CN115327122A

CN115327122A - Matrix metalloproteinase-3 detection kit and preparation method thereof

Info

Publication number: CN115327122A
Application number: CN202210950186.1A
Authority: CN
Inventors: 万蒙; 陈银芳
Original assignee: Jiangxi Lecheng Biological Medical Co ltd
Current assignee: Jiangxi Lecheng Biological Medical Co ltd
Priority date: 2022-08-09
Filing date: 2022-08-09
Publication date: 2022-11-11

Abstract

The invention discloses a matrix metalloproteinase-3 detection kit and a preparation method thereof, wherein the detection kit comprises an R1 reagent and an R2 reagent. The R1 reagent comprises: 160-200 mmol/L HEPES buffer solution, 60-80 mmol/L sodium chloride, 10-15 mmol/L calcium chloride, 1-5% PEG-6000,0.1-0.5% Triton X-100,0.09-0.12% sodium azide, and the pH value of R1 reagent is 7.0-7.5; r2 reagent the method comprises the following steps: 30 to 50mmol/L HEPES buffer solution, 15 to 20 percent of cane sugar, 0.1 percent of latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.1 to 0.5 percent of Tween-20, 0.04 to 0.08 percent of PC-300, and the pH value of the R2 reagent is 7.0 to 7.5. The latex microspheres coated with the human matrix metalloproteinase-3 antibody in the R2 reagent are prepared by mixing 100-150 nm and 300-400 nm latex microspheres according to a certain proportion. Both guaranteed that low high value detection area all has higher sensitivity and precision, guaranteed again that reagent long-term storage has sufficient stability.

Description

Matrix metalloproteinase-3 detection kit and preparation method thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a matrix metalloproteinase-3 detection kit and a preparation method thereof.

Background

Matrix metalloproteinase-3 (MMP-3) is a protease which is mainly produced by synovial cells and chondrocytes, can destroy collagen and degrade articular cartilage, and plays an important role in bone destruction of rheumatoid arthritis. Studies have shown that serum MMP-3 levels are correlated with the degree of bone destruction in patients with Rheumatoid Arthritis (RA).

Studies have shown that MMP-3 levels in early RA patients are significantly elevated, even in RA patients with normal C-reactive protein (CRP) and/or Erythrocyte Sedimentation Rate (ESR), over Osteoarthritis (OA) and healthy controls. CRP and ESR are common laboratory indicators for assessing RA disease activity, but these indicators do not change significantly in early RA patients with a short course of disease. In RA patients with short disease course, MMP-3 has higher positive rate, MMP-3 positive rate of RA patients in very early stage within 6 months of disease course is higher than CRP, MMP-3 has value for auxiliary diagnosis and evaluation of patients with normal CRP, and especially under the condition that common laboratory indexes such as CRP or ESR are normal, the higher positive rate of MMP-3 indicates that the detection of MMP-3 level can provide help for diagnosis of RA patients in early stage.

The detection method commonly used for MMP-3 is as follows: enzyme linked immunosorbent assay (ELISA), latex particle enhanced immunoturbidimetry (PETIA), latex agglutination, etc. The ELISA detection method is complex and complex in operation process, and high-throughput sample detection cannot be carried out. Although the PETIA or latex agglutination method is used, the problem of high-throughput sample detection is overcome, the conventional latex turbidimetric detection reagent has low discrimination on a low-value range, a normal reference interval of a clinical sample is in the low-value range, and the low-value range detection accuracy often influences result judgment. Some reagents need to use latex microspheres with larger particle size in order to achieve higher specificity and sensitivity, and the problem of poor reagent stability exists.

Disclosure of Invention

The invention aims to provide a matrix metalloproteinase-3 detection kit and a preparation method thereof, so as to ensure that low and high detection regions have higher sensitivity and accuracy and ensure that the reagent has enough stability after being stored for a long time.

