CN112362866A

CN112362866A - High-throughput and rapid semi-quantitative immunoassay method and detection reagent

Info

Publication number: CN112362866A
Application number: CN202011024123.0A
Authority: CN
Inventors: 张国英; 曹宁
Original assignee: Chuzhou Ruigu Biotechnology Co ltd
Current assignee: Chuzhou Ruigu Biotechnology Co ltd
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2021-02-12

Abstract

The invention discloses a high-throughput and quick semi-quantitative immunoassay method, which comprises the steps of coating a polyclonal antibody or at least two monoclonal antibodies of an object to be detected on the surface of microspheres, carrying out immunoassay on the object to be detected, and calculating the reduction amount or reduction ratio of the number of the microspheres with the primary particle size to estimate the concentration of the object to be detected. According to the invention, the microsphere polymer is formed by coating the antibody on the surface of the microsphere and the object to be detected, so that the particle size of the formed microsphere is changed, and the object to be detected is rapidly and semi-quantitatively detected.

Description

High-throughput and rapid semi-quantitative immunoassay method and detection reagent

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a high-throughput and rapid semi-quantitative immunodetection method and a detection reagent adopting the method.

Background

The immunoassay is widely applied in the fields of in-vitro diagnosis, food safety and the like, and has very important significance in the aspects of disease screening diagnosis, food quality control and the like. In general, immunoassay refers to detection of a target antigen or antibody by means of specific binding of the antigen or antibody, and methods are classified into latex agglutination, ELISA, and the like. Latex agglutination is subdivided into manual methods, latex turbidimetry using spectrophotometric readings. ELISA methods are subdivided into radioimmunoassays, enzyme-linked immunoassays, fluorogenic immunoassays, luminescent immunoassays, etc., depending on the detection substrate used. In the process of practical application, the methods have advantages and disadvantages, such as simple manual method, but poor sensitivity and accuracy. Latex turbidimetry improves sensitivity but is less accurate than ELISA. The ELISA method improves the sensitivity, but the detection time is long, and the result can be reported only after at least 30 minutes. Whereas ELISA methods based on dry paper strips, although increasing the speed of detection, are difficult to perform at high throughput screening.

For some detection items, the requirement on accurate quantification is not high, and only the existence or nonexistence of the object to be detected or semi-quantification is required to be confirmed. Such as screening of various viruses, detection of various contraband substances in food, etc. The existing method has expensive instruments and equipment, slow detection time or incapability of simultaneously detecting a plurality of items, so that a semi-quantitative immunoassay method suitable for rapid and high-throughput detection needs to be developed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide an immunoassay method capable of quickly realizing semi-quantitative detection.

In order to achieve the above object, the present invention provides a high-throughput and rapid semi-quantitative immunoassay method, which comprises coating a polyclonal antibody or at least two monoclonal antibodies of an analyte on the surface of microspheres for immunoassay of the analyte, and calculating the reduction amount or reduction ratio of the number of microspheres with the original particle size for estimation of the concentration of the analyte in a sample.

The method for estimating the concentration of the substance to be detected comprises the following steps:

(1) preparing a standard concentration curve of the particle size reduction ratio: preparing standard solutions of the object to be detected with different concentration gradients, respectively carrying out immunoreaction with microspheres coated with the antibody on the surfaces, determining the reduction ratio of the microspheres in a corresponding particle size range, and preparing a standard curve of the concentration-particle size reduction ratio of the object to be detected;

(2) substituting the reduction ratio of the number of the microspheres with the primary particle size after the sample is subjected to the immunodetection into the standard curve prepared in the step (1) to obtain the concentration gradient range of the object to be detected corresponding to the reduction ratio, namely the estimated concentration of the object to be detected in the sample.

When a plurality of objects to be detected are detected simultaneously, the adopted semi-quantitative immunoassay method comprises the steps of preparing a plurality of groups of microspheres with different particle sizes, coating one polyclonal antibody or at least two monoclonal antibodies of the corresponding different objects to be detected on the surfaces of the groups of microspheres, and then simultaneously detecting the plurality of objects to be detected, and calculating the reduction amount or reduction ratio of the number of the microspheres with different particle sizes to estimate the concentration of the corresponding objects to be detected in a sample.

