CN115236325A - Kit for determining CTX (CTX) by latex enhanced immunoturbidimetry and preparation and detection method thereof - Google Patents

Abstract

The invention provides a kit for determining CTX by using a latex enhanced immunoturbidimetry, which comprises a reagent R1 and a reagent R2; the reagent R1 comprises: r1 buffer solution, stabilizer, preservative and sensitizer; the reagent R2 comprises: r2 buffer solution, surfactant, stabilizer, CTX antibody, 100-150 nm latex microsphere particles, 160-200 nm latex microsphere particles, preservative, activation buffer solution and coupling buffer solution. The invention also provides a preparation method and a determination method of the kit. The invention has the advantages that: the method has the advantages of simple operation, short detection time, low detection cost, and high sensitivity, precision and accuracy.

Description

Kit for determining CTX (CTX) by latex enhanced immunoturbidimetry and preparation and detection method thereof

Technical Field

The invention relates to the field of immunological determination, in particular to a kit for determining CTX by a latex enhanced immunoturbidimetry method and a preparation and detection method thereof.

Background

Currently, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA, IAIR) and immunoturbidimetry (INA, ITA) are examples of methods for detecting type I collagen C-terminal peptides (CTX). In enzyme-linked immunosorbent assay, the detection sensitivity is high, but the operation process is slightly complicated, the assay time is long, the influence factors are many, and the enzyme-linked immunosorbent assay cannot be popularized in primary hospitals and medical institutions. In the radioactive immunoassay method, the method has the advantages of high sensitivity and strong specificity, but has the disadvantages of radioactive contamination, poor repeatability and high nonspecific binding rate, and the method is mainly applied to scientific research and cannot be used for clinical diagnosis. The immunoturbidimetry can be divided Into Nephelometry (INA) and transmission turbidimetry (ITA), and the methods have the advantages of rapidness, simplicity, high precision, easy automation, suitability for simultaneous detection of a large quantity of samples, capability of measuring a small quantity of samples and emergency samples and strong applicability. Among them, the nephelometry (INA) is usually superior to the turbidimetry in sensitivity and speed measurement of the test result due to the characteristics of its light wave receiving mode, but requires a nephelometer or a special protein meter of a special instrument, and is higher in measurement cost with a special matching reagent, and is not easy to be popularized and used in primary hospitals; the transmission turbidimetry (ITA) is simple and convenient to operate and high in applicability, a common automatic biochemical analyzer and a spectrophotometer can be used, the ITA is easier to be adopted by conventional analysis, almost all laboratories and primary hospitals can develop the ITA, and the ITA is not ideal in sensitivity and precision.

The latex enhanced immunoturbidimetry (PETIA) utilizes the specific combination of antigen and antibody to crosslink or physically adsorb the antibody on the surface of nano-scale latex microsphere, and after the microsphere crosslinked with the antibody in the reagent reaction solution is combined with the antigen in a sample, the microsphere can be rapidly gathered together in a short time to form turbidity, thereby changing the light scattering performance or the light transmission performance of the reaction solution and reflecting the concentration of the detected antigen in a linear range. The latex enhanced immunoturbidimetry has the advantages of low cost, simple operation and capability of obtaining results in minutes. In addition, the simplification of the operation steps of the full-automatic immunoturbidimetry correspondingly avoids the interference of a plurality of human operation factors and external factors such as reagents, environment and the like, has better stability and repeatability, and can accurately reflect the content of the measured substance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a kit for determining CTX by a latex enhanced immunoturbidimetry and a preparation and detection method thereof; according to the invention, the antibody-antigen reaction is amplified, and a stabilizer, a surfactant and the like are added to optimize a reaction system, so that the diameter of an immune complex is increased, and the detection sensitivity and the analysis sensitivity and precision of a determination reagent are improved; through the combination of latex microspheres with different particle sizes, the performance of the I type collagen C-terminal peptide measuring reagent reaches a wider linear range, and the accurate detection of the I type collagen C-terminal peptide is realized; meanwhile, the device is suitable for various full-automatic biochemical analyzers, is suitable for simultaneous detection of a large quantity of samples, can also be used for measuring a small quantity of samples and emergency samples, has wide universality, does not improve the research and development cost of reagents, and is more suitable for the development of modern clinical inspection medicine.