A matrix metalloproteinase-3 detection kit comprises an R1 reagent, an R2 reagent:

the R1 reagent comprises: 160-200 mmol/L HEPES buffer solution, 60-80 mmol/L sodium chloride, 10-15 mmol/L calcium chloride, 1-5% PEG-6000,0.1-0.5% Triton X-100,0.09-0.12% sodium azide, and the pH value of R1 reagent is 7.0-7.5;

the R2 reagent comprises: 30 to 50mmol/L HEPES buffer solution, 15 to 20 percent of cane sugar, 0.1 percent of latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.1 to 0.5 percent of Tween-20, 0.04 to 0.08 percent of PC-300, and the pH value of the R2 reagent is 7.0 to 7.5. (ii) a

The matrix metalloproteinase-3 detection kit is prepared by mixing 100-150 nm and 300-400 nm of latex microspheres coated with the human matrix metalloproteinase-3 antibody according to a certain proportion.

The matrix metalloproteinase-3 detection kit comprises latex microspheres coated with human matrix metalloproteinase-3 antibody, wherein the latex microspheres with two specifications of 100-150 nm and 300-400 nm are as follows: 3 to 3:1, and mixing the components in a ratio of 1.

The matrix metalloproteinase-3 detection kit is characterized in that the latex microspheres coated with the human matrix metalloproteinase-3 antibody are formed by mixing latex microspheres of 120nm and 360nm in a certain proportion.

The matrix metalloproteinase-3 detection kit comprises latex microspheres coated with human matrix metalloproteinase-3 antibody, wherein the latex microspheres with two specifications of 120nm and 360nm are as follows: 1, and mixing the components in a ratio of 1.

A preparation method of a matrix metalloproteinase-3 detection kit comprises the following steps:

step 1, dissolving HEPES, sodium chloride, calcium chloride and PEG-6000 in distilled water, and stirring and mixing uniformly; adding triton X-100, stirring for dissolving, performing ultrasonic homogenization, adding sodium azide, continuing ultrasonic dissolution to ensure that the solution is uniform, adjusting the pH value of the uniform solution to 7.0-7.5, and quantitatively metering to 1L to obtain an R1 reagent;

and 2, dissolving HEPES in distilled water, adjusting the pH value of the solution to 7.0-7.5, adding sucrose, tween-20 and PC-300 into the buffer solution, stirring and dissolving uniformly, removing solution foams by ultrasound to obtain R2 reagent buffer preservation solution, placing the R2 reagent buffer preservation solution on a low-speed magnetic stirrer, slowly adding latex microspheres coated with a human matrix metalloproteinase-3 antibody to the concentration of 0.1%, mixing uniformly, and finally quantitatively fixing the volume of the solution containing the latex microspheres to 1L to obtain the R2 reagent.

In the preparation method, in the step 2, the step of preparing the latex microsphere coated with the human matrix metalloproteinase-3 antibody comprises the following steps:

step 2.1, preparing carboxyl latex microspheres with the particle size of 100-150 nm, adding a 2-morpholine ethanesulfonic acid solution, uniformly mixing, centrifuging to remove supernatant, and repeating for multiple times to obtain the cleaned latex microspheres; preparing carboxyl latex microspheres with the particle size of 300-400 nm, adding a 2-morpholine ethanesulfonic acid solution, centrifuging to remove supernatant, and repeating for multiple times to obtain the cleaned latex microspheres;

step 2.2, adding 2-morpholine ethanesulfonic acid solution into the washed carboxyl latex microspheres with the particle size of 100-150 nm, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding an activating agent to activate the latex, and performing oscillation reaction for 10-20 min to obtain activated latex with the particle size of 100-150 nm; adding 2-morpholine ethanesulfonic acid solution into the washed carboxyl latex microspheres with the particle size of 300-400 nm, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding an activating agent to activate the latex, and performing oscillation reaction for 10-20 min to obtain activated latex with the particle size of 300-400 nm;

