CN115639366A - β2-microglobulin fluorescence immunochromatography assay kit and its detection method - 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

β2-microglobulin fluorescence immunochromatography assay kit and its detection method

technical field

The invention belongs to the field of in vitro diagnosis and immunochromatography detection, and relates to a β2-microglobulin fluorescence immunochromatography assay kit and a detection method thereof, in particular to a method for quantitatively detecting human serum by using the technical principle of fluorescence immunochromatography β2-microglobulin content.

Background technique

β2-microglobulin is a small molecular globulin produced by lymphocytes, platelets, and polymorphonuclear leukocytes, with a molecular weight of 11,800 and a single-chain polypeptide composed of 99 amino acids. It is part of the β chain (light chain) of the human leukocyte antigen (HLA) on the cell surface. The molecule contains a pair of disulfide bonds and does not contain sugar. It is similar to the structure of the stable region of immunoglobulins. Widely present in plasma, urine, cerebrospinal fluid, saliva and colostrum. The synthesis rate of normal human β2-microglobulin and the amount released from the cell membrane are quite constant, β2-microglobulin can be freely filtered from the glomerulus, 99.9% is absorbed in the proximal renal tubule, and is absorbed in the renal tubular epithelial cells Decomposition and destruction, so under normal circumstances, the excretion of β2-microglobulin is very small, and the increase of serum β2-microglobulin can reflect the impairment of glomerular filtration function or whether the filtration load has increased.

β2-microglobulin in the blood of kidney transplant patients is significantly increased, suggesting that the body has a rejection reaction. Because the synthesis of β2-microglobulin is accelerated, although the renal clearance increases, the blood β2-microglobulin is still elevated. Generally, blood β2-microglobulin rises to the peak 2 to 3 days after transplantation, and then gradually decreases. Continuous measurement of blood and urine β2-microglobulin after kidney transplantation can be used as a sensitive indicator of glomerular and tubular lesions; β2-microglobulin can also be used as a simple and accurate method to measure mild renal dysfunction and curative effect observation in diabetic patients It is a sensitive method, and its determination has multiple clinical values.

At present, the common methods for detecting β2-microglobulin, such as: chemiluminescence method/enzyme-linked immunoassay/immune-enhanced turbidimetric method/colloidal gold method, all have the disadvantages of low sensitivity, long detection time, complicated process and high detection cost. This not only plagues the diagnosis link, but also exists in the rehabilitation link. Without accurate diagnosis, it is difficult to have accurate treatment. Therefore, how to accurately screen cases is an urgent problem that needs to be solved.

Contents of the invention

The purpose of the present invention is to provide a β2-microglobulin fluorescent immunochromatographic assay kit with good specificity, high sensitivity, accurate and stable results.

The technical scheme provided by the present invention is as follows: a β2-microglobulin fluorescent immunochromatography assay kit, comprising a reagent card and a diluent, the reagent card comprising a PVC board and a sample pad sequentially arranged on the PVC board, Combining pads, NC membranes and absorbent paper, the NC membranes are provided with detection lines and quality control lines;

The detection line is coated with β2-MG monoclonal antibody, and the coating concentration is 0.8-1.2mg/ml;

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

The antibody solution labeled with fluorescent microspheres is sprayed on the binding pad, and the antibody solution labeled with fluorescent microspheres is specifically a mixture of another binding site β2-MG antibody labeled with fluorescent microspheres and goat anti-chicken IgY antibody labeled with fluorescent microspheres.

Preferably, the fluorescent microspheres for labeling are Eu-fluorescent nanospheres with a particle size of 300 nm from Nanjing Weice Biological Co., Ltd.

Preferably, the labeling amount of the β2-MG-labeled antibody is 10 μg, and the labeling amount of the goat anti-chicken IgY-labeled antibody is 16 μg.

Preferably, the spray volume of the labeled antibody solution is 4-6 μL/cm.

Preferably, the spray volume of the coating working solution is 0.8-1.2 μL/cm.