The invention adopts the following technical scheme to solve the technical problems:

a kit for determining CTX by a latex enhanced immunoturbidimetry method comprises a reagent R1 and a reagent R2; the reagent R1 comprises: 5-20 mmol/L of R1 buffer solution, 8-10 g/L of stabilizer, 1-1.5 g/L of preservative and 15-25 g/L of sensitizer; the reagent R2 comprises: 50-100 mmol/L of R2 buffer solution, 0.05-1 g/L of surfactant, 6-8 g/L of stabilizer, 10-20 mg/L of CTX antibody, 5-10 ml/L of 100-150 nm latex microsphere particles, 5-10 ml/L of 160-200 nm latex microsphere particles, 1-1.5 g/L of preservative, 50-100 mmol/L of activation buffer solution and 20-100 mmol/L of coupling buffer solution.

In a preferred embodiment of the present invention, the R1 buffer, the R2 buffer, the activation buffer and the coupling buffer are one of a boric acid buffer, a sodium borate buffer, a glycine buffer, a phosphate buffer, a Hepes buffer and a Tris-HCl buffer.

In a preferred embodiment of the present invention, the stabilizer is at least one of sucrose, disodium edetate, trehalose, glycerol, gelatin, bovine serum albumin, and casein.

In a preferred embodiment of the present invention, the preservative is at least one of sodium azide, gentamicin, thimerosal, proclin300, and isothiazolinone.

In a preferred embodiment of the present invention, the sensitizer is at least one of polyethylene glycol 6000 and sodium fluoride.

In a preferred embodiment of the present invention, the surfactant is at least one of tween-20, triton X-100, and glycerol.

In a preferred embodiment of the present invention, the CTX antibody is at least one of a monoclonal antibody against a C-terminal peptide of human type I collagen and a monoclonal antibody against a C-terminal peptide of rat type I collagen, or at least one of a polyclonal antibody against a C-terminal peptide of human type I collagen and a polyclonal antibody against a C-terminal peptide of rat type I collagen.

As one of the preferable modes of the invention, the CTX antibody is respectively coupled and combined with 100-150 nm latex microsphere particles and 160-200 nm latex microsphere particles in a coupling reaction manner to form two latex microspheres coated with the CTX antibody and with different particle diameters.

A preparation method of the kit for determining CTX by the latex enhanced immunoturbidimetry comprises the following steps:

(1) Preparing a reagent R1:

according to the component content of the reagent R1, mixing all the components in the same container, and uniformly mixing to obtain a reagent R1;

(2) Preparing a dilution buffer solution of the reagent R2:

mixing an R2 buffer solution, a surfactant, a stabilizer and a preservative according to the component content of the reagent R2 to obtain a dilution buffer solution of the reagent R2;

(3) Preparing a reagent R2:

(1) taking latex microspheres with the particle size of 100-150 nm and latex microspheres with the particle size of 160-200 nm according to the proportion of 1;

(2) adding an activating agent, uniformly mixing, and placing in a constant-temperature shaking table to react for 20-30 min, wherein the temperature range is as follows: 35 to 40 ℃;

(3) adding a coupling buffer solution;

(4) adding the CTX antibody, mixing uniformly, and placing in a constant-temperature shaking table for reacting for 2-3 h, wherein the temperature range is as follows: 30 to 37 ℃;

(5) adding the confining liquid, mixing uniformly, and placing the mixture in a constant temperature shaking table for reacting for 1-2 h, wherein the temperature range is as follows: 30 to 37 ℃;

(6) after the reaction is finished, centrifuging and removing supernatant, wherein the rotating speed is 16000rpm, and the centrifuging time is 35min;

(7) removing the supernatant, re-dissolving with the dilution buffer solution in the step (2), and re-suspending the latex microspheres; and in the process of resuspension, a cell crusher is used for assisting resuspension to prepare a reagent R2.

As one of preferred embodiments of the present invention, the activator: NHS25mg/mL, EDC9.0mg/mL, ready for use; sealing liquid: BSA10g/L.

The detection method of the kit for determining CTX by the latex enhanced immunoturbidimetry comprises the following steps:

(1) Sucking 10 mu L of sample, adding 240 mu L of reagent R1, and incubating for 3-5 min at 37 ℃;

(2) Adding 60 mu L of reagent R2, and incubating at 37 ℃;

(3) After incubation for 20s, measuring a light absorption value A1 by using a full-automatic biochemical analyzer at a wavelength of 470 nm; incubating for 5min, and detecting absorbance value A2 at the same wavelength;

(4) And calculating the absorbance change value delta A according to delta A = A2-A1, and calculating the CTX content in the sample according to the delta A.