step 2.3, centrifuging the activated latex of 100-150 nm, removing supernatant, adding phosphate buffer solution for resuspending microspheres, centrifuging, removing supernatant, adding phosphate buffer solution for resuspending microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding stop solution for continuous oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding confining solution for continuous oscillation reaction for 30 minutes, and obtaining a first part of coupled antibody latex; centrifuging the activated latex of 300-400 nm, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding confining solution to continue oscillation reaction for 30 minutes, and obtaining a second part of coupled antibody latex;

step 2.4, the first and second portions of conjugated antibody latex are mixed on a low speed magnetic stirrer according to a ratio of 1:3 to 3:1, uniformly mixing, centrifuging to remove supernatant, using R2 reagent buffer preservation solution for resuspension, and ultrasonically dispersing into uniform solution to obtain the latex microsphere coated with the human matrix metalloproteinase-3 antibody.

The preparation method comprises the steps of controlling the concentration of the 2-morpholine ethanesulfonic acid solution to be 20-50 mmol/L and controlling the pH value to be 6.0-6.5.

The preparation method comprises the following step of adding 10-20 mg/L of the active agent, wherein the active agent is carbodiimide and N-hydroxysuccinimide in a mass ratio of 1:1.

The preparation method comprises the steps of preparing a stop solution, wherein the stop solution is an ethanolamine, lysine or glycine solution with the concentration of 50-100mg/mL, and the blocking solution is bovine serum albumin with the concentration of 10-30 mg/mL.

The preparation method comprises the steps of preparing phosphate buffer solution by using monopotassium phosphate and disodium hydrogen phosphate, wherein the concentration of the phosphate buffer solution is 20-60 mmol/L, and the pH value is 6.5-7.2.

The preparation method comprises the steps of performing water bath ultrasound for 2s and stopping the water bath ultrasound for 4s in a circulating manner, wherein the duration time of the water bath ultrasound is 2-5 min, and the ultrasound is performed at 40-50 Hz.

The invention has the beneficial effects that:

the invention provides a matrix metalloproteinase-3 detection kit and a preparation method thereof, which can ensure that low and high detection areas have higher sensitivity and precision, can ensure that reagents have enough stability for long-term storage, and can detect clinical samples on a full-automatic biochemical analyzer in a high throughput manner.

Drawings

FIG. 1 is a calibration curve of a kit prepared in example 1 of the present invention and a kit prepared in comparative example 1;

FIG. 2 is a calibration curve of the kit prepared in example 2 of the present invention and the kit prepared in comparative example 2;

FIG. 3 is a calibration curve of the kit prepared in example 3 of the present invention and the kit prepared in comparative example 4;

FIG. 4 is a calibration curve of the kit prepared in example 4 of the present invention and the kit prepared in comparative example 4;

FIG. 5 is a graph showing the results of the 37 ℃ heat-labile stability test of the kit prepared in example 1 of the present invention;

FIG. 6 is a graph showing the results of the test of thermal rupture stability at 37 ℃ of the kit prepared in comparative example 1;

FIG. 7 shows the correlation between the results of the latex agglutination turbidimetry test and the test kit prepared in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A matrix metalloproteinase-3 detection kit comprises an R1 reagent and an R2 reagent.

Wherein the R1 reagent comprises: 200mmol/L HEPES buffer solution, 80mmol/L sodium chloride, 10mmol/L calcium chloride, 5% PEG-6000, 0.1% triton X, 0.09% sodium azide and 7.0 pH value.

Wherein the R2 reagent comprises: HEPES buffer 50mmol/L, 20% sucrose, 0.1% latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.1% Tween-20, 0.04% PC-300, pH 7.0.

The detection kit comprises latex microspheres coated with the human matrix metalloproteinase-3 antibody, wherein the latex microspheres with the sizes of 100nm and 300nm are prepared according to the proportion of 1:1 proportion of the components.

The preparation method of the kit comprises the following steps:

step 1, dissolving 200mmol of HEPES, 80mmol of sodium chloride, 10mmol of calcium chloride and 8978 g of PEG-600050g in a proper amount of distilled water, and stirring and mixing uniformly; adding 0.1 percent of triton X-100, stirring and dissolving, performing ultrasonic uniform treatment, adding 0.09 percent of sodium azide, continuing ultrasonic dissolution to ensure that the solution is uniform, adjusting the pH value of the uniform solution to 7.0, and performing constant volume to 1L to obtain the R1 reagent.

