CN115639366A - Beta 2-microglobulin fluorescence immunochromatography assay kit and detection method thereof - Google Patents

Abstract

The application provides a beta 2-microglobulin fluoroimmunoassay method determination kit and a preparation method thereof, which belong to the field of in vitro diagnosis immunochromatography detection, the adopted fluoroimmunoassay method takes a beta 2-MG monoclonal antibody and a chicken IgY antibody as detection lines and quality control lines respectively for coating the antibodies, and the beta 2-MG antibody and goat anti-chicken IgY are mixed as a fluorescent microsphere labeled antibody, through selecting appropriate raw materials, reasonably designing the material dosage and selecting the optimal reaction conditions, the detection time is greatly shortened, the detection cost is reduced, the detection sensitivity is also improved, compared with the similar methods, the kit has the advantages of good specificity, high sensitivity, accurate and stable result and the like, the product is simple and convenient to use, the cost is low, and the kit is suitable for popularization.

Description

Beta 2-microglobulin fluorescence immunochromatography assay kit and detection method thereof

Technical Field

The invention belongs to the field of in vitro diagnosis immunochromatography detection, relates to a beta 2-microglobulin fluorescence immunochromatography determination kit and a detection method thereof, and particularly relates to a method for realizing quantitative detection of the content of beta 2-microglobulin in human serum by using the technical principle of fluorescence immunochromatography.

Background

The beta 2-microglobulin is a small molecular globulin produced by lymphocyte, platelet and polymorphonuclear leukocyte, has molecular mass of 11800, and is a single-chain polypeptide consisting of 99 amino acids. It is the beta chain (light chain) part of cell surface Human Leukocyte Antigen (HLA), contains a pair of disulfide bonds in the molecule, does not contain sugar, and has a structure similar to that of an immunoglobulin stable region. Widely present in plasma, urine, cerebrospinal fluid, saliva and colostrum. The synthesis rate and release amount of normal human beta 2-microglobulin from cell membrane are quite constant, beta 2-microglobulin can be freely filtered from glomerulus, 99.9% of beta 2-microglobulin can be absorbed by proximal tubules and decomposed and destroyed in the epithelial cells of the tubules, so that the discharge of beta 2-microglobulin is trace under normal condition, and the increase of serum beta 2-microglobulin can reflect the condition of impaired glomerular filtration function or increased filtration load.

The beta 2-microglobulin in the blood of the kidney transplant patient is obviously increased, which indicates that the organism has rejection reaction, and the beta 2-microglobulin in the blood is increased although the kidney clearance is increased due to the accelerated synthesis of the beta 2-microglobulin. Blood beta 2-microglobulin typically rises to a peak 2 to 3 days after transplantation and then gradually falls. After kidney transplantation, continuously measuring beta 2-microglobulin in blood and urine as sensitive index of glomerulus and renal tubule lesion; the beta 2-microglobulin can also be used for measuring the mild renal hypofunction of the diabetic patient and observing the curative effect, and the measurement has a plurality of values in clinic.

At present, methods for detecting beta 2-microglobulin are common, such as: the chemiluminescence method, the enzyme-linked immunosorbent assay, the immunoenhancement turbidimetry method and the colloidal gold method have the defects of low sensitivity, long detection time, complex process and high detection cost, which not only troubles the diagnosis link but also exists in the rehabilitation link, and accurate treatment is difficult to realize without accurate diagnosis, so that how to accurately screen cases is an urgent problem to be solved.

Disclosure of Invention

The invention aims to provide a beta 2-microglobulin fluorescence immunochromatography assay kit which is good in specificity, high in sensitivity, accurate in result and stable.

The technical scheme provided by the invention is as follows: the beta 2-microglobulin fluorescence immunochromatography assay kit comprises a reagent card and diluent, wherein the reagent card comprises a PVC plate, and a sample pad, a combination pad, an NC membrane and absorbent paper which are sequentially arranged on the PVC plate, and the NC membrane is provided with a detection line and a quality control line;

the detection line is coated with a beta 2-MG monoclonal antibody, and the coating concentration is 0.8-1.2MG/ml;

the quality control line is coated with chicken IgY antibody with the coating concentration of 0.8-1.2mg/ml;

the binding pad is sprayed with an antibody solution marked by fluorescent microspheres, wherein the antibody solution marked by the fluorescent microspheres is a mixture of a beta 2-MG antibody containing another binding site marked by the fluorescent microspheres and a goat anti-chicken IgY antibody marked by the fluorescent microspheres.

Preferably, the fluorescent microsphere for marking is Eu-fluorescent nano microsphere with the particle size of 300nm of Nanjing micro-measuring biology company.

Preferably, the labeled amount of the beta 2-MG labeled antibody is 10 mug, and the labeled amount of the goat anti-chicken IgY labeled antibody is 16 mug.

Preferably, the amount of the labeled antibody solution sprayed is 4 to 6. Mu.L/cm.

Preferably, the spraying amount of the coating working solution is 0.8-1.2 muL/cm.

Preferably, in the other binding site β 2-MG antibody labeled by the fluorescent microsphere on the binding pad, the ratio of the fluorescent microsphere to the β 2-MG antibody is 20.

Preferably, in the goat anti-chicken IgY antibody marked by the fluorescent microsphere on the conjugate pad, the feeding ratio of the fluorescent microsphere to the goat anti-chicken IgY antibody is 25.