As one of preferable modes of the present invention, the CTX content is determined by substituting Δ a into "linear relation formula of absorbance change value and CTX concentration"; wherein, the "calibration curve of the absorbance change value and the CTX concentration" is obtained by the following method:

(1) Preparing a calibrator buffer:

50-150 mmol/L of phosphate buffer solution;

NaCl 30g/L；

1g/L sodium azide;

(2) Preparing calibrators with various concentration gradients

Diluting the purified CTX antigen by using a calibrator buffer solution, and preparing calibrator concentration points in sequence as follows: 0g/L, 0.5g/L, 1g/L, 2g/L, 4g/L, 8g/L;

(3) Drawing a calibration curve and obtaining a corresponding formula:

respectively adding 240 mu L of reagent R1 into the obtained 10 mu L of CTX standard substance, uniformly mixing, and incubating for 3-5 min at 37 ℃; then, 60. Mu.L of reagent R2 was added and incubated at 37 ℃; after incubation for 20s, measuring the light absorption value Al by using a full-automatic biochemical analyzer under the wavelength of 470 nm; incubating for 5min, and detecting absorbance value A2 at the same wavelength; respectively calculating the absorbance change value delta A = A2-A1 of the standard substance, and drawing a calibration curve; the calibration curve uses a multi-point non-linear fit.

The CTX determination kit is based on latex enhanced immunoturbidimetry (PETIA), and the principle is that a chemical coupling method is adopted to combine specific antibodies on the surfaces of latex particles with certain particle sizes, and when microspheres crosslinked with the antibodies are combined with antigens, the microspheres are rapidly gathered together in a short time, so that the absorbance of a reaction solution is changed. According to the absorbance increment, the turbidity of the immune complex can be measured at a specific wavelength, and the CTX content in the serum can be quantitatively detected.

Compared with the prior art, the invention has the advantages that:

(1) According to the invention, based on latex enhanced immune turbidimetry, after the reaction of antigen and antibody, the absorbance value of the reaction liquid is directly measured, so that the complicated operation steps of repeated incubation and plate washing of an ELISA method are omitted, the result can be obtained in a few minutes, and time and labor are saved; meanwhile, compared with a chemiluminescence immunoassay method, the method has the advantages of low cost, strong applicability and convenient test, and can be widely applied to clinical detection;

(2) The kit can ensure that the performance of the CTX determination reagent reaches a wider linear range through the combination of the latex microspheres with different particle sizes, and can realize the accurate detection of CTX;

(3) The kit can be used for detecting the content of CTX in blood on a full-automatic biochemical analyzer with the wavelength of 400-800 nm, is directly used on a computer, is rapid and accurate, has high automation degree, and greatly improves the working efficiency; and the detection sample amount is small, and the detection of a small amount of samples and emergency samples can be carried out.

Drawings

FIG. 1 is a graph of a linear fit of the kit of the invention of example 6.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

The kit for determining CTX by using the latex enhanced immunoturbidimetry comprises a reagent R1, a reagent R2 and a calibrator.

The reagent R1 comprises: boric acid buffer 5mmol/L (pH7.0), sucrose 8g/L, sodium azide 1g/L, polyethylene glycol 600015g/L.

The reagent R2 comprises: 50mmol/L (pH 7.0) of boric acid buffer solution, 200.05g/L of Tween-1, 6g/L of sucrose, 10mg/L of rat type I collagen C-terminal peptide monoclonal antibody, 5ml/L of 100nm latex microsphere particles, 5ml/L of 160nm latex microsphere particles, 1g/L of sodium azide, 50mmol/L (pH 3.0) of sodium borate buffer solution and 20mmol/L (pH 4.0) of glycine buffer solution.

The calibration article comprises: 50mmol/L (pH7.0) of phosphate buffer solution, 30g/L of NaCl, 1g/L of sodium azide and 0-8 g/L of CTX antigen.

In the reagent R2, the CTX antibody is respectively coupled and combined with 100nm latex microsphere particles and 160nm latex microsphere particles through coupling reaction to form two latex microspheres coated with the CTX antibody and with different particle sizes. In the process, a sodium borate buffer solution is used as an activation buffer solution, and a glycine buffer solution is used as a coupling buffer solution.

The preparation method comprises the following steps:

(1) Preparing a reagent R1:

according to the component content of the reagent R1, all the components are mixed in the same container, and the reagent R1 is prepared after uniform mixing.

(2) Preparing a dilution buffer solution of the reagent R2:

and mixing the R2 buffer solution, the surfactant, the stabilizer and the preservative according to the component content of the reagent R2 to obtain the dilution buffer solution of the reagent R2.

