CN117368466A - Kit for measuring urine binding globin by using biochemical analyzer and preparation method thereof - Google Patents
Kit for measuring urine binding globin by using biochemical analyzer and preparation method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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
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Abstract
The invention discloses a preparation method of a kit for measuring urine binding globin by using a biochemical analyzer, which is prepared by screening paired specific monoclonal antibodies with balanced binding force against Hp three protein phenotypes, respectively coupling double-particle-size latex microspheres, and further performing turbidimetry platform reactivity verification. The kit prepared by the preparation method is used for performing transmittance turbidity analysis by a biochemical analyzer, meets the requirement of simultaneously providing creatinine ratio HCR, can be complementarily combined with urine microalbumin creatinine ratio ACR on biochemistry, can avoid missing detection of patients with positive genotypes, can also consider sensitivity and wider linear range under a transmittance turbidity scheme, and improves the detection rate of diabetes kidney diseases.
Description
Technical Field
The invention relates to the technical field of biological protein detection, in particular to a kit for measuring urine-binding globin by using a biochemical analyzer and a preparation method thereof.
Background
The binding globin (Hp) is also called Haptoglobin, and the gene is located on human chromosome 16q22.Hp molecules are tetramers composed of two beta and two alpha chains linked by disulfide bonds, an acidic glycoprotein that is widely found in human and many mammalian blood and other body fluids. The concentration in human plasma circulation is 0.3-3 mg/mL. Human Hp has two alleles Hp1 and Hp2, which can form 3 major genotypes: hp1-1, hp2-1 and Hp2-2, the 3 genotypes show multimers of different structures, individuals of the Hp1-1 genotype are dimers, individuals of the Hp2-1 genotype are linear polymers, and individuals of the Hp2-2 genotype are cyclic polymers.
Comprehensive clinical related research results, urine Hp may be the first biomarker to be a predictor of kidney disease progression risk over proteinuria, providing a more accurate method for predicting Diabetes Kidney Disease (DKD) progression. The combined diagnostic and prognostic role of urine HP and proteinuria may be the direction of further investigation. Related studies have shown that the combined use of ACR (urinary microalbumin creatinine ratio) and HCR (conjugated globin creatinine ratio) increases the sensitivity of predicting early renal decline in diabetic patients.
The existing urine Hp detection reagent on the market at present adopts an immunochromatography method, but the immunochromatography method is low in repeatability, is unfavorable for accurate detection, is narrow in linearity, is 20-300 ng/ml, is generally high in urine Hp of a patient with the diagnosis of DKD, accounts for most of the urine Hp, is even 1000ng/ml, and is too narrow in linear interval in the scheme, so that clinical application is unfavorable.
Other methodologies are not yet marketed in the research at present, but some manufacturers are researching the scattering nephelometry, but a specific protein analyzer is required to be equipped, so that project development is not facilitated, and the polyclonal antibody (the serum detection order of magnitude is mg/ml) for detecting serum haptoglobin is adopted in the current research, so that the similar binding force on Hp three protein phenotypes at the ng/ml detection level cannot be ensured, and the difference of multiple antibodies in each batch cannot be ensured. The chemiluminescence method has good detection precision, but can only be used for detection by an immunoassay method, can not be combined with creatinine to obtain HCR ratio, has inaccurate diagnosis result, can not be combined with ACR for detection, and can not achieve the effect of improving DKD detection rate by combined detection.
If urine Hp can be detected on a biochemical analyzer, the method not only meets the requirement of high precision, but also can be combined with a creatine oxidase method which is an optimal creatinine test method to obtain HCR ratio, and is combined with an protogenic project ACR to improve the detection rate of DKD, so that the method is a platform which is most suitable for urine Hp detection.
