ for 7 days 45.93 179.18 472.16
Reducing the amplitude at 37 ℃ for 1 day -5.98％ -4.62％ -2.10％
Reducing the amplitude at 37 ℃ for 3 days -8.09％ -8.65％ -6.14％
Reducing the amplitude at 37 ℃ for 5 days -9.27％ -11.58％ -8.11％
Reducing the amplitude at 37 ℃ for 7 days -12.04％ -13.60％ -10.12％
TABLE 7
It should be understood that: although the above embodiments describe the design concept of the present invention in more detail, these descriptions are merely descriptions of the design concept of the present invention, and not limitations on the design concept of the present invention, and any combination, addition or modification not exceeding the design concept of the present invention falls within the scope of the present invention.

Claims (10)

1. The Gd-IgA1 chemiluminescent quantitative determination kit is characterized by comprising a magnetic particle reagent, an enzyme conjugate reagent, a calibrator, a quality control product, a substrate solution and a cleaning solution which are respectively prepared, wherein the magnetic particle reagent, the enzyme conjugate reagent, the calibrator, the quality control product, the substrate solution and the cleaning solution are independently arranged in a packaging container; the magnetic particle reagent is prepared by coupling a Gd-IgA1 capture antibody with magnetic particles, wherein the Gd-IgA1 capture antibody is coupled on the surfaces of the magnetic particles through a chemical crosslinking agent in a crosslinking buffer solution; the enzyme conjugate reagent is prepared by coupling a Gd-IgA1 detection antibody with alkaline phosphatase, and the Gd-IgA1 detection antibody is coupled with the alkaline phosphatase in a crosslinking buffer solution through a chemical crosslinking agent.
2. The Gd-IgA1 chemiluminescent quantification kit of claim 1 wherein the chemical cross-linking agent is at least one or a mixture of two or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester; the cross-linking buffer solution is one of MES buffer solution, PBS buffer solution, HEPES buffer solution and boric acid buffer solution which do not contain amino groups, and the pH value of the cross-linking buffer solution is 5.0-8.0.
3. The Gd-IgA1 chemiluminescent quantification kit of claim 1 wherein the magnetic particle reagent is prepared by the following method:
(1) Adding magnetic particles containing carboxyl into 50mmol-200mmol of crosslinking buffer solution, wherein the pH value of the crosslinking buffer solution is 5.0-8.0, enabling the concentration of the magnetic particles to be 0.1% -2.0%, adding EDC with the concentration of 0.02% -0.1% and NHS with the concentration of 0.05% -0.2%, stirring at room temperature for 10 min-15 min, standing in a magnetic separator for 1 min-3 min, discarding the supernatant, and resuspending with the crosslinking buffer solution, and obtaining an activated magnetic particle solution with the concentration of 0.05% -0.5% after resuspension;
(2) Adding a Gd-IgA1 capture antibody into the activated magnetic particle solution, wherein the mass ratio of the added Gd-IgA1 capture antibody to the magnetic particles is 0.5-2.0 mg/10 mg;
(3) Fully and uniformly mixing magnetic particles and Gd-IgA1 capture antibody, incubating at room temperature for 2-4 hours, adding 1-5% bovine serum albumin solution, incubating at room temperature for 1-2 hours, standing in a magnetic separator for 1-3 minutes, discarding supernatant, washing with magnetic particle preservation solution for one time, re-suspending and preserving with magnetic particle preservation solution, and diluting with diluent to the working concentration to obtain the magnetic particle reagent.
4. The Gd-IgA1 chemiluminescent quantification assay kit of claim 1 wherein the enzyme conjugate reagent is prepared by the following method:
(1) Dissolving alkaline phosphatase with 50-200 mmol of cross-linking buffer solution, wherein the pH value of the cross-linking buffer solution is 5.0-8.0, adding SMCC, uniformly mixing, keeping the temperature at 25 ℃ in the uniformly mixing process for 20-60 min, desalting for 2-3 times with a 30kD ultrafiltration tube, and finally re-dissolving with the cross-linking buffer solution;