(3) Preparing a reagent R2:

(1) taking latex microspheres with the particle size of 100nm and latex microspheres with the particle size of 160nm according to the proportion of 1;

(2) adding activating agent (NHS 25mg/mL, EDC9.0mg/mL, prepared for use now), mixing, placing in constant temperature shaking table for reaction for 20min, at 35 deg.C;

(3) adding a coupling buffer solution, and adjusting the pH value of the solution to 4.0;

(4) adding CTX antibody, mixing uniformly, placing in a constant temperature shaking table for reacting for 2h at the temperature of 30 ℃;

(5) adding blocking solution (BSA 10 g/L), mixing well, placing in a constant temperature shaking table for reacting for 1h at the temperature of 30 ℃;

(6) after the reaction is finished, centrifuging and removing supernatant, wherein the rotating speed is 16000rpm, and the centrifuging time is 35min;

(7) removing the supernatant, re-dissolving with the dilution buffer solution in the step (2), and re-suspending the latex microspheres; and in the process of resuspension, a cell crusher is used for assisting resuspension to prepare a reagent R2.

(4) Preparing a calibrator:

and (3) mixing the components in the same container according to the component content of the calibrator, and uniformly mixing to obtain the calibrator.

Example 2

The kit for determining CTX by using the latex enhanced immunoturbidimetry comprises a reagent R1, a reagent R2 and a calibrator.

The reagent R1 comprises: 10mmol/L (pH 7.2) of sodium borate buffer solution, 9g/L of ethylene diamine tetraacetic acid, 1.2g/L of gentamicin and 20g/L of sodium fluoride.

The reagent R2 comprises: 70mmol/L (pH 7.2) of sodium borate buffer solution, 1000.08g/L of triton X, 7g/L of trehalose, 15mg/L of human type I collagen C-terminal peptide polyclonal antibody, 8ml/L of 110nm latex microsphere particles, 8ml/L of 170nm latex microsphere particles, 1.2g/L of thimerosal, 70mmol/L (pH 4.0) of glycine buffer solution and 70mmol/L (pH 5.0) of phosphate buffer solution.

The calibration article comprises: 50-150 mmol/L (pH7.2) of phosphate buffer solution, 30g/L of NaCl, 1g/L of sodium azide and 0-8 g/L of LCTX antigen.

In the reagent R2, the CTX antibody is respectively coupled with 110nm latex microsphere particles and 170nm latex microsphere particles through coupling reaction to form two latex microspheres coated with the CTX antibody and with different particle sizes. In the process, glycine buffer is used as an activation buffer, and phosphate buffer is used as a coupling buffer.

The preparation method comprises the following steps:

(1) Preparing a reagent R1:

according to the component content of the reagent R1, all the components are mixed in the same container, and the reagent R1 is prepared after uniform mixing.

(2) Preparing a dilution buffer solution of the reagent R2:

and mixing the R2 buffer solution, the surfactant, the stabilizer and the preservative according to the component content of the reagent R2 to obtain the dilution buffer solution of the reagent R2.

(3) Preparing a reagent R2:

(1) taking latex microspheres with the particle size of 110nm and latex microspheres with the particle size of 170nm according to the proportion of 1;

(2) adding activating agent (NHS 25mg/mL, EDC9.0mg/mL, ready for use), mixing, and reacting in constant temperature shaking table for 25min at 37 deg.C;

(3) adding a coupling buffer solution;

(4) adding CTX antibody, mixing well, placing in constant temperature shaking table to react for 2.5h at 37 deg.C;

(5) adding blocking solution (BSA 10 g/L), mixing well, placing in a constant temperature shaking table for reacting for 1.5h at 37 ℃;

(6) after the reaction is finished, centrifuging and removing supernatant, wherein the rotating speed is 16000rpm, and the centrifuging time is 35min;

(7) removing the supernatant, re-dissolving with the dilution buffer solution in the step (2), and re-suspending the latex microspheres; and in the process of resuspension, a cell crusher is used for assisting resuspension to prepare a reagent R2.

(4) Preparing a calibrator:

and (3) mixing the components in the same container according to the component content of the calibrator, and uniformly mixing to obtain the calibrator.

Example 3

The kit for determining CTX by using the latex enhanced immunoturbidimetry comprises a reagent R1, a reagent R2 and a calibrator.

The reagent R1 comprises: 10mmol/L phosphate buffer (pH8.0), 9g/L glycerol, 1g/L sodium azide and 600020g/L polyethylene glycol.