Preferably, the fluorescent microsphere is marked with a beta 2-MG marked antibody, and the steps are as follows:

a. adding 1mL of marking buffer MES into 2mL of a centrifuge tube; 20 mu L of the fluorescent microspheres with the solid content of 1 percent are uniformly mixed in the marking buffer solution and are uniformly mixed again in a vortex manner;

b. adding 100 μ L of labeled activation solution, mixing, rotating, mixing, and reacting for 30min;

c. centrifuging for 30min at 10000rpm/min; discarding the supernatant, adding 1000 mu L of labeling buffer solution, and performing ultrasonic treatment;

d. adding 10 mu g of beta 2-MG labeled antibody, uniformly mixing, rotating, uniformly mixing and reacting for 30min;

e. adding 100 mu L of labeled confining liquid, performing ultrasonic treatment, rotating, uniformly mixing and reacting for 2h;

f. centrifuging for 30min; removing supernatant, adding 1mL of labeled preservation solution, and performing ultrasonic treatment;

g. transferring the marker into a new 10mL centrifuge tube, sucking 4mL of the marker preservation solution, washing 2mL of the centrifuge tube used for the marker, transferring the washed marker into the 10mL centrifuge tube, and mixing the marker and the centrifuge tube in a vortex mode.

Preferably, the fluorescent microsphere is used for marking the goat anti-chicken IgY antibody, and the steps are as follows:

a. adding 1mL of marking buffer MES into 2mL of a centrifuge tube; 20 mu L of the fluorescent microspheres with the solid content of 1 percent are uniformly mixed in the marking buffer solution and are uniformly mixed again in a vortex manner;

b. adding 100 μ L of labeled activation solution, mixing, rotating, mixing, and reacting for 30min;

c. centrifuging for 30min at 10000rpm/min; discarding supernatant, adding 1000. Mu.L of labeling buffer solution, and performing ultrasonic treatment;

d. adding 16 mu g of goat anti-chicken IgY antibody, mixing uniformly, rotating, mixing uniformly and reacting for 30min;

e. adding 100 mu L of labeled confining liquid, performing ultrasonic treatment, rotating, uniformly mixing and reacting for 2h;

f. centrifuging for 30min; removing supernatant, adding 1mL of labeled preservation solution, and performing ultrasonic treatment;

g. transferring the marker into a new 10mL centrifuge tube, sucking 4mL of the marker preservation solution, washing 2mL of the centrifuge tube used for the marker, transferring the washed marker into the 10mL centrifuge tube, and mixing the marker and the centrifuge tube in a vortex mode.

Preferably, the formula of the diluent is as follows: 0.01M PBS, pH7.4+0.05% of Proclin 300.

The invention aims to provide a preparation method of a beta 2-microglobulin fluorescence immunochromatography assay kit.

The technical scheme provided by the invention is as follows: the preparation method of the beta 2-microglobulin fluorescence immunochromatography assay kit comprises the following steps of:

step 1: arranging a detection line and a quality control line on an NC membrane, preparing coating working solution by respectively using the beta 2-MG monoclonal antibody and the chicken IgY antibody according to the concentration of 0.8-1.2MG/mL, coating the coating working solution by using the coating amount of 0.8-1.2MG/mL, and scribing the coating working solution on the detection line and the quality control line on the NC membrane by using a gold marking scribing machine and then drying the coating working solution;

step 2: spraying the antibody solution marked by the fluorescent microspheres onto the cut bonding pads by a gold spraying and scribing machine according to the spraying amount of 4-6 mu L/cm, and drying;

and step 3: the sample pad is dried after being treated by the sample pad treatment liquid;

and 4, step 4: and sequentially adhering the processed sample pad, the processed combination pad, the processed NC membrane and the processed absorbent paper to a PVC plate to assemble the reagent card.

Preferably, the sample pad treatment fluid formulation is prepared from the following components: 10mg of anti-human RBC antibody, 20mg of blocker 001, 10g of bovine serum albumin, 5mL of Tween-20 and 1000mL of 0.2M borax borate buffer solution with pH8.0.

The principle of the kit comprises the following steps: adopts a double-antibody sandwich method and utilizes the technical principle of fluorescence immunochromatography. During testing, a sample and diluent are uniformly mixed, the mixture is dripped into a sample adding hole of a reagent card, chromatography is carried out under a capillary effect, a beta 2-MG antigen in the sample is combined with a fluorescence labeling beta 2-MG antibody, the diffusion is carried out to a testing area, the detection line is coated with the beta 2-MG monoclonal antibody for capturing, and an antibody-antigen-fluorescence antibody compound is formed; the concentration of the beta 2-MG in the sample is in direct proportion to the fluorescence intensity of the complex, and the dry type fluorescence immunoassay analyzer converts the fluorescence signal value into the concentration of the beta 2-MG in the sample according to a set standard curve. When the function of glomerulus and renal tubule of the body is damaged, the content of beta 2-MG in the body is abnormally increased, and when the content of beta 2-MG is more than or equal to 2.7MG/L, the patient is suggested to be possibly at risk of the damaged renal function.