Preferably, in the other binding site β2-MG antibody labeled with fluorescent microspheres on the binding pad, the feeding ratio of fluorescent microspheres to β2-MG antibody is 20:1.

Preferably, in the goat anti-chicken IgY antibody labeled with fluorescent microspheres on the binding pad, the feeding ratio of fluorescent microspheres to goat anti-chicken IgY antibody is 25:2.

Preferably, fluorescent microspheres are labeled with β2-MG-labeled antibody, and the steps are as follows:

a. Take a 2mL centrifuge tube and add 1mL of labeling buffer MES; take 20μL of fluorescent microspheres with a solid content of 1% in the labeling buffer, and vortex again;

b. Add 100 μL of labeled activation solution, mix well, rotate and mix for 30 minutes;

c. Centrifuge for 30 minutes at a speed of 10,000 rpm/min; discard the supernatant, add 1,000 μL of labeling buffer, and sonicate;

d. Add 10 μg of β2-MG labeled antibody, mix well, rotate and mix for 30 minutes;

e. After adding 100 μL of labeling blocking solution, sonicate, rotate and mix for 2 hours;

f. Centrifuge for 30 minutes; discard the supernatant, add 1 mL of labeled preservation solution and sonicate;

g. Transfer the marker into a new 10mL centrifuge tube, draw up 4mL of marker preservation solution to clean the 2mL centrifuge tube used for the marker, transfer it into a 10mL centrifuge tube after cleaning, and vortex to mix.

Preferably, fluorescent microspheres are labeled with goat anti-chicken IgY antibody, and the steps are as follows:

a. Take a 2mL centrifuge tube and add 1mL of labeling buffer MES; take 20μL of fluorescent microspheres with a solid content of 1% in the labeling buffer, and vortex again;

b. Add 100 μL of labeled activation solution, mix well, rotate and mix for 30 minutes;

c. Centrifuge for 30 minutes at a speed of 10,000 rpm/min; discard the supernatant, add 1,000 μL of labeling buffer, and sonicate;

d. Add 16 μg of goat anti-chicken IgY antibody, mix well, rotate and mix for 30 minutes;

e. After adding 100 μL of labeling blocking solution, sonicate, rotate and mix for 2 hours;

f. Centrifuge for 30 minutes; discard the supernatant, add 1 mL of labeled preservation solution and sonicate;

g. Transfer the marker into a new 10mL centrifuge tube, draw up 4mL of marker preservation solution to clean the 2mL centrifuge tube used for the marker, transfer it into a 10mL centrifuge tube after cleaning, and vortex to mix.

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

The purpose of the present invention is to provide a preparation method of a β2-microglobulin fluorescence immunochromatography assay kit.

The technical scheme provided by the present invention is as follows: a method for preparing the above-mentioned β2-microglobulin fluorescence immunochromatography assay kit, comprising the steps of preparing a reagent card:

Step 1: Set the detection line and quality control line on the NC membrane, prepare the coating working solution with β2-MG monoclonal antibody and chicken IgY antibody at a concentration of 0.8-1.2 mg/mL, and coat at 0.8-1.2 mg/mL Use a gold marking machine to draw it to the detection line and quality control line on the NC membrane and dry it;

Step 2: Spray the fluorescent microsphere-labeled antibody solution with a gold-spraying scribing machine at an amount of 4-6 μL/cm, spray it on the cut binding pad and dry it;

Step 3: After the sample pad is treated with the sample pad treatment solution, it is dried;

Step 4: Paste the above-mentioned treated sample pad, binding pad, NC membrane and absorbent paper on the PVC board in sequence to assemble a reagent card.

Preferably, the formula of the sample pad treatment solution is prepared from the following components: 10 mg of anti-human RBC antibody, 20 mg of blocking agent 001, 10 g of bovine serum albumin, 5 mL of Tween-20, and 1000 mL of 0.2M pH8.0 boric acid borax buffer.