And 2, dissolving 50mmol of HEPES in a proper amount of distilled water, adjusting the pH value of the solution to 7.0, adding 20% of sucrose, 0.1% of Tween-20 and 0.04% of PC-300 into the buffer solution, uniformly stirring and dissolving, and removing the foam of the solution by ultrasonic waves to obtain the R2 reagent buffer preservation solution. And (3) placing the buffer preservation solution on a low-speed magnetic stirrer, slowly adding the latex microspheres coated with the human matrix metalloproteinase-3 antibody to a concentration of 0.1%, uniformly mixing, and finally quantitatively metering the volume of the solution containing the latex microspheres to 1L to obtain the R2 reagent.

In step 2, the preparation of the latex microsphere coated with the human matrix metalloproteinase-3 antibody comprises the following steps:

step 2.1, preparing 10ml of carboxyl latex microspheres with the particle size of 100nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, uniformly mixing, centrifuging to remove supernatant, and repeating for multiple times to obtain the cleaned latex microspheres; preparing 10ml of carboxyl latex microspheres with the particle size of 300nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, centrifuging to remove supernatant, and repeating for multiple times to obtain the washed latex microspheres;

step 2.2, adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 100nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator to activate the latex, and performing oscillation reaction for 15min to obtain 100nm activated latex; adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 300nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator activated latex, and performing oscillation reaction for 15min to obtain 300nm activated latex;

step 2.3, centrifuging the 100nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining first coupled antibody latex; centrifuging the 300nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining a second part of coupled antibody latex;

step 2.4, the first and second conjugated antibody latexes are mixed on a low speed magnetic stirrer according to a ratio of 1:1, uniformly mixing, centrifuging to remove supernatant, using an R2 reagent buffer preservation solution to resuspend to 0.1% concentration, and ultrasonically dispersing to obtain a uniform solution to obtain the latex microsphere coated with the human matrix metalloproteinase-3 antibody.

Wherein the concentration of the solution of the 2-morpholine ethanesulfonic acid is 50mmol/L, and the pH value is 6.0.

Wherein the dosage of the active agent is 10mg/L, the active agent is carbodiimide and N-hydroxysuccinimide, and the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 1:1.

Wherein the phosphate is potassium dihydrogen phosphate and disodium hydrogen phosphate, the concentration is 60mmol/L, and the pH is 7.0.

Wherein the stop solution is ethanolamine solution of 50mg/mL, and the blocking solution is bovine serum albumin of 30 mg/mL.

Wherein the water bath ultrasound is circulating ultrasound for 2s and stopping ultrasound for 4s, the duration time is 2min, and the ultrasound is carried out by adopting 50 Hz.

Example 2

Wherein the R1 reagent comprises: 160mmol/L of HEPES buffer solution, 60mmol/L of sodium chloride, 15mmol/L of calcium chloride, PEG-6000, 0.5% of triton X, 0.12% of sodium azide and pH value of 7.5.

Wherein the R2 reagent comprises: HEPES buffer 30mmol/L, 15% sucrose, 0.1% latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.5% Tween-20, 0.08% PC-300, pH 7.5.

The detection kit comprises latex microspheres coated with human matrix metalloproteinase-3 antibody, wherein the latex microspheres with the sizes of 150nm and 400nm are as follows: 1 proportion of the components.