The reagent R2 comprises: 50mmol/L of glycine buffer solution (pH 8.0), 200.5g/L of tween-200, 7g/L of calf serum albumin, 15mg/L of human type I collagen C-terminal peptide monoclonal antibody, 8ml/L of 150nm latex microsphere particles, 8ml/L of 180nm latex microsphere particles, 3001g/L of Proclin, 100mmol/L of boric acid buffer solution (pH 6.0) and 100mmol/L of sodium borate buffer solution (pH 7.0).

The calibration article comprises: phosphate buffer solution 100mmol/L (pH7.2), naCl 30g/L, sodium azide 1g/L, 0-8 g/L CTX antigen.

In the reagent R2, the CTX antibody is respectively coupled and reacted with 150nm latex microsphere particles and 180nm latex microsphere particles to form two latex microspheres coated with the CTX antibody and with different particle sizes. In the process, a boric acid buffer solution is used as an activation buffer solution, and a sodium borate buffer solution is used as a coupling buffer solution.

The preparation method comprises the following steps:

(1) Preparing a reagent R1:

according to the component content of the reagent R1, all the components are mixed in the same container, and the reagent R1 is prepared after uniform mixing.

(2) Preparing a dilution buffer solution of the reagent R2:

and mixing the R2 buffer solution, the surfactant, the stabilizer and the preservative according to the component content of the reagent R2 to obtain the dilution buffer solution of the reagent R2.

(3) Preparing a reagent R2:

(1) taking latex microspheres with the particle size of 150nm and latex microspheres with the particle size of 180nm according to the proportion of 1;

(2) adding activating agent (NHS 25mg/mL, EDC9.0mg/mL, ready for use), mixing, placing in constant temperature shaking table for reaction for 25min at 37 deg.C;

(3) adding a coupling buffer solution;

(4) adding CTX antibody, mixing well, placing in constant temperature shaking table to react for 2.5h at 37 deg.C;

(5) adding blocking solution (BSA 10 g/L), mixing uniformly, and placing in a constant temperature shaking table for reacting for 1.5h at the temperature of 37 ℃;

(6) after the reaction is finished, centrifuging and removing supernatant, wherein the rotating speed is 16000rpm, and the centrifuging time is 35min;

(7) removing the supernatant, re-dissolving with the dilution buffer solution in the step (2), and re-suspending the latex microspheres; and in the process of resuspension, a cell crusher is used for assisting resuspension to prepare a reagent R2.

(4) Preparing a calibrator:

and (3) mixing the components in the same container according to the component content of the calibrator, and uniformly mixing to obtain the calibrator.

Example 4

The kit for determining CTX by using the latex enhanced immunoturbidimetry comprises a reagent R1, a reagent R2 and a calibrator.

The reagent R1 comprises: hepes buffer solution 20mmol/L (pH9.0), gelatin 10g/L, isothiazolinone 1.5g/L, sodium fluoride 25g/L.

The reagent R2 comprises: 100mmol/L Tris-HCl buffer (pH9.0), 1g/L glycerol, 8g/L casein, 20mg/L rat type I collagen C-terminal peptide polyclonal antibody, 10 ml/L150 nm latex microsphere particles, 10 ml/L200 nm latex microsphere particles, 1.5g/L isothiazolinone, 100mmol/L phosphate buffer (pH6.0), and 100mmol/L Good's buffer (pH7.0).

The calibration article comprises: 150mmol/L phosphate buffer (pH7.2), 30g/L NaCl, 1g/L sodium azide and 0-8 g/LCTX antigen.

In the reagent R2, the CTX antibody is respectively coupled and reacted with 150nm latex microsphere particles and 200nm latex microsphere particles to form two latex microspheres coated with the CTX antibody and with different particle sizes. In this process, phosphate buffer was used as the activation buffer, and Good's buffer was used as the coupling buffer.

The preparation method comprises the following steps:

(1) Preparing a reagent R1:

according to the component content of the reagent R1, all the components are mixed in the same container, and the reagent R1 is prepared after uniform mixing.

(2) Preparing a dilution buffer solution of the reagent R2:

and mixing the R2 buffer solution, the surfactant, the stabilizer and the preservative according to the component content of the reagent R2 to obtain the dilution buffer solution of the reagent R2.