The determination principle of this kit: adopt the double-antibody sandwich method, and utilize the technical principle of fluorescence immunochromatography. During the test, the sample is mixed with the diluent and added dropwise to the sample hole of the reagent card. Chromatography is performed under the capillary effect. The β2-MG antigen in the sample binds to the fluorescently labeled β2-MG antibody and diffuses to the test area , is captured by the β2-MG monoclonal antibody coated with the detection line, and forms an "antibody-antigen-fluorescent antibody" complex; the concentration of β2-MG in the sample is proportional to the fluorescence intensity of the complex, according to the set standard curve, The dry fluorescent immunoassay analyzer converts the fluorescent signal value into the concentration of β2-MG in the sample. When the glomerular and renal tubular functions of the body are damaged, the content of β2-MG in the body increases abnormally. When β2-MG ≥ 2.7mg/L, it indicates that the patient may suffer from the risk of impaired renal function.

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

This application provides a β2-microglobulin fluorescent immunochromatography assay kit, using β2-MG monoclonal antibody and chicken IgY antibody as the detection line and quality control line respectively as the coating antibody, β2-MG antibody and sheep Anti-chicken IgY is mixed as a fluorescent microsphere-labeled antibody, and the fluorescent immunochromatography method used is the same as the current common detection methods of β2-microglobulin, such as: chemiluminescence method/enzyme-linked immunosorbent method/immune-enhanced turbidimetric method/colloidal gold Compared with the method, it not only greatly shortens the detection time and reduces the detection cost, but also improves the detection sensitivity. Compared with similar methods, it has the advantages of good specificity, high sensitivity, accurate and stable results, etc.

This application provides a method for preparing a β2-microglobulin fluorescent immunochromatography assay kit. By selecting suitable raw materials, rationally designing the dosage of each material, and selecting the most suitable reaction conditions, the preparation process is simple, and the prepared product has good specificity, High sensitivity, accurate and stable results, low detection cost, easy to use, low cost, and suitable for promotion.

Description of drawings

Fig. 1 is the structural representation of reagent card in the embodiment of the present application;

Figure 2 is the gradient ratio results of different T-line and C-line labeled antibody feeding combinations in the examples of the present application;

Fig. 3 is the scatter diagram of β2-MG standard curve in the embodiment of the present application;

Fig. 4 is the clinical comparison result of the embodiment of the present application and the control reagent test.

Detailed ways

The specific technical solutions of the present invention will be described below in conjunction with specific embodiments.

Example 1:

A β2-microglobulin fluorescent immunochromatography assay kit, comprising a reagent card and a diluent, the reagent card comprising a PVC plate and a sample pad, a binding pad, an NC membrane and a water-absorbing material sequentially arranged on the PVC plate paper, the NC membrane is provided with a detection line and a quality control line;

The detection line is coated with β2-MG monoclonal antibody, and the coating concentration is 0.8-1.2mg/ml;

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

The antibody solution labeled with fluorescent microspheres is sprayed on the binding pad, and the antibody solution labeled with fluorescent microspheres is specifically a mixture of another binding site β2-MG antibody labeled with fluorescent microspheres and goat anti-chicken IgY antibody labeled with fluorescent microspheres.

Example 2:

A method for preparing a β2-microglobulin quantitative fluorescence immunochromatography assay kit, comprising the steps of preparing a reagent card:

Step 1: Set the T and C lines on the NC membrane, prepare the coating working solution with β2-MG coating antibody and chicken IgY coating antibody at a concentration of 1.0 mg/mL respectively, and use gold to coat the volume at 1.0 μL/cm The marking machine draws it on the NC film, and places the NC film in a humidity ≤ 30%, (37±2) ℃ electric blast drying oven to dry (16 ~ 24h);

Step 2: Spray the reconstituted fluorescent microsphere-labeled antibody solution at 4 μL/cm with a gold-spraying scribe machine, and spray it onto the cut-out binding pad in an environment with a humidity of ≤30% and (37±2)°C Under drying (16 ~ 24h);

Step 3: Set the height of the wheel axle of the paper soaking pad press machine, pour the sample pad treatment solution, place the processed sample pad on the drying rack with a humidity ≤ 30%, and dry overnight (16-24h) at (37±2)°C;

Step 4: As shown in Figure 1, paste the above-mentioned treated sample pad, bonding pad, NC film and absorbent paper on the PVC sticky bottom in order, with the NC film at the bottom, and each component needs to overlap by about 2mm or more. Assemble, assemble the prepared experimental materials into a reagent card for use.