The procedure of the preparation method of the kit was the same as in example 1, except that the latex microspheres coated with the human matrix metalloproteinase-3 antibody in the R2 reagent were prepared:

the latex microspheres coated with the human matrix metalloproteinase-3 antibody in the R2 reagent are prepared as follows:

step 2.1, preparing 10ml of carboxyl latex microspheres with the particle size of 150nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, uniformly mixing, centrifuging to remove supernatant, and repeating for multiple times to obtain the cleaned latex microspheres; preparing 10ml of carboxyl latex microspheres with the particle size of 400nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, centrifuging to remove supernatant, and repeating for multiple times to obtain the washed latex microspheres;

step 2.2, adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 150nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator to activate the latex, and performing oscillation reaction for 15min to obtain 150nm activated latex; adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 400nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator activated latex, and performing oscillation reaction for 15min to obtain 400nm activated latex;

step 2.3, centrifuging the 150nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining first coupled antibody latex; centrifuging the activated latex with the particle size of 400nm, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining a second part of coupled antibody latex;

Wherein the concentration of the solution of the 2-morpholine ethanesulfonic acid is 20mmol/L, and the pH value is 6.0.

Wherein the dosage of the active agent is 20mg/L, the active agent is carbodiimide and N-hydroxysuccinimide, and the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 1:1.

Wherein the phosphate is potassium dihydrogen phosphate and disodium hydrogen phosphate, the concentration is 20mmol/L, and the pH is 7.0.

Wherein the stop solution is 100mg/mL glycine solution, and the blocking solution is 10mg/mL bovine serum albumin.

Wherein the water bath ultrasound is circulating ultrasound for 2s and stopping for 4s, the duration is 5min, and the ultrasound is carried out at 40 Hz.

Example 3

Wherein the R1 reagent comprises: 180mmol/L HEPES buffer solution, 70mmol/L sodium chloride, 12mmol/L calcium chloride, 2.5% PEG-6000, 0.2% Triton X-100, 0.10% sodium azide and 7.2 pH value.

Wherein the R2 reagent comprises: HEPES buffer 40mmol/L, 18% sucrose, 0.1% latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.2% Tween-20, 0.06% PC-300, pH 7.2.

The detection kit comprises latex microspheres coated with the human matrix metalloproteinase-3 antibody, wherein the latex microspheres have the sizes of 120nm and 360nm and are prepared according to the proportion of 1:3, mixing the components in proportion.

The procedure of the preparation method of the kit is the same as that of example 1, except that the latex microspheres coated with the human matrix metalloproteinase-3 antibody in the R2 reagent are prepared:

step 2.1, preparing 10ml of carboxyl latex microspheres with the particle size of 120nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, uniformly mixing, centrifuging to remove supernatant, and repeating for multiple times to obtain cleaned latex microspheres; preparing 10ml of carboxyl latex microspheres with the particle size of 360nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, centrifuging to remove supernatant, and repeating for multiple times to obtain the washed latex microspheres;

step 2.2, adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 120nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator to activate the latex, and performing oscillation reaction for 20min to obtain 120nm activated latex; adding 50ml of 2-morpholine ethanesulfonic acid solution into the cleaned 360nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator activated latex, and performing oscillation reaction for 20min to obtain 360nm activated latex;

step 2.3, centrifuging the 120nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining first coupled antibody latex; centrifuging the 360nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining a second part of coupled antibody latex;

step 2.4, the first and second portions of conjugated antibody latex are mixed on a low speed magnetic stirrer according to a ratio of 1:3, uniformly mixing, centrifuging to remove supernatant, using R2 reagent buffer preservation solution to resuspend to 0.1% concentration, and ultrasonically dispersing to obtain a uniform solution.

Wherein the concentration of the solution of the 2-morpholine ethanesulfonic acid is 25mmol/L, and the pH value is 6.5.

Wherein the dosage of the active agent is 15mg/L, the active agent is carbodiimide and N-hydroxysuccinimide, and the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 1:1.

Wherein the phosphate is potassium dihydrogen phosphate and disodium hydrogen phosphate, the concentration is 50mmol/L, and the pH is 6.8.

Wherein the stop solution is 70mg/mL ethanolamine solution, and the blocking solution is 20mg/mL bovine serum albumin.

Wherein the water bath ultrasound is circulating ultrasound for 2s and stopping for 4s, the duration is 4min, and the ultrasound is carried out at 40 Hz.