(3) Preparing a reagent R2:

(1) taking latex microspheres with the particle size of 150nm and latex microspheres with the particle size of 200nm according to the proportion of 1;

(2) adding activating agent (NHS 25mg/mL, EDC9.0mg/mL, prepared for use now), mixing, placing in constant temperature shaking table for reaction for 30min, at 40 deg.C;

(3) adding a coupling buffer solution;

(4) adding CTX antibody, mixing uniformly, placing in a constant temperature shaking table to react for 3h at 37 ℃;

(5) adding blocking solution (BSA 10 g/L), mixing well, placing in a constant temperature shaking table for reacting for 2h at 37 ℃;

(6) after the reaction is finished, centrifuging and removing supernatant, wherein the rotating speed is 16000rpm, and the centrifuging time is 35min;

(7) removing the supernatant, re-dissolving with the dilution buffer solution in the step (2), and re-suspending the latex microspheres; and in the process of resuspension, a cell crusher is used for assisting resuspension to prepare a reagent R2.

(4) Preparing a calibrator:

and (3) mixing the components in the same container according to the component content of the calibrator, and uniformly mixing to obtain the calibrator.

Comparative example 1

The components and the preparation process of the kit for measuring CTX by the latex-enhanced immunoturbidimetry are substantially the same as those of the kit in example 3, and the main differences are as follows: the two microspheres were used in a ratio of 3 (150 nm microspheres) to 1 (180 nm microspheres).

Comparative example 2

The components and the preparation process of the kit for measuring CTX by the latex-enhanced immunoturbidimetry are substantially the same as those of the kit in example 3, and the main differences are as follows: after blocking, the solution was diluted directly with diluent.

Example 5

The detection method of the kit for measuring CTX by latex enhanced immunoturbidimetry of the embodiment comprises the following steps:

(1) Sucking 10 mu L of sample, adding 240 mu L of reagent R1, and incubating for 3-5 min at 37 ℃;

(2) Adding 60 mu L of reagent R2, and incubating at 37 ℃;

(3) After incubation for 20s, measuring the light absorption value Al by using a full-automatic biochemical analyzer under the wavelength of 470 nm; incubating for 5min, and detecting absorbance value A2 at the same wavelength;

(4) The absorbance change value Δ a was calculated as Δ a = A2-A1, and Δ a was substituted into "calibration curve of absorbance change value versus CTX concentration" to determine the CTX content.

Wherein, the linear relation formula of the absorbance change value and the CTX concentration is obtained by the following method:

(1) Preparing a calibrator buffer:

50-150 mmol/L of phosphate buffer solution;

NaCl 30g/L；

1g/L of sodium azide;

(2) Preparing calibrators with various concentration gradients

Diluting the purified CTX antigen by using a calibrator buffer solution, and preparing calibrator concentration points in sequence as follows: 0g/L, 0.5g/L, 1g/L, 2g/L, 4g/L, 8g/L;

(3) Drawing a calibration curve and obtaining a corresponding formula:

respectively adding 240 mu L of reagent R1 into the obtained 10 mu L of CTX standard substance, uniformly mixing, and incubating for 3-5 min at 37 ℃; then, 60. Mu.L of reagent R2 was added and incubated at 37 ℃; after incubation for 20s, measuring the light absorption value Al by using a full-automatic biochemical analyzer under the wavelength of 470 nm; incubating for 5min, and detecting absorbance value A2 at the same wavelength; respectively calculating the absorbance change value delta A = A2-A1 of the standard substance, and drawing a calibration curve; the calibration curve uses a multi-point non-linear fit.

The samples were measured using the test method of example 5 using the kits of example 3 and comparative examples 1 and 2 (the measurement results of the kit for measuring type I collagen C-terminal peptide from a certain manufacturer are shown in tables 1 and 2).

TABLE 1 test results (unit: ng/mL) of samples of example 3 and comparative examples 1 and 2 using the kit

TABLE 2 CTX serum sample test results

The results show that according to the detection results of the example 3, the comparative example 1 and the comparative example 2, the average values of the inspection results of the 20 samples are calculated to be 71.72, 71.83 and 71.82 respectively, and the calculated relative deviations are 0.08%, 0.24% and 0.22% respectively; according to the detection results of the example 3, the comparative example 1, the comparative example 2 and the control reagent, the average values of the 3 times of the detection results of the quality control product with the target value of 25ng/mL are respectively 25.067, 24.967, 25.067 and 25.133, and the calculated relative deviations are respectively 0.27%, -0.13%, 0.27% and 0.53%, which shows that the detection result of the type I collagen C-terminal peptide detection kit has no obvious difference from the result of the control kit and has higher accuracy (conformity), but the result of the kit is the best.

Based on the above results, the ratio of the two microspheres used in the preparation process of the kit of the present invention and the preparation process of the latex microspheres coated with the monoclonal antibody are both critical.