Wherein, the reconstituted fluorescent microsphere-labeled antibody solution includes a 1:1 mixture of fluorescent microsphere-labeled β2-MG-labeled antibody and fluorescent microsphere-labeled goat anti-chicken IgY antibody.

Fluorescent microspheres label β2-MG-labeled antibody, including the following steps:

a. Take a 2mL centrifuge tube and add 1mL of labeling buffer MES; take 20μL of fluorescent microspheres with a solid content of 1% in the labeling buffer, and vortex again;

b. Add 100 μL of labeled activation solution, mix well, rotate and mix for 30 minutes;

c. Centrifuge for 30 minutes at a speed of 10,000 rpm/min; discard the supernatant, add 1,000 μL of labeling buffer, and sonicate;

d. Add 10 μg of β2-MG labeled antibody, mix well, rotate and mix for 30 minutes;

e. After adding 100 μL of labeling blocking solution, sonicate, rotate and mix for 2 hours;

f. Centrifuge for 30 minutes; discard the supernatant, add 1 mL of labeled preservation solution and sonicate;

g. Transfer the marker into a new 10mL centrifuge tube, draw up 4mL of marker preservation solution to clean the 2mL centrifuge tube used for the marker, transfer it into a 10mL centrifuge tube after cleaning, and vortex to mix.

Fluorescent microspheres labeled goat anti-chicken IgY antibody, the steps are as follows:

a. Take a 2mL centrifuge tube and add 1mL of labeling buffer MES; take 20μL of fluorescent microspheres with a solid content of 1% in the labeling buffer, and vortex again;

b. Add 100 μL of labeled activation solution, mix well, rotate and mix for 30 minutes;

c. Centrifuge for 30 minutes at a speed of 10,000 rpm/min; discard the supernatant, add 1,000 μL of labeling buffer, and sonicate;

d. Add 16 μg of goat anti-chicken IgY antibody, mix well, rotate and mix for 30 minutes;

e. After adding 100 μL of labeling blocking solution, sonicate, rotate and mix for 2 hours;

f. Centrifuge for 30 minutes; discard the supernatant, add 1 mL of labeled preservation solution and sonicate;

g. Transfer the marker into a new 10mL centrifuge tube, draw up 4mL of marker preservation solution to clean the 2mL centrifuge tube used for the marker, transfer it into a 10mL centrifuge tube after cleaning, and vortex to mix.

Further, chicken IgY and goat anti-chicken IgY from Shanghai Lingchao were selected as quality control line coating antibodies for the quality control line (i.e. C line) coating antibody and quality control line labeling antibody, and their T/C precision is relatively high. Well, the quality control line (i.e. C line) coating antibody and quality control line marker antibody are selected as follows:

Use the mouse IgG, goat anti-mouse IgG and chicken IgY, goat anti-chicken IgY antibodies from Shanghai Lingchao Biotechnology Co., Ltd. to dilute the β2-MG antibody pair with ultrapure water at the usual concentration, and make a reagent card for use Reference samples were tested. The specific operation is basically the same as that of the antibody selection operation of the detection line. The test results (line C) of antibody preparation reagent cards from different manufacturers are shown in Table 1:

Table 1 Test results of antibody preparation reagent cards from different manufacturers (line C)

It can be seen from Table 1 that there are certain differences in the linear gradient of the reference products of the antibody preparation reagent cards of various manufacturers, among which Shanghai Lingchao (chicken IgY + sheep anti-chicken IgY) > Shanghai Lingchao (rat IgG + sheep anti-mouse IgG); T The precision of /C Shanghai Lingchao (chicken IgY + sheep anti-chicken IgY) is better. Chicken IgY and goat anti-chicken IgY from Shanghai Lingchao were selected as quality control lines for coating and labeling antibodies.