Wherein, the microsphere adopts polymer microsphere, such as polystyrene microsphere, polystyrene/polymethyl methacrylate microsphere, polystyrene/polyacrylic acid microsphere.

In some embodiments, the microspheres have a particle size of 0.5 to 5 μm.

In some embodiments, polystyrene microspheres are preferred.

The invention also provides a method for coating the antibody on the surface of the semi-microsphere with high flux and rapidness, which adopts carboxyl covalent crosslinking and physical adsorption.

In some embodiments, the method for coating the surface of the microsphere with the antibody preferably employs a carboxyl covalent crosslinking method.

When the microsphere adopts a polystyrene microsphere, the surface antibody coating is carried out by the following method: firstly, preparing carboxyl modified polystyrene microspheres, then activating the microspheres, then performing antibody crosslinking, and finally sealing.

A quantitative immunoassay reagent prepared by the method comprising:

(1) carboxyl modified polystyrene microspheres: mixing polystyrene microspheres with relatively consistent particle sizes into an alcohol-water solution, uniformly mixing, adding a mixed solution of azodiisobutyronitrile, styrene and divinylbenzene under the protection of nitrogen, introducing nitrogen for 30 minutes, heating to 70 ℃, stirring, condensing, refluxing for 24 hours, centrifuging the product at a high speed, removing supernatant, washing with the alcohol-water solution, and drying at a constant temperature of 50 ℃ to constant weight;

(2) activation of microspheres: dissolving the microspheres modified in the step (1) in water to prepare a solution with the concentration of 0.1% (W/V), adding an EDAC (electronic design automation) and NHS (polyethylene glycol succinate) aqueous solution, mixing, standing at room temperature for 4 hours, centrifuging at a high speed, removing supernatant, and taking the precipitated microspheres for re-suspension by using MES (MES) buffer solution;

(3) cross-linking of antibodies: adding the antibody corresponding to the substance to be detected into the microsphere solution prepared in the step (2), stirring in water bath at 30 ℃ for 4 hours, centrifuging at high speed to remove supernatant, and re-suspending the precipitated crosslinked microspheres with MES buffer solution;

(4) and (3) sealing: and (4) adding a BSA (bovine serum albumin) solution into the crosslinked microsphere solution obtained in the step (3), stirring at room temperature for 1 hour, centrifuging to remove a supernatant, taking a precipitate, and re-suspending the precipitate by using MES (MES) to obtain the semi-quantitative immunoassay reagent.

When the semi-quantitative immunoassay reagent can be used for simultaneously performing immunoassay of a plurality of objects to be detected, a plurality of microspheres with different particle sizes corresponding to the objects to be detected one by one are respectively selected in the step (1), the microspheres with different particle sizes are sequentially subjected to cross-linking sealing of different antibodies corresponding to the objects to be detected in the subsequent steps (1), (2), (3) and (4), and finally, all microsphere solutions after cross-linking the antibodies are mixed to obtain the semi-quantitative immunoassay reagent.

Particularly, when the semi-quantitative immunoassay reagent can simultaneously perform immunoassay of three objects to be detected, corresponding microspheres with three different particle sizes are respectively selected in the step (1), the three particle sizes are respectively controlled to be 1 mu m, 2 mu m and 3 mu m, the microspheres with different particle sizes sequentially pass through the subsequent steps (1), (2), (3) and (4) to respectively perform cross-linking and sealing of different antibodies of the corresponding objects to be detected, and finally, all microsphere solutions after cross-linking the antibodies are mixed to obtain the semi-quantitative immunoassay reagent.

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

the invention uses the surface of the microsphere to coat polyclonal antibodies or a plurality of monoclonal antibodies, and the polyclonal antibodies or the monoclonal antibodies are combined with each other through immunoreaction between the object to be measured (antigen) and the antibodies on the microsphere, and the microspheres are polymerized together through formed antigen/antibody conjugates to form a microsphere polymer, so that the particle size distribution of the microspheres is changed, the data of the microspheres with original particle size is reduced, and the concentration estimation (semi-quantitative) of the object to be measured is carried out by calculating the reduction amount or reduction ratio of the microspheres according to the counting result of the particle size instrument on the corresponding microspheres, which is faster; meanwhile, the surface of the microsphere with different particle size specifications can be coated with corresponding antibodies of different objects to be detected, and the change of the number of the microspheres with different particle sizes is counted, so that the simultaneous detection and concentration estimation of the different objects to be detected are realized, and the aim of high-throughput detection is fulfilled.