Therefore, there is a need to provide a novel kit for measuring urine-binding globin using a biochemical analyzer and a preparation method thereof to solve the above problems.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit for measuring urine-binding globin by using a biochemical analyzer and a preparation method thereof, which can be used for testing on the biochemical analyzer, meet the requirement of simultaneously having creatinine ratio HCR, and can be complementarily combined with urine microalbumin creatinine ratio ACR on biochemistry, thereby improving the detection rate of diabetic kidney diseases.
In order to solve the technical problems, the invention adopts a technical scheme that: the kit is prepared by screening paired specific monoclonal antibodies with balanced binding force for Hp three protein phenotypes, respectively coupling the paired specific monoclonal antibodies with double-particle-size latex microspheres, and further performing turbidimetry platform reactivity verification.
In a preferred embodiment of the invention, the preparation method comprises the following steps:
s1: preparing an anti-Hp monoclonal antibody;
s2: phenotypic response primary screening:
s201: cross pairing the obtained anti-Hp monoclonal antibodies, detecting mixed phenotype human Hp antigen by ELISA, and screening out positive pairing;
s202: testing the pure proteins of three phenotypes of Hp1-1, hp2-1 and Hp2-2 respectively for the screened positive pairing, and screening out the related pairing with the binding force closest to the proteins of the three phenotypes according to the reaction result;
s3: preparation of urine Hp biochemical reagent:
s301: coupling the paired antibodies screened in the step S202 with double-particle-diameter latex microspheres to obtain a plurality of sensitization latices with different particle diameters;
s302: mixing the paired size particle size sensitization latex two by two, preparing turbidimetric reagent R2, and preparing a biochemical kit by the turbidimetric reagent R1 for providing a reaction environment;
s4: and (3) verifying the reaction effect:
and verifying the reactivity of the biochemical kit to each Hp phenotype, screening out an optimal pairing scheme after coupling the latex microspheres, and optimizing the reagent prepared by the pairing scheme to ensure that the biochemical kit meets the optimal performance requirement of urine Hp detection.
In a preferred embodiment of the present invention, the specific steps of step S1 include:
s101: mixing phenotype human Hp pure antigen to immunize female BALB/c mice, taking spleen of the immunized mice to prepare cell suspension, fusing myeloma cell SP2/0, and re-suspending HAT culture solution;
s102: selectively culturing hybridoma cells, coating mixed phenotype human Hp antigen by indirect ELISA to measure positive holes, subcloning until the positive hole rate is 100%, screening positive holes with high antibody secretion for injecting female BALB/c mice, collecting peritoneal effusion, and extracting and purifying anti-Hp monoclonal antibodies.
In a preferred embodiment of the present invention, in step S3, the dual-particle latex microspheres are 180nm to 240nm large-particle latex microspheres and 60nm to 80nm small-particle latex microspheres.
In a preferred embodiment of the present invention, in step S4, the turbidimetric reagent R2 includes the following components in the optimized reagent:
10mM, pH7.5 HEPES, 0.2% Hp-sensitized latex, 0.05% Tween-20, 2% sucrose, 1% NaCl, 1% BSA and 0.05% ProClin300.
In a preferred embodiment of the present invention, in step S4, the optimized reagent R1 includes the following components:
10mM, pH7.5 HEPES, 3% PEG6000, 1% NaCl, 0.05% ProClin300.
In order to solve the technical problems, the invention adopts another technical scheme that: there is provided a kit prepared by the method for preparing a kit for biochemical analyzer for assaying urine-binding globin as described in any one of the above.
In a preferred embodiment of the invention, the kit further comprises a calibrator comprising a base solution of 10mM, pH7.4 PBS containing 1% BSA, 3% sucrose, 1% trehalose, 0.05% ProClin300, hp mix antigen.
The beneficial effects of the invention are as follows: the preparation method of the biochemical kit for determining urine-binding globin, which is described by the invention, not only can avoid the omission of detection on patients with positive genotypes, but also can give consideration to sensitivity and a wider linear range under a transmittance turbidity scheme, the detection range can reach at least 20ng/ml to 2000ng/ml, the wide range of clinical urine-binding globin detection is satisfied, and the requirement of simultaneously having creatinine ratio HCR is satisfied by adopting a transmission scheme on a biochemical analyzer, and the biochemical kit can be complementarily combined with urine microalbumin creatinine ratio ACR on biochemistry, thereby improving the detection rate of diabetic kidney diseases.