Further, the fluorescent microspheres of the present application choose the Eu-fluorescent nanospheres of Nanjing Weicei Biotechnology Co., Ltd. 300nm as the fluorescent microspheres for labeling. The linear gradient is 51.41, and the T/C precision is <5%. Choose as follows:

For the Eu-fluorescent nanospheres of Nanjing Weice Biotechnology Co., Ltd. with a particle size of about 200nm and 300nm and the hydroxylated fluorescent nanospheres of Changsha Meiniu Biotechnology Co., Ltd. of 200nm and 300nm, four test particle sizes, fluorescence intensity and PDI coefficient, Then they were labeled with antibodies in turn, prepared binding pads to make reagent cards, and tested with enterprise reference products. The test results of reagent cards prepared by fluorescent microspheres from different manufacturers are shown in Table 2:

Table 2 Test results of reagent cards prepared by fluorescent microspheres from different manufacturers

It can be seen from the above table 2 that the linear gradient of 51.41 at 300nm is the best, and the T/C precision is less than 5%. Therefore, Eu-fluorescence nanospheres 300nm of Nanjing Weice Biotech were selected as fluorescent microspheres for labeling.

Further, the labeling preservation solution formula is prepared according to 1000mL: bovine serum albumin 20g, sucrose 20g, casein sodium salt 5g, casein sodium 50g, 1mL Proclin 300, 0.02M pH8.0 PBS buffer 1000mL, the labeling buffer is determined as follows :

Prepare 4 different labeling and preservation solutions to label and store markers, and prepare binding pads, and prepare different reagent cards according to the biological materials determined in previous experiments. Observe the storage status of the markers, and prepare reference products for the binding pad assembly reagent card test enterprise. According to the test results, formula 4 (prepared by 1000mL: bovine serum albumin 20g, sucrose 20g, casein sodium salt 5g, casein sodium 50g, 1mLProclin 300, 0.02M pH8.0 PBS buffer 1000mL) was finally selected as the application β2 -The labeling preservation solution formula of the microglobulin (β2-MG) assay kit (fluorescence immunochromatography).

Further, 0.01M PBS containing 1% sucrose pH 7.4 is selected as the coating solution. After the reagent strip is made, the coating solution is selected as a clear liquid, no crystal precipitation and a good linear gradient. The pH of the coating solution is 7.4 and the linear gradient is high. , the choice of coating solution is as follows:

Dilute the coating antibody β2-MG and chicken IgY with four formulations of coating solutions (0.01MpH7.4 and 8.0PBS, 0.02MpH7.4 and 8.0PBS solutions) to the coating concentration, and after making reagent strips, select The coating liquid is a clear liquid without crystal precipitation and a buffer solution with a good linear gradient as the coating liquid.

Table 3 Test results of reagent cards prepared by different coating solutions

The pH value of the coating solution has a great influence on the linear gradient. When the pH value of the coating solution is 7.4, the linear gradient is high, so the pH value of the selected coating solution is 7.4; comprehensive consideration, choose 0.01 containing 1% sucrose pH7.4 M PBS was used as the final formulation of the coating solution.