Drawings

FIG. 1 is a schematic diagram showing the structure of microspheres of a surface-coated antibody in example 1 of the present invention;

FIG. 2 is a schematic structural view of polymerization between microspheres in example 1 of the present invention;

in FIGS. 1 and 2, 1-microsphere, 5-polyclonal antibody, 4-analyte;

FIG. 3 is a graph comparing the particle size distribution of microspheres before and after examination in example 1 of the present invention;

FIG. 4 is a standard curve between the concentration of clenbuterol and the percentage of particles lost in example 1 of the present invention;

FIG. 5 is a schematic diagram showing the structure of microspheres of the antibody coating of the bread in example 2 of the present invention;

FIG. 6 is a schematic structural view of polymerization between microspheres in example 2 of the present invention;

in FIGS. 5 and 6, 1-microsphere, 2-antibody I, 3-antibody II, 4-analyte;

FIG. 7 is a standard curve of the concentration of salbutamol versus the percentage of particles lost in example 2 of the present invention;

FIG. 8 is a standard curve of myoglobin concentration versus percent particles lost in example 3 of the present invention;

FIG. 9 is a standard curve between the concentration of creatine kinase MB-type isozyme and the percentage of lost particles in example 3 of the present invention;

fig. 10 is a standard curve between troponin I concentration and the percentage of particles lost in example 3 of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

Clenbuterol, salbutamol and ractopamine detection reagent

1. Preparation of polystyrene microspheres

Adding alcohol-water solution of polyvinylpyrrolidone into a flask, fully stirring, adding mixed solution of azodiisobutyronitrile, styrene and divinylbenzene under the protection of nitrogen, introducing nitrogen for 30 minutes, heating to 70 ℃, stirring, condensing and refluxing for 12 hours. The size of polystyrene microspheres generated by the reaction is controlled by adjusting the addition amount of styrene and divinylbenzene, and microspheres with average particle diameters of about 1 μm, 2 μm and 3 μm are prepared in sequence.

2. Preparation of carboxyl modified polystyrene microspheres

Sequentially mixing the prepared polystyrene microspheres with different particle sizes into a certain amount of alcohol-water solution, uniformly mixing, adding into a flask, adding a mixed solution of azodiisobutyronitrile, styrene and divinylbenzene under the protection of nitrogen, introducing nitrogen for 30 minutes, heating to 70 ℃, and stirring, condensing and refluxing for 24 hours. After the obtained product was centrifuged at high speed, the supernatant was discarded, and the washed with an alcohol-water solution repeatedly. Drying at constant temperature of 50 ℃ to constant weight to respectively obtain the carboxyl modified polystyrene microspheres with average particle diameters of 1 mu m, 2 mu m and 3 mu m.

3. Microsphere activation

The microspheres with different particle sizes prepared in the above way are respectively dissolved in water to prepare a solution with the concentration of 0.1% (W/V). Then, an aqueous solution of EDAC (carbodiimide) and NHS (N-N-hydroxysuccinimide) was added thereto, mixed, and then left at room temperature for 4 hours, followed by high-speed centrifugation to discard the supernatant. The precipitated activated microspheres were then resuspended in MES buffer.

4. Cross-linked antibodies

Adding 1mg of each of clenbuterol polyclonal antibody (Abnova, PAB8240), salbutamol polyclonal antibody (Abnova, PAB8355) and ractopamine polyclonal antibody (Abnova, PAB8350) into the activated microsphere solution with the particle size of 1 μm, 2 μm and 3 μm, and stirring in water bath at 30 ℃ for 4 hours. The supernatant was removed by high speed centrifugation and the precipitated microspheres were resuspended in MES buffer. According to the method, microspheres with the sizes of 1 micron, 2 microns and 3 microns are crosslinked with different antibodies, and the three are uniformly mixed. As shown in fig. 1, the surface of each microsphere 1 is coated with a corresponding polyclonal antibody 5.