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

the application provides a beta 2-microglobulin fluorescence immunochromatography assay kit, which takes a beta 2-MG monoclonal antibody and a chicken IgY antibody as a detection line and a quality control line respectively for coating the antibodies, takes a mixture of the beta 2-MG antibody and goat anti-chicken IgY as a fluorescence microsphere labeled antibody, adopts a fluorescence immunochromatography method, and is similar to the current common method for detecting the beta 2-microglobulin as follows: compared with a chemiluminescence method, an enzyme-linked immunosorbent assay, an immune enhanced turbidimetry method and a colloidal gold method, the method not only greatly shortens the detection time, reduces the detection cost, but also improves the detection sensitivity, and compared with the similar methods, the method has the advantages of good specificity, high sensitivity, accurate and stable result and the like.

The application provides a preparation method of a beta 2-microglobulin fluorescence immunochromatography assay kit, which is simple in preparation process by selecting appropriate raw materials, reasonably designing the using amount of each material and selecting the optimal reaction condition, and the prepared product is good in specificity, high in sensitivity, accurate and stable in result, low in detection cost, simple and convenient to use, low in cost and suitable for popularization.

Drawings

FIG. 1 is a schematic diagram of the structure of a reagent card in an embodiment of the present application;

FIG. 2 shows the results of different combinations of gradient ratios of T-line labeled antibodies and C-line labeled antibodies in the examples of the present application;

FIG. 3 is a scatter plot of a standard curve of β 2-MG in the examples of the present application;

FIG. 4 shows the clinical alignment of the test of the examples of the present application with the control reagents.

Detailed Description

The following describes a specific embodiment of the present invention with reference to specific embodiments.

Example 1:

the beta 2-microglobulin fluorescence immunochromatography assay kit comprises a reagent card and diluent, wherein the reagent card comprises a PVC plate, and a sample pad, a combination pad, an NC membrane and absorbent paper which are sequentially arranged on the PVC plate, and the NC membrane is provided with a detection line and a quality control line;

the detection line is coated with a beta 2-MG monoclonal antibody, and the coating concentration is 0.8-1.2MG/ml;

the quality control line is coated with chicken IgY antibody with the coating concentration of 0.8-1.2mg/ml;

the binding pad is sprayed with an antibody solution marked by fluorescent microspheres, wherein the antibody solution marked by the fluorescent microspheres is a mixture of a beta 2-MG antibody containing another binding site marked by the fluorescent microspheres and a goat anti-chicken IgY antibody marked by the fluorescent microspheres.

Example 2:

a method for preparing a beta 2-microglobulin quantitative fluorescence immunochromatography assay kit comprises the following steps:

step 1: arranging T and C lines on an NC membrane, preparing coating working solution by beta 2-MG coated antibody and chicken IgY coated antibody according to the concentration of 1.0MG/mL respectively, marking the coating working solution on the NC membrane by a gold mark scribing machine according to the coating amount of 1.0 mu L/cm, placing the NC membrane in an electrothermal blowing dry box with the humidity of less than or equal to 30 percent and the temperature of (37 +/-2) DEG C for drying for 16-24 h;

and 2, step: spraying the redissolved fluorescent microsphere labeled antibody solution onto the cut bonding pad by a gold spraying and scribing machine according to the spraying amount of 4 mu L/cm, and drying at the temperature of (37 +/-2) DEG C for 16-24 h under the condition that the humidity is less than or equal to 30%;

and step 3: setting the height of the wheel shaft of the paper-soaking press pad, pouring the sample pad treatment solution, placing the treated sample pad on an air-drying rack, drying the sample pad overnight (16-24 h) at the temperature of (37 +/-2) DEG C;

and 4, step 4: the processed sample pad, the bonding pad, the NC film and the absorbent paper are sequentially attached to a PVC adhesive base plate as shown in fig. 1, the NC film is at the bottom, each component is overlapped by about 2mm or more to be assembled, and the prepared experimental material is assembled into a reagent card for standby.

The redissolved fluorescent microsphere labeled antibody solution comprises a fluorescent microsphere labeled beta 2-MG labeled antibody and a fluorescent microsphere labeled goat anti-chicken IgY antibody which are mixed by 1:1.

The fluorescent microsphere labeled beta 2-MG labeled antibody comprises the following steps:

a. adding 1mL of marking buffer MES into 2mL of a centrifuge tube; 20 mu L of the fluorescent microspheres with the solid content of 1 percent are uniformly mixed in the marking buffer solution and are uniformly mixed again in a vortex manner;

b. adding 100 μ L of labeled activation solution, mixing, rotating, mixing, and reacting for 30min;

c. centrifuging for 30min at 10000rpm/min; discarding the supernatant, adding 1000 mu L of labeling buffer solution, and performing ultrasonic treatment;

d. adding 10 mu g of beta 2-MG labeled antibody, uniformly mixing, rotating, uniformly mixing and reacting for 30min;

e. adding 100 mu L of labeled confining liquid, performing ultrasonic treatment, rotating, uniformly mixing and reacting for 2h;

f. centrifuging for 30min; removing supernatant, adding 1mL of labeled preservation solution, and performing ultrasonic treatment;

g. transferring the marker into a new 10mL centrifuge tube, sucking 4mL of the marker preservation solution, washing 2mL of the centrifuge tube used for the marker, transferring the washed marker into the 10mL centrifuge tube, and mixing the marker and the centrifuge tube in a vortex mode.