Further, the formula of sample pad treatment solution is prepared in 1000mL: anti-human RBC antibody 10mg, blocking agent 00120mg, bovine serum albumin 10g, 5mL Tween-20, 0.2M pH8.0 boric acid borax buffer 1000mL, the anti-interference ability of this configuration Stronger, determination of the formulation of the sample pad treatment solution:

Prepare 4 different formulations (treatment solution 1: anti-human RBC antibody 10mg, blocking agent 001 10mg, bovine serum albumin 10g, 5mL Tween-20, 0.2M pH8.0 borate borax buffer 1000mL; treatment solution 2: anti-human RBC antibody 20mg, blocking agent 001 20mg, bovine serum albumin 10g, 5mL Tween-20, 0.2M pH8.0 borate borax buffer 1000mL; treatment solution 3: anti-human RBC antibody 10mg, blocking agent 001 20mg, bovine serum Albumin 10g, 5mL Tween-20, 0.2M pH8.0 boric acid borax buffer 1000mL; treatment solution 4: anti-human RBC antibody 20mg, blocking agent 001 10mg, bovine serum albumin 10g, 5mLTween-20, 0.2M pH8. 0 boric acid borax buffer solution 1000mL) of the sample pad treatment solution, using the reference substance of β2-MG concentration of 0.3mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L respectively to detect, the test results are shown in the table 4 shown;

Table 4 Four kinds of sample pad treatment liquid test the effect of adding interfering substances to the healthy person sample

It can be seen from the above table 4 that the T/C of the 10mg blocker 001 test of the reference product added with the interfering substances doubled, but it was significantly lower than the T/C of the untreated sample pad reagent card, which has a certain resistance Interference ability; 20mg blocker 001 test has no significant difference in T/C between the reference product with added interferents and the reference product without added interferents, and has strong anti-interference ability. Sample pad treatment solution 2 and sample pad treatment solution 3 all meet the requirements , comprehensive cost factors, finally select the sample pad treatment solution 3 (prepared by 1000mL: anti-human RBC antibody 10mg, 20mg blocker 001, bovine serum albumin 10g, 5mL Tween-20, 0.2M pH8.0 borate borax buffer 1000mL ) is the sample pad treatment solution formula.

Further, the formula of the sample diluent 4 is: 0.01M PBS, pH7.4+0.05% Proclin 300, its T/C precision is less than 6%, overall better, the formula of the sample diluent is determined as follows:

Prepare 4 kinds of sample dilutions (0.01M pH6.8, 7.0, 7.2, 7.4 four kinds of PBS buffers) with different formulations, and use reference substances to detect respectively, and the results are shown in Table 5:

Table 5 Test results of different sample dilutions

It can be seen from the above Table 5 that the T/C precision of sample dilution 4 is all <6%. , the overall is better, so sample diluent 4 is selected, the formula is: 0.01M PBS, pH7.4+0.05% Proclin 300.

Further, the present application chooses 10 μg of β2-MG labeled antibody and 16 μg of goat anti-chicken IgY labeled antibody, that is, fluorescent microsphere:antibody=20:1 is the T line feeding ratio; fluorescent microsphere:antibody=25:2 The feed ratio of the C-line is the C-line feed ratio. The concentration gradient ratio of this configuration is 66.1, which is relatively the best, with high sensitivity, good linearity, and precision <6%. The T-line labeled antibody feed ratio and the C-line labeled antibody feed ratio are determined as follows:

Set the amount of T-line antibody labeling to 5 μg, 10 μg, and 20 μg, and the amount of C-line antibody labeling to 8 μg, 16 μg, and 32 μg for positive crossover experiments, and prepared 9 sets of different reagent cards to detect the concentration of β2-MG at 0.3 mg/L. , 1mg/L, 5mg/L, 10mg/L, 20mg/L β2-MG reference substance.

Table 6 T line, C line feeding combination

Table 7 T/C precision results of different T-line and C-line labeled antibody feed ratios

From Table 7 and Figure 2, it can be seen that the concentration gradient ratio of the reference products of each enterprise in Combination 5 is 66.1, which is relatively the best, with high sensitivity, good linearity, and precision <6%. Select β2-MG labeled antibody feeding 10μg, goat anti-chicken IgY labeled antibody concentration 16μg combined feeding, that is, fluorescent microspheres: antibody = 20:1 is the T line feeding ratio; fluorescent microspheres: antibody = 25:2 is the C line feeding ratio .

