CN111239422A - Serum lipoprotein (a) latex enhanced turbidimetric detection kit - Google Patents
Serum lipoprotein (a) latex enhanced turbidimetric detection kit Download PDFInfo
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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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- G01N33/54313—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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses a liquid double-reagent kit for measuring lipoprotein (a) in serum by using lipoprotein (a) antibody and a latex enhanced immunoturbidimetry method. The kit provided by the invention comprises a reagent R1, a reagent R2 and a lipoprotein (a) standard, wherein the reagent R1 comprises a high-molecular accelerator, a preservative, a surfactant, inorganic salt and a buffer solution; the reagent R2 comprises polystyrene latex particles combined with anti-human lipoprotein (a) antibody, the diameter of the latex particles is 120-160nm, and the reagent also comprises a preservative, a surfactant, an inorganic salt and a buffer solution; the standard lipoprotein (a) comprises preservative, purified human lipoprotein (a), inorganic salt and buffer solution. The kit has the advantages of strong specificity, high sensitivity, wide linear range and simple and convenient operation, and is suitable for various full-automatic biochemical analyzers.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a serum lipoprotein (a) latex enhanced turbidimetry detection kit and a using method thereof.
Background
The principle of the detection method in human serum is that LP (a) in human serum is combined with corresponding monoclonal antibody to immediately form antigen-antibody complex, and turbidity is generated, and the turbidity is proportional to the content of antigen when a certain amount of antibody exists. The lipoprotein (a) is macromolecular lipoprotein and consists of two parts of lipid and protein; wherein the lipid part is composed of cholesterol, phospholipid, etc., and is located in the core of the granule; the protein part is formed by connecting apo (a) and apoB100 through a disulfide bond and is positioned on the periphery of the particle; in each LP (a), apo (a) and apoB100 are in a 1:1 relationship, and possibly 1-2 apos (a) are bound to 1-2 apoBs 100, and free apos (a) are present. apo (a) is glycoprotein-rich, so lp (a) is also a structurally complex glycoprotein. apo (a) contains a Kringle structure, wherein the number of Kringle 4-2(K4-2) repeats is different (3-40), and the relative molecular weight per Kringle-4 is about 12.7kDa, thereby dominating apo (a) to show significant size polymorphism (relative molecular weight is from 18.7kDa to 66.2 kDa). The size polymorphism of apo (a) in turn determines the LP (a) particle size polymorphism. Due to the extremely different molecular sizes of apo (a), lp (a) with different particle sizes generates inconsistent light scattering and light absorption, which brings certain difficulties for determining lp (a) by using monoclonal antibodies.
The monoclonal antibody or polyclonal antibody used by the currently used immunoturbidimetry (ITA, INA) in clinical laboratories mainly acts on K4-2, and produces larger deviations on apo (a) molecular size-sensitive methods. The reactivity of the antibody acting at the K4-2 site depends on apo (a) molecular size, i.e., the number of K4-2 repeats, e.g., the number of K4-2 repeats of the sample lipoprotein (a) is less than the number of K4-2 repeats of the assay calibrator lipoprotein (a), and the results of the assay lipoprotein (a) are underestimated; in contrast, the number of K4-2 repeats in the sample lipoprotein (a) was greater than the number of K4-2 repeats in the calibrator lipoprotein (a), and the assay results were overestimated. In summary, the heterogeneity of apo (a) molecular size affects the immunochemical assay of LP (a) to varying degrees. The features of the apo (a) structure present a serious challenge to immunoassays. The content determination of LP (a) is still based on the mass table of the whole particle, and the influence of the size polymorphism of apo (a) on the current determination of LP (a) is inevitable. For this reason, the standardized procedure for LP (a) assay has yet to be further investigated. The lipemia is the most serious interference factor in the current immunoturbidimetric assay, the reaction system of the invention strengthens the action of the surfactant and adopts automatic blank deduction, so that the nonspecific reaction is balanced before the antigen-antibody specific reaction, and the influence of hemolysis, high bilirubin or lipemia on the assay result can be corrected within a certain range.
Disclosure of Invention
The present invention is provided to solve the above problems.