Example 4

Wherein the R1 reagent comprises: 170mmol/L HEPES buffer solution, 75mmol/L sodium chloride, 12mmol/L calcium chloride, PEG-6000, 0.3% triton X, 0.11% sodium azide and pH value of 7.4.

Wherein the R2 reagent comprises: HEPES buffer 45mmol/L, 16% sucrose, 0.1% latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.2% Tween-20, 0.05% PC-300, pH 7.4.

The detection kit comprises latex microspheres coated with human matrix metalloproteinase-3 antibody, wherein the latex microspheres have sizes of 130nm and 340nm according to a ratio of 3:1 proportion of the components.

step 2.1, preparing 10ml of carboxyl latex microspheres with the particle size of 130nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, uniformly mixing, centrifuging to remove supernatant, and repeating for multiple times to obtain the cleaned latex microspheres; preparing 10ml of carboxyl latex microspheres with the particle size of 340nm, adding 30ml of 2-morpholine ethanesulfonic acid solution, centrifuging to remove supernate, and repeating for multiple times to obtain cleaned latex microspheres;

step 2.2, adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 130nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activating agents to activate the latex, and performing oscillation reaction for 20min to obtain 130nm activated latex; adding 50ml of 2-morpholine ethanesulfonic acid solution into the washed 340nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding carbodiimide and N-hydroxysuccinimide activator activated latex, and performing oscillation reaction for 20min to obtain 340nm activated latex;

step 2.3, centrifuging the 130nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining first coupled antibody latex; centrifuging the 340nm activated latex, removing supernatant, adding phosphate buffer solution to resuspend microspheres, centrifuging, removing supernatant, adding 50mL of phosphate buffer solution to resuspend microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, dropwise adding 4mL of 5mg/mL human matrix metalloproteinase-3 antibody solution, performing oscillation reaction at 37 ℃ for 2 hours, adding 1mL of ethanolamine stop solution to continue oscillation reaction for 30 minutes, centrifuging to remove supernatant, adding 1mL of bovine serum albumin confining solution to continue oscillation reaction for 30 minutes, and obtaining a second part of coupled antibody latex;

step 2.4, the first and second conjugated antibody latexes are mixed on a low speed magnetic stirrer according to a ratio of 3:1, uniformly mixing, centrifuging to remove supernatant, using an R2 reagent buffer preservation solution to resuspend to 0.1% concentration, and ultrasonically dispersing to obtain a uniform solution to obtain the latex microsphere coated with the human matrix metalloproteinase-3 antibody.

Wherein the concentration of the solution of the 2-morpholine ethanesulfonic acid is 40mmol/L, and the pH value is 6.2.

The dosage of the active agent is 15mg/L, the active agent is carbodiimide and N-hydroxysuccinimide, and the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 1:1.

Wherein the phosphate is potassium dihydrogen phosphate and disodium hydrogen phosphate, the concentration is 40mmol/L, and the pH is 7.0.

Wherein the stop solution is a lysine solution of 70mg/mL, and the blocking solution is bovine serum albumin of 18 mg/mL.

Wherein the water bath ultrasound is circulating ultrasound for 2s and stopping ultrasound for 4s, the duration time is 3min, and the ultrasound is carried out by adopting 50 Hz.

Comparative example 1

The conjugated antibody latex was prepared using only 100nm latex microspheres, and the rest was the same as in example 1.

Comparative example 2

The conjugated antibody latex was prepared using only 300nm latex microspheres, and the rest was the same as in example 2.

Comparative example 3

The conjugated antibody latex was prepared using only 150nm latex microspheres, and the rest was the same as in example 3.

Comparative example 4

The conjugated antibody latex was prepared using only 400nm latex microspheres, and the rest was the same as in example 4.