Furthermore, the concentration of β2-MG coating antibody is 1.0 mg/mL, and the concentration of chicken IgY coating antibody is 1.0 mg/mL as the final T line and C line coating concentration. This configuration has higher detection sensitivity, better linearity, and precision. All < 6%, the determination of the concentration of the coating antibody in this application is as follows:

Set the concentration of T-line and C-line coating antibodies to 0.8mg/mL, 1.0mg/mL, and 1.2mg/mL, prepare 9 different combinations in the table below, and detect the concentrations of 0.3mg/L, 1mg/L, 5mg/L, 10mg/L, and 20mg/L β2-MG reference substances, the test results are shown in Table 9;

Table 8 T line, C line coating concentration combination

Table 9 T/C precision results of different T-line and C-line coating concentration combinations

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%

It can be seen from the above table 9 that the detection sensitivity of combination 5 is higher, the linearity is better, and the precision is less than 6%. Select β2-MG coating antibody concentration of 1.0mg/mL, chicken IgY coating antibody concentration of 1.0mg/mL as the final T line, C line coating concentration.

Further, the injection volume of the labeled antibody solution is 4 μL/cm, and the T/C precision is less than 6%, which is relatively optimal, and there is no obvious difference in the linear gradient of each injection volume. The injection volume of the labeled antibody solution is determined as follows:

Load fluorescent microsphere-labeled antibody solutions at 3 μL/cm, 4 μL/cm, and 5 μL/cm into the gold-sprayed scribing machine, and spray it onto the cut-out binding pad; dry overnight (16-24 h), and prepare After the experimental materials were assembled into reagent cards, they were evaluated with enterprise reference products.

Table 10 T/C precision results of different injection volumes

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 spray volume 4μL/cm was less than 6%, relatively optimal, and there was no significant difference in the linear gradient of each spray volume. Taking cost and performance into consideration, 4μL/cm was selected as the spray volume of the labeled antibody solution.

Further, the spray volume of the coating working fluid is 1.0 μL/cm, and the T/C precision is less than 6%. The spray volume of the coating working fluid is determined as follows:

Coat the T-line and C-line with the working solution, draw the coating working solution on the NC membrane at 0.8μL/cm, 1.0μL/cm, and 1.2μL/cm respectively with a gold marking machine, and place the NC membrane on Dry in an electric blast drying oven (16-24 hours), assemble the prepared experimental materials into reagent cards, and evaluate with enterprise reference products.

Table 11 T/C precision results of different injection 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%

After coating, the diffusion traces expand with the increase of spray volume, and the precision of 1.0μL/cm T/C is less than 6%, which is relatively optimal. Considering the appearance, performance and cost, we choose 1.0μL/cm for coating work Liquid spray volume.

Further, the optimal time required for the reaction is 10mim, and the determination of the reaction time is as follows:

The reagent card is prepared according to the established production process, and the prepared test card is tested according to the set reaction time (5min, 10min, 15min, 20min), and the detection concentration is 0.3mg/L, 1mg/L, 5mg/L respectively , 10mg/L, 20mg/L β2-MG reference substance. According to the linear level of the detection result, the optimal time required to screen out the reaction is 10mim.

Further, the sample volume is 100 μL as the optimal sample volume for the β2-microglobulin (β2-MG) assay kit (fluorescence immunochromatography). This design has a fast chromatographic process, absorbent paper without fluorescent microspheres, linear If the gradient is good and the sample does not overflow, the amount of sample added is determined as follows:

Prepare the reagent card according to the established production process, and use the prepared test card according to the set sample volume (80μL, 90μL, 100μL, 110μL, 120μL) to detect the concentration of 0.3mg/L, 1mg/L, 5mg respectively /L, 10mg/L, 20mg/L β2-MG reference substance. The chromatography process is fast, the absorbent paper has no fluorescent microspheres, the linear gradient is good, and the sample does not overflow. The final sample volume was 100 μL as the optimal sample volume for the β2-microglobulin (β2-MG) assay kit (fluorescence immunochromatography).