The fluorescent microsphere is used for marking the anti-chicken IgY antibody of sheep, and the steps are as follows:

a. adding 1mL of marking buffer MES into 2mL of a centrifuge tube; 20 mu L of the fluorescent microspheres with the solid content of 1 percent are uniformly mixed in the marking buffer solution and are uniformly mixed again in a vortex manner;

b. adding 100 μ L of labeled activation solution, mixing, rotating, mixing, and reacting for 30min;

c. centrifuging for 30min at 10000rpm/min; discarding the supernatant, adding 1000 mu L of labeling buffer solution, and performing ultrasonic treatment;

d. adding 16 mu g of goat anti-chicken IgY antibody, mixing uniformly, rotating, mixing uniformly and reacting for 30min;

e. adding 100 mu L of labeled confining liquid, performing ultrasonic treatment, rotating, uniformly mixing and reacting for 2h;

f. centrifuging for 30min; removing supernatant, adding 1mL of labeled preservation solution, and performing ultrasonic treatment;

g. transferring the marker into a new 10mL centrifuge tube, sucking 4mL of the marker preservation solution, washing 2mL of the centrifuge tube used for the marker, transferring the washed marker into the 10mL centrifuge tube, and mixing the marker and the centrifuge tube in a vortex mode.

Furthermore, the control line (i.e. C line) coated antibody and the control line labeled antibody of the present application select shanghai choo chicken IgY and goat anti-chicken IgY as the control line coated antibody, which has better T/C precision, and the control line (i.e. C line) coated antibody and the control line labeled antibody are selected as follows:

mouse IgG, goat anti-mouse IgG, chicken IgY and goat anti-chicken IgY antibodies of Shanghai tide Biotech Co., ltd are used, and the beta 2-MG antibody pair is diluted with ultrapure water according to the concentration commonly used, and a reagent card is prepared and detected by using a reference product. The specific operation is basically consistent with the antibody selection operation of the detection line, and the detection results (line C) of the reagent cards prepared by antibodies of different manufacturers are shown in Table 1:

TABLE 1 preparation of antibody reagent card test results from different manufacturers (C line)

As can be seen from Table 1, there are some differences in the linear gradients of the antibody preparation reagent card test enterprise reference products of each manufacturer, wherein Shanghai collar tide (chicken IgY + goat anti-chicken IgY) > Shanghai collar tide (mouse IgG + goat anti-mouse IgG); the T/C precision Shanghai collar tide (chicken IgY + sheep anti-chicken IgY) is better. Selecting chicken IgY and goat anti-chicken IgY of Shanghai collar tide as quality control line coated antibody and labeled antibody.

Further, the fluorescent microsphere selects Nanjing micro-biological Eu-fluorescent nano-microsphere 300nm as the fluorescent microsphere for marking, the linear gradient 51.41 is optimal, the T/C precision is less than 5%, and the fluorescent microsphere is selected as follows:

the particle size of Eu-fluorescent nano microsphere of Nanjing micro-measuring Biotechnology Limited is about 200nm, 300nm and the particle size, fluorescence intensity and PDI coefficient of hydroxylated fluorescent nano microsphere of Changsha Mei Niu Biotechnology Limited are tested, then the Eu-fluorescent nano microsphere is marked with antibody in turn, the binding pad is prepared to be made into a reagent card, the reagent card is detected by enterprise reference products, and the detection results of the reagent card prepared by fluorescent microspheres of different manufacturers are shown in Table 2:

table 2 detection results of reagent cards prepared by fluorescent microspheres of different manufacturers

From the above table 2, it can be seen that the linear gradient 51.41 of Nanjing micro-assay 300nm is the most preferable, and the T/C precision is less than 5%, so that the Nanjing micro-assay biological Eu-fluorescent nano-microsphere 300nm is selected as the fluorescent microsphere for marking.

Further, the formula of the marking preservation solution is prepared according to 1000 mL: bovine Serum Albumin (BSA) 20g, sucrose (sucrose) 20g, sodium caseinate (Casein salt) 5g, sodium caseinate (Casein) 50g, 1mL Proclin 300, 0.02M (pH8.0), and PBS (PBS) 1000mL, wherein the labeling buffer is determined as follows:

4 different marking preservation liquids are prepared for marking preservation of the markers, a combination pad is prepared, and different reagent cards are prepared according to the biological materials determined by the previous experiment. And observing the storage state of the marker, and preparing a reference product of a test enterprise of the reagent card assembled by the bonding pad. According to the detection result, a formula 4 (prepared according to 1000 mL: 20g of bovine serum albumin, 20g of sucrose, 5g of casein sodium salt, 50g of casein sodium, 1mL of procolin 300 and 1000mL of 0.02M PBS buffer solution with the pH value of 8.0) is finally selected as a formula of the marked preservation solution of the beta 2-microglobulin (beta 2-MG) determination kit (fluorescence immunochromatography) of the application.

Further, 0.01M PBS containing 1% sucrose and pH7.4 is selected as coating liquid, and after the coating liquid is prepared into a reagent strip, the coating liquid is selected to be clear liquid, no crystal precipitation exists, the linear gradient is good, the pH of the coating liquid is 7.4, and the linear gradient is high, and the coating liquid is selected as follows:

respectively diluting coating antibodies beta 2-MG and chicken IgY with 4 formulas of coating solutions (0.01MpH7.4 and 8.0PBS, 0.02MpH7.4 and 8.0 PBS) to coating concentration, preparing into reagent strips, and selecting the coating solution as a clear solution, a buffer solution without crystal precipitation and with good linear gradient as the coating solution.