(2) Dissolving Gd-IgA1 detection antibody with 50mmol-200mmol of crosslinking buffer solution, wherein the pH value of the crosslinking buffer solution is 5.0-8.0, adding Traut's Reagent, and uniformly mixing, wherein the temperature in the uniformly mixing process is kept at 25 ℃ for 20-60 min, desalting for 2-3 times by using a 100kD ultrafiltration tube, and finally re-dissolving by using the crosslinking buffer solution;
(3) Mixing 2 reaction liquids, reacting for 16-18 hours at the temperature of 2-8 ℃ after uniform mixing, adding a preservation solution, namely an enzyme conjugate stock solution, and diluting to the working concentration by using a diluent to obtain the enzyme conjugate reagent.
5. The Gd-IgA1 chemiluminescent quantification kit of claim 1 wherein the calibrator is prepared by the method comprising:
1.0ug of Gd-IgA1 protein is taken and added into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 1000ug/L, and the Gd-IgA1 protein solution is used as a calibrator S7; then, the calibrator S7 was diluted with a dilution gradient to prepare a calibrator S6 having a concentration of 500ug/L, a calibrator S5 having a concentration of 250ug/L, a calibrator S4 having a concentration of 125ug/L, a calibrator S3 having a concentration of 62.5ug/L, a calibrator S2 having a concentration of 31.25ug/L, and a calibrator S1 having a concentration of 15.625ug/L, and the dilution was used as a calibrator S0 having a concentration of 0.
6. The Gd-IgA1 chemiluminescent quantification assay kit according to claim 1 wherein the quality control product is prepared by the following method:
adding 0.5ug of Gd-IgA1 protein into 1.0ml of diluent to prepare a Gd-IgA1 protein solution with the concentration of 500ug/L as a quality control product C1; then, the quality control product C1 is diluted into a quality control product C2 with the concentration of 200ug/L and a quality control product C3 with the concentration of 50ug/L by using a diluent in a gradient way.
7. The Gd-IgA1 chemiluminescent quantitative determination kit according to claim 3, 4, 5 or 6 wherein the diluent is a PBS buffer containing 10-100mM of bovine serum albumin, tween-20 and Proclin300, wherein the PBS buffer has a pH of 6.0-8.0, wherein the bovine serum albumin is 0.5-1.0% of the total mass of the diluent, wherein the Tween-20 is 0.02-0.1% of the total mass of the diluent, and wherein the Proclin300 is 0.02-0.1% of the total mass of the diluent.
8. The Gd-IgA1 chemiluminescent quantification assay kit of claim 1 wherein the substrate solution is formulated from AMPPD dissolved in a substrate buffer solution; the substrate buffer comprises the following components: purified water, tris (hydroxymethyl) aminomethane, 2-amino-2-methyl-1-propanol, magnesium sulfate and zinc chloride and sodium dodecyl sulfonate, wherein the concentration of tris (hydroxymethyl) aminomethane in the substrate buffer is 10mM-100mM, the concentration of 2-amino-2-methyl-1-propanol in the substrate buffer is 0.01M-0.5M, the concentration of magnesium sulfate in the substrate buffer is 0.02-0.2M, the concentration of zinc chloride in the substrate buffer is 0.02-0.2M, and the concentration of sodium dodecyl sulfonate in the substrate buffer is 0.05-0.5M.
9. The Gd-IgA1 chemiluminescent quantification kit according to claim 1 wherein the cleaning solution is prepared by the following method:
1000g of purified water is weighed, 60.7g of tris (hydroxymethyl) aminomethane and 5.0g of sodium chloride are added, stirring and dissolving are carried out, HCl is used for regulating the pH value to 8.0, 0.5ml of Tween-20 and 0.25ml of Proclin300 are added, and stirring is carried out uniformly, thus obtaining the cleaning liquid.
10. The Gd-IgA1 chemiluminescent quantification assay kit according to claim 1 wherein the magnetic particles are immunomagnetic particles and the surface of the immunomagnetic particles contains carboxyl groups, the immunomagnetic particles having a particle size of 1um to 3um.
CN202310982989.XA 2023-08-07 2023-08-07 Gd-IgA1 chemiluminescence quantitative determination kit Pending CN117031012A (en)

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