Test conditions and methods

The instrument comprises the following steps: hitachi 7180 full-automatic biochemical analyzer

Parameters are as follows:

dominant wavelength	570℃	Sample (I)	3μL
				Sub-wavelength
	800℃	Reagent 1	150μL
				Reaction temperature	37℃	Reagent 2	50μL
Reaction direction	Liter reaction	Type of reaction	End point method

Reaction step

Calculate Δ a = A2-A1. And determining a working curve by adopting a multipoint nonlinear calibration mode according to the concentration of the multipoint calibrator and the corresponding absorbance change value delta A, wherein the concentration value of the absorbance change of the sample on the working curve is the measured concentration.

Test 1: the linear range of the detection kit of each embodiment and the detection kit of the comparative example of the invention

The test method comprises the following steps: the calibrator solutions with the concentration gradients of 0ng/ml,100ng/ml,200ng/ml,400ng/ml,800ng/ml and 1600ng/ml were taken, the detection kits prepared in examples 1 to 4 of the present invention and the detection kits prepared in comparative examples 1 to 4 were used to perform detection, and calibration working curves of the detection kits were drawn.

And (3) test results: referring to fig. 1 to 4, it can be seen from the calibration working curves plotted according to the detection results of the kits that the linear states of the detection kits of examples 1 to 4 are significantly better than those of the detection kits of comparative examples 1 to 4; in addition, the test kits of examples 1 to 4 of the present invention maintained good linearity in comparison with the test kits of comparative examples 1 to 4, whether in the high concentration value region or the low concentration value region.

Test 2: the accuracy of sample detection of the detection kit of each embodiment and the detection kit of the comparative example

The test method comprises the following steps: 20 samples with different concentrations are prepared, after the accurate value determination of the standard reagent, the detection kit prepared in the embodiment 1-4 of the invention and the detection kit prepared in the comparative example 1-4 are used for detecting, and the deviation of the detection result is calculated.

TABLE 1 accuracy of detection of samples by the detection kit of the present invention in examples 1 to 4

TABLE 2 accuracy of samples detected by the detection kit of comparative examples 1-4

And (3) test results: as is clear from the results in tables 1 and 2, the test samples of the test kits prepared in examples 1 to 4 of the present invention have a small deviation from the set value, which is less than 7%. The deviation of the detection result of the detection kit of the comparative examples 1 to 4 from the fixed value is larger, basically more than 10%, and larger in the low value and high value areas, which indicates that the sensitivity of the detection kit of the comparative examples is lower in the low value range, and the linearity is poorer in the high value area, resulting in obvious deviation of the detection result. The accuracy of the detection kit is obviously superior to that of the detection kit of the comparative example.

Test 3 Heat burst stability test of the kit of example 1 of the invention and the kit of comparative example 1

The test method comprises the following steps: the detection kit prepared in example 1 of the present invention and the detection kit prepared in comparative example 1 were respectively heat-treated at 37 ℃ for 0 day, 3 days, 5 days and 7 days, and the calibrators were measured after different treatment times, respectively, and the Δ a values thereof were recorded, and the change curves thereof were drawn.

And (3) test results: the test result of the 37 ℃ thermal stability of the test kit of the embodiment 1 is shown in figure 5, and the test result of the 37 ℃ thermal stability of the test kit of the comparative example 1 is shown in figure 6. As can be seen from the results shown in FIGS. 5 and 6, the detection kit prepared in example 1 of the present invention has good stability of heat treatment at 37 ℃ and reliable stability, thus ensuring the accuracy of the measured value, and being more suitable for clinical diagnosis.

Test 4 correlation between the detection kit of example 1 of the present invention and the results of the latex agglutination turbidimetry test

The test method comprises the following steps: the content of matrix metalloproteinase-3 in a serum sample was measured using the detection kit prepared in example 1 of the present invention and a commercially available matrix metalloproteinase-3 detection reagent (latex agglutination turbidimetry, sekisui Medical Co., ltd.), and the results measured by the two kits were subjected to regression analysis to examine the correlation between the detection results of the two kits.

And (3) test results: as shown in FIG. 7, it was found from the results of the test that the detection kit prepared in example 1 of the present invention correlated well with the value measured by the commercial matrix metalloproteinase-3 latex agglutination turbidimetry.