The invention is realized by the following technical scheme:
the invention provides a liquid double-reagent kit for measuring lipoprotein (a) in serum by a latex enhanced immunoturbidimetry method by using an anti-lipoprotein (a) camelid single-domain antibody, which comprises a reagent R1, a reagent R2 and a lipoprotein (a) antigen calibrator with known concentration, wherein the reagent R1 comprises a high-molecular accelerator, a preservative, a surfactant, inorganic salt and a buffer solution; the reagent R2 comprises polystyrene latex particles combined with anti-human lipoprotein (a) antibody, the diameter of the latex particles is 120-160nm, and the reagent also comprises a preservative, a surfactant, an inorganic salt and a buffer solution; the standard lipoprotein (a) antigen of known concentration comprises preservative, purified human lipoprotein (a), inorganic salt and buffer, and the antibody coupled to the latex is camelid single domain anti-lipoprotein (a) antibody.
7-8% of high molecular accelerator in the reagent R1, 0.01-0.05% of preservative, 0.3-0.5% of surfactant, 1.0-2.0% of inorganic salt and 10-50mmol/L of buffer solution; the content percentage of latex particles in the reagent R2 is 0.4-0.9%, the content of camelid single domain anti-lipoprotein (a) polyclonal antibody is 0.9-1.5 mg/mL, the mass percentage of preservative is 0.01-0.05%, the mass percentage of surfactant is 0.3-0.5%, the mass percentage of inorganic salt is 1.0-2.0%, and the concentration of buffer solution is 10-50 mmol/L; the content of the purified lipoprotein (a) antigen in the lipoprotein (a) antigen standard with known concentration is 75ng/mL-1200ng/mL, the mass percent of the preservative is 0.01-0.05%, the mass percent of the inorganic salt is 1.0-2.0%, and the concentration of the buffer solution is 10-50 mmol/L.
Wherein the buffer solution is 4-hydroxyethyl piperazine ethanesulfonic acid (HAPES) buffer solution, and the pH value of the buffer solution is 4.7-6.5, preferably 5.0-6.0; wherein the polymeric accelerator in the reagent R1 is selected from PEG200, PEG600 and PEG800, preferably PEG 200; wherein the preservative is selected from proclin 200; wherein the surfactant in the reagents R1 and R2 is selected from dodecafluoroheptyl methacrylate; wherein the inorganic salt is sodium chloride or calcium chloride, preferably sodium chloride; wherein the concentration of lipoprotein (a) in the antigen standard with known concentration is 75, 150, 300, 600 and 1200ng/mL respectively.
In the liquid double reagent kit for measuring lipoprotein (a) in serum by latex-enhanced immunoturbidimetry using camelidae lipoprotein (a) antibody of the present invention, the lipoprotein (a) antibody is derived from camel or alpaca, preferably from alpaca. The polyclonal antibody can specifically recognize a plurality of antigenic sites of the lipoprotein (a), so that the polyclonal antibody can specifically bind to the lipoprotein (a) with different molecular weights, and simultaneously eliminates the influence of factors such as rheumatoid factors and the like in the traditional IGG type antibody on a measured value. Therefore, the polyclonal antibody can not only ensure the high-efficiency affinity of the antigen and the antibody, but also ensure the precision of the kit, improve the stability of the measured value of the kit and optimize the reaction time.
The invention adopts a latex particle enhanced transmission immunoturbidimetry, and the principle is that latex particles coated with high-specificity antibodies are specifically combined with corresponding antigens in a sample to form an antigen-antibody-latex particle compound, the compound forms a certain turbidity in a specific buffer solution, the increase degree of the turbidity is in direct proportion to the antigen concentration in the sample, and the turbidity is measured under a certain wavelength, so that the content of the detected antigens in the sample can be measured.
In the present invention, the lipoprotein (a) antigen in serum is conjugated with a specific camel anti-human lipoprotein (a) polyclonal antibody bound to the surface of latex particles, an antigen-antibody reaction occurs to form an immune complex, a fixed turbidity is formed, the absorbance at a wavelength of 570nm of the turbidity is measured, and the content of lipoprotein (a) in a sample is determined by referring to a standard curve.
The liquid double-reagent kit for determining inches by using the camelidae single-domain antibody and determining the lipoprotein (a) in the serum through a latex enhanced immunoturbidimetry method is adopted to determine inches, a sample and a reagent Rl are mixed uniformly, the absorbance Al is read, then the reagent R2 is added, the absorbance A2 is read, the reaction absorbance change value is calculated, and the content of the lipoprotein (a) in the sample is obtained by contrasting a standard curve.