At present, no urine Hp reagent which can be measured on a biochemical analyzer is found on the market, so the invention has the following advantages:
(1) The method has balanced detection rates for three Hp phenotypes, and avoids missed detection of Hp patients with different genotypes;
(2) The linear range can reach at least 20ng/ml to 2000ng/ml, and the wide range of detection of normal people and DKD (direct current) people is satisfied;
(3) Can be directly used on a biochemical analyzer, can meet the requirement of simultaneously measuring the HCR ratio of creatinine, can be complementarily combined with ACR in biochemistry, and improves the detection rate of DKD.
Drawings
FIG. 1 is a graph showing correlation coefficients of Hp high value sample solution and diluted sample solution calculated test values and dilution ratios.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
A kit for measuring urine-binding globin by biochemical analyzer is prepared through screening the paired specific monoclonal antibodies with balanced binding force to Hp three protein phenotypes, respectively coupling with dual-particle-diameter latex microspheres, and further verifying the reactivity of turbidimetric platform. The preparation method specifically comprises the following steps:
s1: preparing an anti-Hp monoclonal antibody:
s101: mixing phenotype human Hp pure antigen to immunize 6-8 Zhou Cixing BALB/c mice, taking spleen of the immunized mice to prepare cell suspension, fusing myeloma cell SP2/0, and re-suspending HAT culture solution;
s102: selectively culturing hybridoma cells, coating mixed phenotype human Hp antigen by indirect ELISA (enzyme-linked immunosorbent assay) to measure positive holes, subcloning until the positive hole is 100%, screening positive holes with high antibody secretion for injecting female BALB/c mice, and collecting peritoneal effusion to extract and purify anti-Hp monoclonal antibodies.
S2: phenotypic reactivity primary screening:
s201: the obtained monoclonal antibodies are crossed and paired, ELISA is used for detecting mixed phenotype human Hp antigen, and pairing with stronger positive is screened out;
s202: and testing the pure proteins of the three phenotypes of Hp1-1, hp2-1 and Hp2-2 respectively on the screened positive pairing, and screening out the related pairing with the binding force closest to the proteins of the three phenotypes according to the reaction result.
S3: preparation of urine Hp biochemical reagent:
s301: coupling large and small latex microspheres with the screened pairing antibodies respectively, wherein each pairing monoclonal antibody is coupled with one large microsphere and one small microsphere to obtain a plurality of sensitization latex with different particle sizes;
preferably, the dual-particle-diameter latex particles adopt 180 nm-240 nm large-particle-diameter latex microspheres and 60 nm-80 nm small-particle-diameter latex microspheres.
S302: the paired size-diameter sensitive latex is mixed two by two to prepare turbidimetric reagent R2, and the turbidimetric reagent R2 and the R1 reagent for providing a reaction environment are prepared into a biochemical kit.
S4: and (3) verifying the reaction effect:
s401: verifying the reactivity of the biochemical kit to each Hp phenotype, and screening out the optimal pairing scheme after coupling the latex microspheres;
s402: the reagent prepared by the pairing scheme is optimized, so that the biochemical kit meets the optimal performance requirement of urine Hp detection.
According to the technical scheme, firstly, anti-Hp monoclonal antibodies are prepared, pairing preliminary screening is carried out, hp1-1, hp2-1 and Hp2-2 phenotype reactivities are checked, and after Hp monoclonal antibodies suitable for turbidimetry platform are screened out, turbidimetry platform reactivities are further verified, mainly because typing determination results are influenced by two aspects: firstly, because the molecular weight of Hp monomers of each phenotype is different, the test results are different when the same protein quantity is combined; secondly, since the Hp phenotypes form various forms such as dimer, linear polymer and cyclic polymer respectively, and the binding efficacy itself is different when a turbidimetric large structure is formed, reverse verification is performed according to the actual effect.