In conclusion, the matrix metalloproteinase-3 detection kit and the preparation method thereof provided by the invention can ensure that the low-value detection area and the high-value detection area have higher sensitivity and precision, can ensure that the reagents have enough stability after being stored for a long time, and can be used for detecting clinical samples in a full-automatic biochemical analyzer in a high-throughput manner.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., 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 invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. The matrix metalloproteinase-3 detection kit is characterized by comprising an R1 reagent, an R2 reagent:

the R1 reagent comprises: 160-200 mmol/L HEPES buffer solution, 60-80 mmol/L sodium chloride, 10-15 mmol/L calcium chloride, 1-5% of PEG-6000,0.1-0.5% of triton X-100,0.09-0.12% of sodium azide, and the pH value of the R1 reagent is 7.0-7.5;

the R2 reagent comprises: 30-50 mmol/L HEPES buffer solution, 15-20% of cane sugar, 0.1% of latex microspheres coated with human matrix metalloproteinase-3 antibody, 0.1-0.5% of Tween-20, 0.04-0.08% of PC-300, and the pH value of R2 reagent is 7.0-7.5.

2. The kit for detecting matrix metalloproteinase-3 as claimed in claim 1, wherein the latex microspheres coated with the antibody of human matrix metalloproteinase-3 are prepared by mixing latex microspheres of 100-150 nm and 300-400 nm in a certain proportion.

3. The kit for detecting matrix metalloproteinase-3 as claimed in claim 2, wherein the latex microspheres coated with the human matrix metalloproteinase-3 antibody are latex microspheres with two specifications of 100-150 nm and 300-400 nm according to a ratio of 1:3 to 3:1, and mixing the components in a ratio of 1.

4. The kit for detecting matrix metalloproteinase-3 as claimed in claim 2, wherein the latex microspheres coated with the antibody of human matrix metalloproteinase-3 are prepared by mixing latex microspheres of 120nm and latex microspheres of 360nm in a certain proportion.

5. The kit for detecting matrix metalloproteinase-3 as claimed in claim 4, wherein the latex microspheres coated with the human matrix metalloproteinase-3 antibody are latex microspheres with two specifications of 120nm and 360nm according to a ratio of 1:1, and mixing the components in a ratio of 1.

6. The method for preparing the matrix metalloproteinase-3 detection kit of claim 1, comprising the steps of:

7. The method for preparing the kit for detecting matrix metalloproteinase-3 as claimed in claim 6, wherein the step of preparing latex microspheres coated with human matrix metalloproteinase-3 antibody in step 2 comprises:

step 2.2, adding 2-morpholine ethanesulfonic acid solution into the washed 100-150 nm carboxyl latex microspheres, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding an activating agent to activate the latex, and performing oscillation reaction for 10-20 min to obtain 100-150 nm activated latex; adding 2-morpholine ethanesulfonic acid solution into the washed carboxyl latex microspheres with the particle size of 300-400 nm, performing water bath ultrasound to enable the microspheres to be in a monodisperse state, adding an activating agent to activate the latex, and performing oscillation reaction for 10-20 min to obtain activated latex with the particle size of 300-400 nm;

8. The method for preparing the matrix metalloproteinase-3 detection kit of claim 7, wherein the concentration of the 2-morpholine ethanesulfonic acid solution is 20 to 50mmol/L, and the pH is 6.0 to 6.5.

9. The method for preparing the matrix metalloproteinase-3 detection kit according to claim 7, wherein the amount of the active agent is 10-20 mg/L, the active agent is carbodiimide and N-hydroxysuccinimide, and the mass ratio of the active agent to the N-hydroxysuccinimide is 1:1.

10. The method for preparing the matrix metalloproteinase-3 detection kit of claim 7, wherein the stop solution is ethanolamine, lysine or glycine solution with a concentration of 50-100mg/mL, and the blocking solution is bovine serum albumin with a concentration of 10-30 mg/mL.