Example 6

This example is intended to evaluate the effect of the use of the kit for measuring CTX by latex-enhanced immunoturbidimetry according to the invention.

1. And (3) accuracy analysis:

the test instrument: hitachi 7180 full-automatic biochemical analyzer.

Detecting a sample: 20 serum samples with higher CTX concentration and without hemolysis, jaundice and turbidity.

Control kit: the CTX detection kit (latex enhanced immunoturbidimetry) of a certain manufacturer approved by the national food and drug administration for marketing (including reagents R1 and R2, but having different compositions from the present invention, hereinafter referred to as a control reagent).

The kit prepared by the invention (taking the kit in example 3 as an example) and the control kit (latex enhanced immunoturbidimetry) are respectively used for simultaneously measuring human serum samples with higher CTX concentration and without hemolysis, jaundice and turbidity, repeating the steps for 3 times, and calculating the mean value, CV and deviation. The interference is considered as if the deviation range is within +/-10%, and the interference is considered as if the deviation range exceeds +/-10%. The results are shown in tables 1 and 2.

The result shows that the detection result of the detection kit has no obvious difference with the result of the contrast kit, and has higher accuracy (conformity).

2. And (3) sensitivity analysis:

the test instrument: hitachi 7180 full-automatic biochemical analyzer.

Detecting a sample: 1 part of purified water, 1 part of type I collagen C-terminal peptide low value sample with concentration of 0.95 ng/mL.

The kit prepared by the invention (taking the kit of the embodiment 3 as an example) and a reference kit (a kit for measuring the type I collagen C-terminal peptide of a certain manufacturer) are simultaneously calibrated by using respective detection methods and repeatedly detected for 20 times for each sample to be detected, the absorbance value is recorded, and the average value and the Standard Deviation (SD) are calculated; the absorbance value corresponding to the lowest detection limit is obtained by adding 2SD to the average absorbance value of water, and since the relationship between absorbance and concentration is basically linear, the concentration of the lowest detection limit, namely the sensitivity, can be calculated by comparing with the average absorbance value of a sample of 0.95 ng/mL. Sensitivity = (water absorbance difference average +2 SD) × sample concentration/sample absorbance difference average. The results are shown in Table 3.

TABLE 3 analysis results of sensitivity of the kit of the present invention and the control kit (unit: ng/mL)

The result shows that the sensitivity of the kit for detecting the type I collagen C-terminal peptide is 0.045ng/mL, and the sensitivity of the contrast reagent is 0.073ng/mL, which indicates that the kit has higher sensitivity.

3. And (3) repeatability analysis:

the test instrument: hitachi 7180 full-automatic biochemical analyzer.

Detecting a sample: 2 clinical serum samples (5.8 ng/mL) (low value samples), (197.6 ng/mL) (high value samples).

The kit prepared by the invention (taking the kit in example 3 as an example) and the control kit (the kit for measuring the type I collagen C-terminal peptide of a certain manufacturer) are used for repeatedly detecting each sample to be detected for 10 times, and the detection results are shown in table 4.

TABLE 4 results of precision analysis of the kit of the present invention (unit: ng/mL)

The results show that the precision of the kit of the invention is: a CV low value of 3.43% and a CV high value of 1.73%, both of which are less than or equal to 10%; the CV low value of the control kit is 4.53 percent, and the CV high value is 3.22 percent, which shows that the kit has higher precision than the control kit.

4. Linear analysis:

the test instrument: hitachi 7180 full-automatic biochemical analyzer.

Detecting a sample: serum samples of high type I collagen C-terminal peptide (800 ng/mL).

High type I collagen C-terminal peptide serum samples (800 ng/mL) were diluted to 6 different concentrations with calibrator dilutions, 0ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, and 800ng/mL, and each concentration of the samples was tested three times using the kit prepared in the present invention (taking example 3 as an example), and correlation coefficient R values were calculated, and the test results are shown in Table 5.

TABLE 5 results of the linear analysis of the kit

The result shows that the regression equation obtained from the detection result of the kit prepared by the invention is y =0.9943x +0.0952, and the correlation coefficient R ² =0.9998, as shown in fig. 1, indicating that the kit of the invention is at 8Has good linearity in the range of 00 ng/mL.