The biochemical kit prepared by the scheme adopts a monoclonal antibody which is specially screened to ensure that the kit has the most similar binding force to three Hp phenotypes and reduces the inter-batch difference; the sensitivity and linearity of the coupled double-particle-size latex microspheres during transmission analysis are ensured; can be directly used on a biochemical analyzer to form a ratio with creatinine and is combined with ACR for clinical application.
The preparation method and the prepared kit performance of the invention are described in the following with a specific example.
1. Preparation of anti-Hp monoclonal antibodies
(1) Human Hp mixed natural protein (from Sigma, H3536), 7 week female BALB/c mice were immunized with Freund's complete adjuvant, 2 weeks later with Freund's incomplete adjuvant for secondary immunization, and 3 weeks later with no adjuvant for tertiary immunization;
(2) The ascites titer is higher than 1:5120000, the spleen of the immunized mouse is taken, the cell suspension is obtained by grinding and sieving, and the RPMI-1640 culture solution is washed twice after centrifugation.
(3) Washing the cell suspension with myeloma cell SP2/0 (from and a subunit) with 1640 culture solution, water-bathing to 40 ℃, slowly dropwise adding 50% PEG4000 fusion cells at 37 ℃ under slight shaking, stopping dropwise adding 2min of the process, stopping adding 1640 culture solution at 37 ℃, centrifuging, and re-suspending with HAT culture solution;
(4) The hybridoma cells were selectively cultured by adding 100. Mu.l of the fused cells per well to a 96-well plate containing feeder cells. Indirect ELISA is used for coating mixed phenotype human Hp antigen to measure positive holes, and subcloning is carried out by a limiting dilution method until the positive porosity is 100%;
(5) Screening positive holes with high antibody secretion to obtain 15 hybridomas, respectively injecting female BALB/c mice, collecting peritoneal effusion, and extracting and purifying the anti-Hp monoclonal antibody.
2. Phenotypic reactivity primary screening
(1) Purified antibodies of each clone number were cross paired and mixed phenotype human Hp was detected by a double antibody sandwich ELISA to screen for available positive pairing: (1) 10H4-16C12; (2) 13C11-4A5; (3) 4A5-16C12; (4) 4B9-4A5; (5) 8H11-16C12; (6) 3C8-4A5; (7) 8B2-16C12; (8) 1E7-16C12; (9) 17A9-16C12; 15F4-16C12;7E9-16C12；/>16C12-4A5；/>11H3-8B2；/>11H3-1E7；/>11H3-7E9；/>11H3-4A5；
(2) The screened pairs were used to test the pure proteins of the three phenotypes Hp1-1, hp2-1 and Hp2-2 (purchased from Athens Research & Technology, verified by electrophoresis), and the pairs with the most similar binding force to the three phenotypes were selected as 16C12-4A5 and 11H3-4A5.
3. Preparation of urine Hp biochemical reagent
(1) Both 200nm and 70nm carboxyl polystyrene microspheres (LP) were prepared according to LP: EDC: sulfonhs=10:1:1, the activation system was 50mm, ph5.5mes;
(2) 200nm activated LP was centrifuged at 15000rpm, 70nm activated LP was hollow fiber ultrafiltered, and the buffer system was replaced with 50mM, pH6.5 MES, according to LP: ab (antibody) =10:1 ratio was conjugated to three monoclonal antibodies 16C12, 11H3 and 4A5 respectively, and then blocked with Blockmaster CE510 to give 6 different conjugates;
(3) 200nmLP-16C12 and 70nmLP-4A5 are mixed and preserved, 200nmLP-4A5 and 70nmLP-16C12 are mixed and preserved, 200nmLP-11H3 and 70nmLP-4A5 are mixed and preserved, 200nmLP-4A5 and 70nmLP-11H3 are mixed and preserved by using R2 preservation solution, and 4R 2 are obtained after curing at 37 ℃.