TABLE 3 preparation of reagent card test results for different coating solutions

The pH value of the coating solution has great influence on the linear gradient, and when the pH value of the coating solution is 7.4, the linear gradient is high, so that the pH value of the selected coating solution is 7.4; taken together, 0.01M PBS containing 1% sucrose, pH7.4, was selected as the final formulation for the coating solution.

Further, the formulation of the sample pad treatment solution was prepared at 1000 mL: 10mg of anti-human RBC antibody, 00120mg of blocking agent, 10g of bovine serum albumin, 5mL of Tween-20 and 1000mL of 0.2M pH8.0 borax borate buffer solution, wherein the preparation has strong anti-interference capability, and the formula of the sample pad treatment solution is determined as follows:

4 sample pad treatment solutions of different formulations (treatment solution 1: 10MG of anti-human RBC antibody, 001 10MG of blocking agent, 10g of bovine serum albumin, 5mL of Tween-20, 0.2M pH8.0 borax borate buffer 1000mL; treatment solution 2: 20MG of anti-human RBC antibody, 001 0MG of blocking agent, 10g of bovine serum albumin, 5mL of Tween-20, 0.2M pH8.0 borax borate buffer 1000mL; treatment solution 3: 10MG of anti-human RBC antibody, 001 MG of blocking agent, 10g of bovine serum albumin, 5mL of Tween-20, 0.2M pH8.0 borax borate buffer 1000mL; treatment solution 4: 20MG of anti-human RBC antibody, 10MG of blocking agent, 10g of bovine serum albumin, 5mL of Tween-20, 0.2M pH8.0 borax borate buffer 1000 mL) were prepared, and the test results were shown in reference samples of beta 2-MG concentrations of 0.3MG/L, 1MG/L, 5MG/L, 10MG/L, and 20MG/L, and 4 MG/L, respectively;

table 4 four sample pad treatment solutions the effect of testing a sample of a healthy person for the addition of interferents

As can be seen from the above table 4, the reference substance added with the interferent in the test of 10mg of the blocker 001 shows T/C doubling increase, but the T/C is obviously lower than that of an untreated sample pad reagent card, and the reference substance has certain anti-interference capability; the 20mg blocker 001 test has no obvious difference between the T/C of the reference product added with the interferent and the T/C of the reference product not added with the interferent, the anti-interference capability is stronger, the sample pad treatment fluid 2 and the sample pad treatment fluid 3 both meet the requirements, the cost factor is synthesized, and the sample pad treatment fluid 3 (prepared according to 1000 mL: 10mg of anti-human RBC antibody, 20mg blocker 001, 10g of bovine serum albumin, 5mL of Tween-20, and 1000mL of 0.2M borax borate buffer solution with pH of 8.0) is finally selected as the formula of the sample pad treatment fluid.

Further, the formulation of the sample diluent 4 is: 0.01M PBS, 0.05 percent of Proclin 300 with pH7.4+ and T/C precision of less than 6 percent, the whole is better, and the formula of a sample diluent is determined as follows:

4 sample dilutions (0.01M pH6.8,7.0,7.2,7.4 four PBS buffers) were prepared for each of the reference samples and tested as shown in Table 5:

TABLE 5 test results for different sample dilutions

As can be seen from Table 5 above, the T/C precision of sample dilution 4 was < 6%. And the whole is better, so the sample diluent 4 is selected, and the formula is as follows: 0.01M PBS, pH7.4+0.05% of Proclin 300.

Furthermore, the application selects 10 mu g of beta 2-MG labeled antibody and 16 mu g of goat anti-chicken IgY labeled antibody to be fed in a combined manner, namely the fluorescent microspheres: antibody =20, 1 is the T-line dosage ratio; fluorescent microspheres: antibody =25, C-line charge ratio, 66.1, which is a relatively best concentration gradient ratio, higher sensitivity, better linearity, and less than 6% precision, and the T-line labeled antibody charge ratio and C-line labeled antibody charge ratio are determined as follows:

orthogonal experiments are carried out with the T-line antibody labeling quantity set as 5 mug, 10 mug and 20 mug, and the C-line antibody labeling quantity set as 8 mug, 16 mug and 32 mug, 9 groups of different reagent cards are prepared, and beta 2-MG reference products with beta 2-MG concentrations of 0.3MG/L, 1MG/L, 5MG/L, 10MG/L and 20MG/L are detected respectively.

TABLE 6 feeding combination of T and C lines

TABLE 7T/C precision results for different T-line and C-line labeled antibody feed ratios

From table 7 and fig. 2, it can be seen that the concentration gradient ratio of the reference substance of each enterprise in combination 5 is 66.1, which is relatively best, the sensitivity is higher, the linearity is better, and the precision is less than 6%. Selecting 10 mu g of beta 2-MG labeled antibody and 16 mu g of goat anti-chicken IgY labeled antibody to be combined and fed, namely fluorescent microspheres: antibody =20, 1 is the T-line dosage ratio; fluorescent microspheres: antibody =25, C-line dosage ratio.