Carry out following performance test with the kit prepared by embodiment:

1. Establishment of standard curve

Prepare β2-MG antigen with fat-free normal human serum as required to prepare reference substances with concentrations of 0.3mg/L, 1mg/L, 5mg/L, 10mg/L, and 20mg/L, and test them. Take the assembled reagent card every The measurement was repeated 3 times, and the relative deviation (B) and linear correlation coefficient (r) between the average value (M) and the theoretical value of the 3 test results of each sample were calculated according to the following formulas (1) and (2).

B=(M-T)/T×100％..........(1)

In the formula:

B - relative deviation;

M - the average value of the test results;

T - theoretical value.

In the formula:

n—the number of samples to be measured;

Xi—measure the concentration of the sample;

Yi—the average value of the measured values corresponding to the measured sample concentration for 4 repeated determinations;

r—linear correlation coefficient;

i——1, 2, 3, ... n.

The test results are as follows:

Table 12 Tests for Linear Reference Products

It can be seen from Table 12 and Figure 3 that there is a correlation between the test results and the theoretical concentration, the correlation r ² =0.9981, and the scatter diagram equation y=1.0379x-0.147, and the correlation is good.

2. Accuracy testing

Determination of β2-microglobulin enterprise reference products 1mg/L, 5mg/L, 10mg/L, according to the steps in the instructions for detection, after each repeated measurement 3 times, each test result is recorded as (Xi), if 3 times the result If the relative deviation does not exceed ±15%, it is judged as qualified. If the results of more than or equal to 2 times are not met, it will be judged as unqualified. If there is one result that does not meet the requirements, it should be tested continuously for 20 times, and the relative deviation should be calculated according to formula (1). If the relative deviation of the results of more than or equal to 19 tests does not exceed ±10%, it is judged as qualified .

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

In the formula:

Bi - relative deviation;

Xi - each measurement concentration;

T - marked concentration.

The test results are as follows:

Table 13 Test of Accuracy Reference Products

Marked 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%

It can be seen from Table 13 above that the relative deviations of the accuracy reference products are all <±10%.

3. Repeatability detection

The same operator tested the β2-MG enterprise reference substance with concentrations of 1.00mg/L, 5.00mg/L and 10.00mg/L on the same instrument, and each sample was measured 10 times. The results are as follows:

Table 14 Tests for Repeatability Reference Products

It can be seen from Table 14 above that the precision of the repeatable reference products is all <10%.

4. Minimum detection limit detection

Use the reagent card to detect the β2-MG enterprise reference product with a concentration of 0.270-0.290 mg/L, and the minimum detection limit should not be greater than 0.3 mg/L. The test results are as follows:

Table 15 detection limit test results

It can be seen from the above table that the minimum detection limit is not more than 0.3mg/L.

5. Evaluation of HAMA effect

Use the reagent card to test the prepared concentration, perform statistical analysis on the test data, and perform statistical analysis on the test results to meet the acceptance criteria of the designed experiment, that is, as the HAMA effect performance index of β2-MG. The test results are as follows:

Table 16 Prepared HAMA Serum Concentrations

group Serum concentration of HAMA (ng/mL) Experimental group 1 90 Experimental group 2 60 Experimental group 3 30 control group 0

Table 17 HAMA test results of different samples

It can be seen from the test data in Table 17 that when the serum concentration of HAMA is ≤60ng/mL, the relative deviations are all ≤±10%, and the serum concentration of HAMA≤60ng/mL has no obvious interference to the test samples.

6. Clinical sample detection of experimental and control reagents

The β2-microglobulin (β2-MG) assay reagent (fluorescence immunochromatography) produced by the assembled reagent card and the control reagent were tested in 81 clinical cases for comparison, and the test results are shown in Figure 4.

It can be seen from Figure 4 that the clinical correlation between the present application and the control reagent test is good, y=0.9813x+1.3467, R ² =0.9878.

The above results show that the product prepared by this application has good specificity, high sensitivity, accurate and stable results, low detection cost, easy to use and low cost, and is suitable for promotion.

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