Wherein, R2 preservation solution is: 10mM, pH7.4 PBS containing 0.05% Tween-20, 2% sucrose, 1% NaCl, 1% BSA, and 0.05% ProClin300.
(4) The preparation of the R1 reagent comprises the following steps: 10mM, pH7.4 PBS containing 5% PEG6000, 0.05% ProClin300. 4 turbidimetric reagents were prepared in combination with 4R 2.
4. Verification of reaction Effect
(1) The biochemical analyzer is set with a dominant wavelength of 578nm, R1/S/R2=150/20/50, langdao IT2691 calibrator (tracing to an international standard substance ERM-DA470 k/IFCC) is adopted to calibrate a calibration curve, hp proteins with three phenotypes are respectively tested, test results are compared, and the optimal pairing is selected to be 200nmLP-4A5 and 70nmLP-16C12 combination;
(2) Turbidimetric reagent formula optimization is performed based on the combination of the screened 200nmLP-4A5 and 70nmLP-16C12, and the formula optimization is as follows:
r1 reagent: 10mM, pH7.5 HEPES, 3% PEG6000, 1% NaCl, 0.05% ProClin300;
r2 reagent: 10mM, pH7.5 HEPES, containing 0.2% Hp priming latex, 0.05% Tween-20, 2% sucrose, 1% NaCl, 1% BSA, and 0.05% ProClin300;
calibration material: the matrix solution was 10mM, PBS pH7.4, 1% BSA, 3% sucrose, 1% trehalose, 0.05% ProClin300, and 20ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml, 2000ng/ml Hp mix antigen was prepared and traced to ERM-DA470k.
The main performance of the kit prepared by the method is verified as follows:
urine Hp biochemical reagent prepared according to the method above, calibrator (20, 100, 500, 1000, 2000) with Hp concentration of 5 levels from low to high were taken, working calibration curve was prepared by test with 0 concentration, and 4PL regression fit was performed.
(1) Accuracy of
100ng/ml and 1000ng/ml standards (traceable to international standard) were tested with relative deviations of 2.4% and 1.1%, respectively.
(2) Precision of
Urine samples with low and high concentrations were tested 10 times each, and coefficient of variation CV was calculated to be 1.3% and 1.1%, respectively.
(3) Linear range
The Hp high-value sample solution with the original concentration of over 2000ng/ml is diluted into 5 low-concentration samples with different gradients, each sample is tested 3 times and the average value is obtained, and the correlation coefficient r=0.9999 of the test value and the dilution ratio is calculated, as shown in fig. 1.
(4) Phenotypic reactivity assessment
And (3) taking three phenotype proteins of Hp1-1, hp2-1 and Hp2-2, diluting to two concentrations of 50ng/ml and 1000ng/ml, and measuring by using the prepared Hp detection reagent on a machine. The reaction results were as follows:
50ng/ml assay
1000ng/ml test results
As can be seen from the above results, the Hp1-1 measured value is higher under the dual factors of molecular weight and phenotypic reactivity, the Hp2-1 reaction result is closest to the mixed international standard substance, and the Hp2-2 measured value is lowest, but under the turbidimetric pairing, the relative deviation with the international standard substance is not more than 15%, and the genotyping reactivity is very close.
In conclusion, the Hp latex enhanced transmittance turbidity reagent prepared by the method can be directly used on a biochemical analyzer, and the accuracy relative deviation is less than 10%, the precision CV is less than 5%, the linear range is not less than 20ng/ml to 2000ng/ml, and the deviation between the three phenotype detection results of Hp1-1, hp2-1 and Hp2-2 and the international standard substance is not more than 15%.