Further, the concentration of the beta 2-MG coated antibody is 1.0MG/mL, the concentration of the chicken IgY coated antibody is 1.0MG/mL, the final T line and C line coated concentrations are obtained, the detection sensitivity of the configuration is high, the linearity is good, and the precision is less than 6%, and the concentration of the coated antibody is determined as follows:

the using concentrations of the T-line and C-line coated antibodies are set to be 0.8MG/mL, 1.0MG/mL and 1.2MG/mL, 9 different combinations are prepared as shown in the following table, beta 2-MG reference products with the respective detection concentrations of 0.3MG/L, 1MG/L, 5MG/L, 10MG/L and 20MG/L are respectively detected, and the test results are shown in the table 9;

TABLE 8 combination of coating concentrations of T thread and C thread

TABLE 9T/C precision results for different combinations of T-line and C-line envelope concentrations

T/C CV	0.3mg/L	1mg/L	5mg/L	10mg/L	20mg/L
						Combination 1	6.77％	9.72％	8.18％	9.95％	9.32％
Combination 2	3.93％	7.99％	3.26％	5.19％	7.07％
						Combination 3	6.03％	8.64％	6.58％	8.21％	6.93％
Combination 4	6.07％	8.06％	7.25％	8.78％	9.92％
						Combination
5	5.72％	2.25％	3.37％	4.43％	3.52％
						Combination 6	6.23％	9.44％	8.68％	5.67％	9.78％
Combination 7	5.04％	7.07％	8.62％	5.37％	8.82％
						Combination 8	6.12％	7.90％	7.54％	7.10％	6.00％
Combination 9	7.76％	9.96％	5.89％	5.24％	7.16％

From table 9 above, it can be seen that the combination 5 has high detection sensitivity and good linearity, and the precision is less than 6%. The concentration of beta 2-MG coating antibody is 1.0MG/mL, and the concentration of chicken IgY coating antibody is 1.0MG/mL, which is the final T line and C line coating concentration.

Furthermore, the spraying amount of the labeled antibody solution is 4 mu L/cm, the T/C precision is less than 6 percent, the relative optimization is realized, the linear gradient of each spraying amount has no obvious difference, and the spraying amount of the labeled antibody solution is determined as follows:

respectively loading 3 muL/cm, 4 muL/cm and 5 muL/cm of fluorescent microsphere labeled antibody solution into a gold spraying scribing machine, and spraying the gold spraying scribing machine onto the cut bonding pads; drying overnight (16-24 h), respectively assembling the prepared experimental materials into reagent cards, and then evaluating the reagent cards by using enterprise reference products.

TABLE 10 different spray T/C accuracy results

T/C CV	0.3mg/L	1mg/L	5mg/L	10mg/L	20mg/L
						3uL/cm	8.89％	7.82％	9.81％	8.37％	5.40％
4uL/cm	4.23％	3.18％	3.45％	5.04％	4.42％
						5uL/cm	8.34％	8.26％	5.91％	9.56％	6.58％

The T/C precision of the spraying amount of 4 mu L/cm is less than 6 percent, the spraying amount is relatively optimal, the linear gradient of each spraying amount has no obvious difference, and 4 mu L/cm is selected as the spraying amount of the labeled antibody solution by comprehensively considering the cost and the performance.

Further, the spraying amount of the coating working solution is 1.0 μ L/cm, the T/C precision is less than 6%, and the spraying amount of the coating working solution is determined as follows:

respectively scribing coating working solution of T thread and C thread on an NC film by using a gold marking scribing machine at 0.8 muL/cm, 1.0 muL/cm and 1.2 muL/cm, drying the NC film in an electrothermal blowing dry box (16-24 h), respectively assembling the prepared experimental materials into a reagent card, and then evaluating by using enterprise reference products.

TABLE 11T/C precision results for different spray volumes

T/C CV	0.3mg/L	1mg/L	5mg/L	10mg/L	20mg/L
						0.8uL/cm	8.55％	6.88％	8.93％	9.16％	6.68％
1.0uL/cm	5.06％	3.26％	4.79％	2.17％	5.03％
						1.2uL/cm	6.42％	5.04％	5.17％	8.03％	9.36％

The diffusion trace after the film scribing is enlarged along with the increase of the spraying amount, the precision of 1.0 mu L/cm T/C is less than 6 percent, the precision is relatively optimal, and 1.0 mu L/cm is selected as the spraying amount of the coating working solution in consideration of the appearance, the performance and the cost.

Further, the optimum time required for the reaction was 10 mm, and the reaction time was determined as follows:

preparing a reagent card according to a determined production process, and detecting beta 2-MG reference substances with concentrations of 0.3MG/L, 1MG/L, 5MG/L, 10MG/L and 20MG/L respectively by using the prepared detection card according to set reaction time (5 min, 10min, 15min and 20 min). The optimum time required for the reaction was 10 mm according to the linear horizontal screening of the results.

Furthermore, the sample dosage is 100 μ L, which is the optimal sample dosage of the beta 2-microglobulin (beta 2-MG) determination kit (fluorescence immunochromatography), the designed chromatography process is fast, the absorbent paper has no fluorescent microspheres, the linear gradient is good, the sample does not overflow, and the sample dosage is determined as follows:

the preparation of the reagent card is carried out according to the determined production process, and the prepared detection card detects beta 2-MG reference substances with the concentrations of 0.3MG/L, 1MG/L, 5MG/L, 10MG/L and 20MG/L according to the set sample adding amount (80 muL, 90 muL, 100 muL, 110 muL and 120 muL). The chromatographic process is fast, the water absorption paper has no fluorescent microspheres, the linear gradient is good, and the sample has no overflow. Finally, the sample dosage of 100 mu L is selected as the optimal sample dosage of the beta 2-microglobulin (beta 2-MG) determination kit (fluorescence immunochromatography).