The technical core of the invention is as follows: the specific camelidae single-domain anti-human lipoprotein (a) antibody is used for identifying a plurality of antigenic sites of the lipoprotein (a) with different molecular weights, and simultaneously eliminating the influence of factors such as rheumatoid factors and the like in the traditional IGG type antibody on the measured value. Therefore, the determination precision and the anti-interference capability of the kit are improved. The method of the invention can ensure the detection of the lipoprotein (a) with great molecular weight difference and has good clinical application value.
The liquid double-reagent kit has the advantages of high accuracy, good specificity, high sensitivity, strong anti-interference capability and the like, can be used for detecting the concentration of the lipoprotein (a) in serum, and is suitable for various clinical full-automatic biochemical analyzers.
Detailed Description
The present invention will be described in further detail with reference to embodiments. The following examples are only exemplary and are intended to illustrate the technical solutions of the present invention in further detail, and it should be understood by those skilled in the art that modifications or substitutions to the technical solutions without departing from the spirit and scope of the technical solutions of the present invention should be covered by the claims of the present invention.
EXAMPLE one kit preparation
The main components and the mixture ratio of the kit are as follows:
reagent Rl:
HAPES buffer 40mmol/L
Proclin 200 0.02％
1.5 percent of sodium chloride
PEG-200 7.5％
0.4 percent of dodecafluoroheptyl methacrylate
Reagent R2:
HAPES buffer 40mmol/L
Proclin 200 0.02％
1.5 percent of sodium chloride
0.4 percent of dodecafluoroheptyl methacrylate
Lipoprotein (a) polyclonal antibody 0.97mg/mL
0.5% w/v of 120-160nm particle size polystyrene latex
Lipoprotein (a) standard:
HAPES buffer 40mmol/L
Proclin 200 0.02％
Sodium chloride (electrolyte) 1.5%
Purified human lipoprotein (a)75-1200ng/mL
The kit determination method comprises the following analysis methods: two-point end-point method, reaction time: 10min, sample size: 7 μ L, R1300 μ L and water 20 μ L are added 90s before the addition of the specimen, R270 μ L and water 10 μ L are added to the specimen and water 10 μ L,180s reads the turbidity value A1 at the first point, 540s reads the turbidity value A2 at the second point, A2-A1 is the turbidity change of the reaction, dominant wavelength: 570nm, secondary wavelength 800nm, reaction direction: ascending reaction, calibration mode: spline, calibration method: 5-point scaling, the first, second, third, fourth and fifth points are lipoprotein (a) standards with concentrations of 75, 150, 300, 600, 1200ng/mL
Experimental example-Standard Curve preparation
The reference serum was diluted with saline to lp (a) levels of 75, 150, 300, 600, 1200ng/mL and the instrument was automatically run using the Logit-LogP nonlinear standard mode. As a result, the lp (a) concentration was linear in the range of 0 to 1200 ng/mL.
Experimental example II recovery test
Taking mixed serum with lp (a) concentration of 85ng/mL, and adding different concentrations of reference serum respectively to make the theoretical values thereof respectively 155, 242 and 398 ng/mL. The actual measurement results were 152, 245 and 392 ng/mL. The recovery rates were 97.3%, 101.3% and 98.3%, respectively.
Experimental example three interference test
1. Hemoglobin interference is realized by diluting 50g/L hemoglobin standard solution into five concentrations of 25, 12.5, 6.25, 5.0 and 2.5g/L, respectively adding the five concentrations into a sample with the same volume of lp (a) and the content of the sample being 250mg L, and the results of the measurement of the lp (a) (the measured value is multiplied by 2) are 344, 299, 275, 253 and 252 mg/L. The above results indicate that hemoglobin concentrations below 2.5g/L had no effect on the measurements. 2. Interference of bilirubin is determined by adding equal volume of bilirubin of 240, 120, 60, 30, 15. mu. mol/L to a sample containing lp (a) of 250 ng/mL, and determining the result of lp (a) (the determined value is multiplied by 2) to be 204, 220, 233, 251, 253 ng/mL. This result indicates that the final bilirubin concentration of 15. mu. mol/L or less had no effect on the assay, and that 15. mu. mol/L or more negatively interfered with the results. The sample preservation, generally considered that the serum sample is preserved in a sealed manner at 4 ℃ for not more than 2 weeks and preserved at-20 ℃ or-80 ℃ for 3 months, has no influence on the measurement result.