The technological parameters of the embodiment in this scheme are only unique, and the obtained monoclonal antibody cell strain has uncertainty due to different immune effects, so that the monoclonal antibody obtained by repeating this embodiment cannot be ensured to be identical with the embodiment, and the parameters in the preparation of reagents by subsequent coupling are inconsistent due to the difference of the monoclonal antibodies.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (8)
1. A preparation method of a kit for measuring urine binding globin by using a biochemical analyzer is characterized in that the kit is prepared by screening paired specific monoclonal antibodies with balanced binding force for Hp three protein phenotypes, respectively coupling double-particle-size latex microspheres, and further performing turbidimetric platform reactivity verification.
2. The method for preparing a kit for measuring urine-binding globin by a biochemical analyzer according to claim 1, comprising the steps of:
s1: preparing an anti-Hp monoclonal antibody;
s2: phenotypic response primary screening:
s201: cross pairing the obtained anti-Hp monoclonal antibodies, detecting mixed phenotype human Hp antigen by ELISA, and screening out positive pairing;
s202: testing the pure proteins of three phenotypes of Hp1-1, hp2-1 and Hp2-2 respectively for the screened positive pairing, and screening out the related pairing with the binding force closest to the proteins of the three phenotypes according to the reaction result;
s3: preparation of urine Hp biochemical reagent:
s301: coupling the paired antibodies screened in the step S202 with double-particle-diameter latex microspheres to obtain a plurality of sensitization latices with different particle diameters;
s302: mixing the paired size particle size sensitization latex two by two, preparing turbidimetric reagent R2, and preparing a biochemical kit by the turbidimetric reagent R1 for providing a reaction environment;
s4: and (3) verifying the reaction effect:
and verifying the reactivity of the biochemical kit to each Hp phenotype, screening out an optimal pairing scheme after coupling the latex microspheres, and optimizing the reagent prepared by the pairing scheme to ensure that the biochemical kit meets the optimal performance requirement of urine Hp detection.
3. The method for preparing a kit for measuring urine-binding globin by a biochemical analyzer according to claim 1, wherein the specific steps of the step S1 include:
s101: mixing phenotype human Hp pure antigen to immunize female BALB/c mice, taking spleen of the immunized mice to prepare cell suspension, fusing myeloma cell SP2/0, and re-suspending HAT culture solution;
s102: selectively culturing hybridoma cells, coating mixed phenotype human Hp antigen by indirect ELISA to measure positive holes, subcloning until the positive hole rate is 100%, screening positive holes with high antibody secretion for injecting female BALB/c mice, collecting peritoneal effusion, and extracting and purifying anti-Hp monoclonal antibodies.
4. The method for preparing a kit for measuring urine-binding globin by a biochemical analyzer according to claim 1, wherein in the step S3, the double-particle-diameter latex microspheres are 180 nm-240 nm large-particle-diameter latex microspheres and 60 nm-80 nm small-particle-diameter latex microspheres.
5. The method for preparing a kit for biochemical analyzer assay of urine-binding globin as defined in claim 1, wherein in step S4, the turbidimetric reagent R2 comprises the following components:
10mM, pH7.5 HEPES, 0.2% Hp-sensitized latex, 0.05% Tween-20, 2% sucrose, 1% NaCl, 1% BSA and 0.05% ProClin300.
6. The method for preparing a kit for biochemical analyzer assay of urine-binding globin as defined in claim 1, wherein in step S4, the R1 reagent comprises the following components:
10mM, pH7.5 HEPES, 3% PEG6000, 1% NaCl, 0.05% ProClin300.
7. A kit prepared by the method for preparing a kit for biochemical analyzer to measure urine-binding globin according to any one of claims 1 to 6.
8. The kit for biochemical analyzer assay of urine binding globin as defined in claim 7, further comprising a calibrator comprising a matrix solution of 10mM, ph7.4 PBS containing 1% bsa, 3% sucrose, 1% trehalose, 0.05% proclin300, hp mixed antigen.
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