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (10)

1. A kit for determining CTX by a latex enhanced immunoturbidimetry is characterized by comprising a reagent R1 and a reagent R2; the reagent R1 comprises: 5-20 mmol/L of R1 buffer solution, 8-10 g/L of stabilizer, 1-1.5 g/L of preservative and 15-25 g/L of sensitizer; the reagent R2 comprises: 50-100 mmol/L of R2 buffer solution, 0.05-1 g/L of surfactant, 6-8 g/L of stabilizer, 10-20 mg/L of CTX antibody, 5-10 ml/L of 100-150 nm latex microsphere particles, 5-10 ml/L of 160-200 nm latex microsphere particles, 1-1.5 g/L of preservative, 50-100 mmol/L of activation buffer solution and 20-100 mmol/L of coupling buffer solution.

2. The kit for determining CTX by latex-enhanced immunoturbidimetry according to claim 1, wherein the R1 buffer, the R2 buffer, the activating buffer and the coupling buffer are one of boric acid buffer, sodium borate buffer, glycine buffer, phosphate buffer, hepes buffer and Tris-HCl buffer, respectively.

3. The kit for determining CTX by latex-enhanced immunoturbidimetry according to claim 1, wherein the stabilizer is at least one of sucrose, disodium edetate, trehalose, glycerol, gelatin, calf serum albumin, casein.

4. The kit for latex-enhanced immunoturbidimetry CTX assay according to claim 1, wherein the preservative is at least one of sodium azide, gentamicin, thimerosal, proclin300, isothiazolinone.

5. The kit for determining CTX by latex enhanced immunoturbidimetry according to claim 1, wherein the sensitizer is at least one of polyethylene glycol 6000 and sodium fluoride.

6. The kit for determining CTX by latex-enhanced immunoturbidimetry according to claim 1, wherein the surfactant is at least one of Tween-20, triton X-100 and glycerol.

7. A kit for latex immunoturbidimetric assay of CTX according to claim 1, wherein said CTX antibody is at least one of a monoclonal antibody against C-terminal peptide of human type I collagen, a monoclonal antibody against C-terminal peptide of rat type I collagen, or a polyclonal antibody against C-terminal peptide of human type I collagen, a polyclonal antibody against C-terminal peptide of rat type I collagen.

8. The kit for determining CTX by latex-enhanced immunoturbidimetry according to claim 1, wherein the CTX antibody is coupled with 100-150 nm latex microspheroidal particles and 160-200 nm latex microspheroidal particles respectively to form two latex microspheres coated with the CTX antibody and having different particle sizes.

9. A method for preparing a kit for latex enhanced immunoturbidimetry CTX determination as claimed in any one of claims 1 to 8, comprising the steps of:

(1) Preparing a reagent R1:

according to the component content of the reagent R1, mixing all the components in the same container, and uniformly mixing to obtain a reagent R1;

(2) Preparing a dilution buffer solution of the reagent R2:

mixing an R2 buffer solution, a surfactant, a stabilizer and a preservative according to the component content of the reagent R2 to obtain a dilution buffer solution of the reagent R2;

(3) Preparing a reagent R2:

(1) taking latex microspheres with the particle size of 100-150 nm and latex microspheres with the particle size of 160-200 nm according to the proportion of 1;

(2) adding an activating agent, mixing uniformly, and placing the mixture in a constant temperature shaking table to react for 20-30 min, wherein the temperature range is as follows: 35 to 40 ℃;

(3) adding a coupling buffer solution;

(4) adding CTX antibody, mixing uniformly, placing in a constant temperature shaking table for reacting for 2-3 h, wherein the temperature range is as follows: 30 to 37 ℃;

(5) adding the confining liquid, mixing uniformly, and placing the mixture in a constant temperature shaking table for reacting for 1 to 2 hours at a temperature range: 30 to 37 ℃;

(6) after the reaction is finished, centrifuging and removing supernatant, wherein the rotating speed is 16000rpm, and the centrifuging time is 35min;

(7) removing the supernatant, re-dissolving with the dilution buffer solution in the step (2), and re-suspending the latex microspheres; and in the process of resuspension, a cell disruptor is used for assisting in resuspension to prepare a reagent R2.

10. A method for detecting a kit for the latex-enhanced immunoturbidimetry determination of CTX according to any of claims 1 to 8, comprising the steps of:

(1) Sucking 10 mu L of sample, adding 240 mu L of reagent R1, and incubating for 3-5 min at 37 ℃;

(2) Adding 60 mu L of reagent R2, and incubating at 37 ℃;

(3) After incubation for 20s, measuring a light absorption value A1 by using a full-automatic biochemical analyzer at a wavelength of 470 nm; incubating for 5min, and detecting absorbance value A2 at the same wavelength;

(4) And calculating the absorbance change value delta A according to the delta A = A2-A1, and calculating the CTX content in the sample according to the delta A.

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