Experimental example four precision experiment
Selecting 3 lipid control products provided by Randox corporation with lot numbers of 1854CH, 1855CH and 1856 CH) and continuously detecting each level of the control product 20 times according to a specimen inspection program, and calculating a mean value, a standard deviation and a coefficient of variation. The paired data t test was performed using Excel2007 software. Precision evaluation is carried out by using recommended parameters, and the minimum value of the maximum value of the CV of 3 concentration levels is the suggestion that lp (a) precision is less than 4% in the suggestions about clinical blood fat determination, so that the clinical detection requirements can be met. Precision test, taking low, medium and high concentration mixed serum to measure. Results the intra-batch CVs (n ═ 20) were 2.8% [ lp (a) mean 85ng/mL ], 2.6% [ lp (a) mean 263ng/mL ], 2.1% [ lp (a) mean 664ng/mL ]; the batch CV (n ═ 20) was 4.2%, 3.5% and 3.7%, respectively.
Claims (10)
1. A liquid double reagent kit for measuring lipoprotein (a) in serum by a latex enhanced immunoturbidimetry method, the kit comprises a reagent R1, a reagent R2 and an antigen standard of lipoprotein (a) with known concentration, wherein the reagent R1 comprises a high molecular accelerator, a preservative, a surfactant, inorganic salt and a buffer solution; the reagent R2 comprises polystyrene latex particles combined with anti-human lipoprotein (a) antibody, the diameter of the latex particles is 120-160nm, and the reagent also comprises a preservative, a surfactant, an inorganic salt and a buffer solution; the standard lipoprotein (a) antigen with known concentration comprises preservative, purified human lipoprotein (a), inorganic salt and buffer solution, and is characterized in that the antibody coupled to the latex is camelid single-domain anti-lipoprotein (a) antibody.
2. A kit according to claim 1, wherein the camelidae single domain lipoprotein (a) antibody is from a camel or alpaca, preferably from an alpaca.
3. The kit of claim 1, comprising reagent R1, reagent R2, and a standard of known concentration of lipoprotein (a) antigen, wherein: 7-8% of high molecular accelerator in the reagent R1, 0.01-0.05% of preservative, 0.3-0.5% of surfactant, 1.0-2.0% of inorganic salt and 10-50mmol/L of buffer solution; the content percentage of latex particles in the reagent R2 is 0.4-0.9%, the content of camelid single domain anti-lipoprotein (a) polyclonal antibody is 0.9-1.5 mg/mL, the mass percentage of preservative is 0.01-0.05%, the mass percentage of surfactant is 0.3-0.5%, the mass percentage of inorganic salt is 1.0-2.0%, and the concentration of buffer solution is 10-50 mmol/L; the content of the purified lipoprotein (a) antigen in the lipoprotein (a) antigen standard with known concentration is 75ng/mL-1200ng/mL, the mass percent of the preservative is 0.01-0.05%, the mass percent of the inorganic salt is 1.0-2.0%, and the concentration of the buffer solution is 10-50 mmol/L.
4. The kit of claim 1 or 3, wherein the buffer is 4-hydroxyethylpiperazine ethanesulfonic acid (HAPES) buffer.
5. The kit according to claim 1 or 3, wherein the buffer has a pH value of 4.7-6.5, preferably 5.0-6.0.
6. The kit according to claim 1 or 3, wherein the polymeric accelerator in reagent R1 is selected from PEG200, PEG600, PEG800, preferably PEG 200.
7. The kit according to claim 1 or 3, wherein the preservative is selected from Proclin 200.
8. The kit according to claim 1 or 3, wherein the surfactant in reagents R1, R2 is selected from dodecafluoroheptyl p-methacrylate.
9. The kit according to claim 1 or 3, wherein the inorganic salt is sodium chloride or calcium chloride, preferably sodium chloride.
10. The kit of claim 1 or 3, wherein the concentration of lipoprotein (a) of the known concentration of lipoprotein (a) antigen standard is 75, 150, 300, 600, 1200ng/mL, respectively.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111766381A (en) * | 2020-06-11 | 2020-10-13 | 武汉生之源生物科技股份有限公司 | Determination kit based on latex immunoturbidimetry and application thereof |
| CN113189341A (en) * | 2021-02-26 | 2021-07-30 | 苏州优诺康生物技术有限公司 | Human heparin binding protein immunoturbidimetry detection reagent and application thereof |
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