The kits prepared in the examples were subjected to the following performance tests:

1. creation of a Standard Curve

According to the requirements, the beta 2-MG antigen is respectively prepared into reference substances with the concentrations of 0.3MG/L, 1MG/L, 5MG/L, 10MG/L and 20MG/L by using degreased normal human serum and detected, the assembled reagent card is taken to repeat the measurement for 3 times, and the relative deviation (B) and the linear correlation coefficient (r) between the average value (M) of the detection results of 3 times and the theoretical value of each sample are calculated according to the following formulas (1) and (2).

B＝(M-T)/T×100％....................(1)

In the formula:

b-relative deviation;

m is the average value of the detection results;

t is a theoretical value.

In the formula:

n-number of samples measured;

xi-determining the concentration of the sample;

yi-mean of measured values corresponding to the concentration of the measured sample for 4 repeated measurements;

r-linear correlation coefficient;

i——1，2，3，……n。

the detection results are as follows:

TABLE 12 testing of Linear references

As can be seen from Table 12 and FIG. 3, the test results showed a correlation with the theoretical concentration, and the correlation r ² =0.9981, scatterplot equation y =1.0379x-0.147, correlation is good.

2. Accuracy detection

Measuring beta 2-microglobulin enterprise reference products of 1mg/L, 5mg/L and 10mg/L, detecting according to the steps in the specification, repeatedly measuring for 3 times, recording the test result of each time as (Xi), and judging as qualified if the relative deviation of the results of 3 times is not more than +/-15%. If the results of more than or equal to 2 times are not met, the product is judged to be unqualified. If the results of 1 test are not satisfactory, the test is repeated for 20 times, the relative deviation is calculated according to the formula (1), and if the relative deviation of the results of 19 tests is not more than +/-10%, the test is judged to be qualified.

Bi＝(Xi-T)/T×100％...................................(1)

In the formula:

bi-relative deviation;

xi-concentration measured at each time;

t-marker concentration.

The detection results are as follows:

TABLE 13 test of accuracy reference

Indicating concentration (mg/L)	1	5.00	10.00
				Measured value 1 (mg/L)	1.08	4.61	9.42
Measured value 2 (mg/L)	0.95	4.82	10.78
				Measured value 3 (mg/L)	1.00	5.19	10.22
Relative deviation B1 (%)	8.00％	-7.80％	-5.80％
				Relative deviation B2 (%)	-5.00％	-3.60％	7.80％
Relative deviation B3 (%)	0.00％	3.80％	2.20％

From Table 13 above, it can be seen that the relative deviations of the accuracy references are all < + > 10%.

3. Repeatability detection

The same operator detects the beta 2-MG enterprise reference products with the concentrations of 1.00MG/L, 5.00MG/L and 10.00MG/L on the same instrument, each sample is repeatedly measured for 10 times, and the results are as follows:

table 14 testing of repetitive references

As can be seen from Table 14 above, the precision of the repetitive references was < 10%.

4. Minimum detection limit detection

The reagent card is used for detecting the beta 2-MG enterprise reference product with the concentration of 0.270-0.290MG/L, the lowest detection limit is not more than 0.3MG/L, and the detection result is as follows:

TABLE 15 detection Limit test results

As can be seen from the above table, the lowest detection limit is not more than 0.3mg/L.

5. HAMA Effect assessment

And testing the prepared concentration by using the reagent card, performing statistical analysis on the test data, and performing statistical analysis on the test result to meet the acceptable standard of a design test, namely the HAMA effect performance index serving as the beta 2-MG. The detection results are as follows:

HAMA serum concentrations formulated in Table 16

Group of	HAMA serum concentration (ng/mL)
		Experimental group 1	90
Experimental group 2	60
		Experimental group 3	30
Control group	0

TABLE 17 HAMA test results for different samples

As can be seen from the test data in Table 17, when the HAMA serum concentration is less than or equal to 60ng/mL, the relative deviation is less than or equal to +/-10%, and the HAMA serum concentration is less than or equal to 60ng/mL, so that the detection sample is not obviously interfered.

6. Clinical sample detection with experimental and control reagents

81 cases of beta 2-microglobulin (beta 2-MG) assay reagents (fluorescence immunochromatography) produced by assembling the reagent card and the contrast reagent were clinically compared, and the detection results are shown in FIG. 4.

As can be seen from FIG. 4, the clinical relevance of the present application to the control reagent test is good, y =0.9813x +1.3467, R ² ＝0.9878。

The results show that the product prepared by the method has the advantages of good specificity, high sensitivity, accurate and stable result, low detection cost, simple and convenient product use, low cost and suitability for popularization.

Claims (10)

1. The utility model provides a beta 2-microglobulin fluorescence immunochromatography assay kit, includes reagent card and diluent, the reagent card includes the PVC board and locates in proper order sample pad, combination pad, NC membrane and the paper that absorbs water on the PVC board, be equipped with detection line and quality control line on the NC membrane, its characterized in that:

the detection line is coated with a beta 2-MG monoclonal antibody, and the coating concentration is 0.8-1.2MG/ml;

the quality control line is coated with chicken IgY antibody with the coating concentration of 0.8-1.2mg/ml;

the binding pad is sprayed with an antibody solution marked by fluorescent microspheres, wherein the antibody solution marked by the fluorescent microspheres is a mixture of a beta 2-MG antibody containing another binding site marked by the fluorescent microspheres and a goat anti-chicken IgY antibody marked by the fluorescent microspheres.

2. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: the fluorescent microsphere for marking is Eu-fluorescent nano microsphere with the particle size of 300nm of Nanjing micro-measuring biology company.

3. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: the labeled amount of the beta 2-MG labeled antibody is 10 mug, and the labeled amount of the goat anti-chicken IgY labeled antibody is 16 mug.

4. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: the spraying amount of the labeled antibody solution is 4-6 mu L/cm;

the spraying amount of the coating working solution is 0.8-1.2 mu L/cm.

5. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: in another binding site beta 2-MG antibody marked by the fluorescent microsphere on the binding pad, the feeding ratio of the fluorescent microsphere to the beta 2-MG antibody is 20;

in the goat anti-chicken IgY antibody marked by the fluorescent microspheres on the combination pad, the feeding ratio of the fluorescent microspheres to the goat anti-chicken IgY antibody is 25.

6. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: the method for labeling the beta 2-MG labeled antibody by the fluorescent microsphere comprises the following steps:

a. adding 1mL of marking buffer MES into 2mL of a centrifuge tube, uniformly mixing 20 mu L of fluorescent microspheres with the solid content of 1% in the marking buffer, and uniformly mixing by vortexing again;

b. adding 100 μ L of labeled activation solution, mixing, rotating, mixing, and reacting for 30min;

c. centrifuging for 30min at 10000rpm/min, discarding supernatant, adding 1000 μ L of labeling buffer solution, and performing ultrasonic treatment;

d. adding 10 mu g of beta 2-MG labeled antibody, uniformly mixing, rotating, uniformly mixing and reacting for 30min;

e. adding 100 mu L of labeled confining liquid, performing ultrasonic treatment, rotating, uniformly mixing and reacting for 2h;

f. centrifuging for 30min, discarding supernatant, adding 1mL of labeled preservation solution, and performing ultrasonic treatment;

g. transferring the marker into a new 10mL centrifuge tube, sucking 4mL of the marker preservation solution, washing 2mL of the centrifuge tube used for the marker, transferring the washed marker into the 10mL centrifuge tube, and mixing the marker and the centrifuge tube in a vortex mode.

7. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: the method for labeling the goat anti-chicken IgY antibody by the fluorescent microspheres comprises the following steps:

a. adding 1mL of marking buffer MES into 2mL of a centrifuge tube, uniformly mixing 20 mu L of fluorescent microspheres with the solid content of 1% in the marking buffer, and uniformly mixing by vortexing again;

b. adding 100 μ L of labeled activation solution, mixing, rotating, mixing, and reacting for 30min;

c. centrifuging for 30min at 10000rpm/min, discarding supernatant, adding 1000 μ L of labeling buffer solution, and performing ultrasonic treatment;

d. adding 16 mu g of goat anti-chicken IgY antibody, mixing uniformly, rotating, mixing uniformly and reacting for 30min;

e. adding 100 mu L of marked confining liquid, performing ultrasonic treatment, and rotating and uniformly mixing to react for 2h;

f. centrifuging for 30min, discarding supernatant, adding 1mL of labeled preservation solution, and performing ultrasonic treatment;

g. transferring the marker into a new 10mL centrifuge tube, sucking 4mL of the marker preservation solution, washing 2mL of the centrifuge tube used for the marker, transferring the washed marker into the 10mL centrifuge tube, and mixing the marker and the centrifuge tube in a vortex mode.

8. The β 2-microglobulin fluorescence immunochromatography assay kit according to claim 1, characterized in that: the formula of the diluent is as follows: 0.01M PBS, pH7.4+0.05% of Proclin 300.

9. A method for preparing a β 2-microglobulin fluorescence immunochromatography assay kit according to any one of claims 1 to 8, comprising preparing a reagent card by the steps of:

step 1: arranging a detection line and a quality control line on an NC membrane, preparing coating working solution by respectively using the beta 2-MG monoclonal antibody and the chicken IgY antibody according to the concentration of 0.8-1.2MG/mL, coating the coating working solution by using the coating amount of 0.8-1.2MG/mL, and scribing the coating working solution on the detection line and the quality control line on the NC membrane by using a gold marking scribing machine and then drying the coating working solution;

step 2: spraying the antibody solution marked by the fluorescent microspheres onto the cut bonding pads by a gold spraying and scribing machine according to the spraying amount of 4-6 mu L/cm, and drying;

and step 3: the sample pad is dried after being treated by the sample pad treatment liquid;

and 4, step 4: and sequentially adhering the processed sample pad, the processed combination pad, the processed NC membrane and the processed absorbent paper to a PVC plate to assemble the reagent card.

10. The method for preparing a β 2-microglobulin fluorescence immunochromatography assay kit according to claim 9, wherein the sample pad treatment solution is prepared from the following components: 10mg of anti-human RBC antibody, 20mg of blocker 001, 10g of bovine serum albumin, 5mL of Tween-20 and 1000mL of 0.2M borax borate buffer solution with pH8.0.

Images