5. Sealing of

BSA (bovine serum albumin) solution was added to the crosslinked microspheres, and after stirring at room temperature for 1 hour, the supernatant was centrifuged off. The pellet was resuspended in MES (2- (N-morpholine) ethanesulfonic acid) buffer. Thus preparing the required reagent.

6. Detection of

0.5ml of the above reagent was taken, and the diameter and the number of particles were measured on a particle size analyzer. After mixing 0.5ml of the reagent with 50ul of the sample and reacting for 5 minutes, the particle diameter and number were measured again as shown in FIG. 3.

After the sample is added, the analyte 4 (antigen) contained therein and the corresponding polyclonal antibody 5 on the surface of the microsphere are subjected to immunoreaction and combined with each other, and the microspheres are polymerized together through the formed antigen/antibody conjugate to form a microsphere polymer, as shown in fig. 2. The concentration of the analyte is related to the amount of polymerization forming the microspheres, resulting in a reduction in the original particle size of the microspheres. The number of particles reduced is compared in the same diameter range. Thereby calculating the concentration of the analyte.

7. Statistics of test results

The clenbuterol term corresponds to a particle loss of 25.7%, indicating that the sample contains clenbuterol, while the other two lost particles are close to 0%, indicating that the sample does not contain salbutamol and ractopamine.

8. Semi-quantitative method

Clenbuterol standard solutions (0ng/ml, 0.2ng/ml, 5ng/ml, 10ng/ml and 20ng/ml) with known concentrations are prepared and are respectively tested on a computer to obtain corresponding percent of lost particles, and a standard curve is drawn by taking the concentration of the clenbuterol standard solution as an X axis and the percent of lost particles as a Y axis, as shown in figure 4. According to the loss particle percentage of 25.7 percent of the sample to be tested, the corresponding concentration is 3.5ng/ml (in the range of 0.2-5 ng/ml) by curve checking. The concentration of the clenbuterol is divided into four groups of <0.2ng/ml, 0.2ng/ml to 5ng/ml, 5ng/ml to 10ng/ml and 10ng/ml to 20ng/ml, which are respectively defined as-, + + and + + +, and the result of the semi-quantitative test of the sample is "+", namely the concentration of the clenbuterol in the sample is between 0.2ng/ml and 5 ng/ml.

Example 2

Clenbuterol, salbutamol and ractopamine detection reagent

1. Preparation of polystyrene/polyacrylic acid microspheres

Then, the surface of the polystyrene microsphere is sulfonated to improve the hydrophilicity: weighing a certain amount of PS seed microspheres, putting the PS seed microspheres into a three-neck flask, slowly adding concentrated sulfuric acid, oscillating for 30 minutes in ultrasonic waves, reacting for 10 hours at 60 ℃, centrifuging at 6000 r/min after sulfonation reaction, and washing for 3 times.

Adding the polystyrene microsphere solution, potassium persulfate, acrylic acid, ethylene glycol dimethacrylate and distilled water into a three-necked bottle, stirring (300 revolutions per minute), introducing nitrogen for 30 minutes, sealing, heating to 70 ℃, and reacting for 24 hours. Preparing the polystyrene/polyacrylic acid microspheres.

2. Adsorption buffer

According to the difference of isoelectric points of different proteins (antibodies), buffer solutions with different pH values are prepared to be used as adsorption buffer solutions for adsorbing the clenbuterol antibody, the salbutamol antibody and the ractopamine antibody respectively. In this example, PBS buffer (pH 7.4) and borate buffer (pH 8.5) were used as adsorption buffers. Microspheres with a particle size of 1um were dissolved in PBS buffer to prepare a solution with a concentration of 0.1% (W/V). Microspheres with a particle size of 2um were dissolved in borate buffer to prepare a solution with a concentration of 0.1% (W/V). Microspheres with a particle size of 3um were dissolved in PBS buffer to prepare a solution with a concentration of 0.1% (W/V).

3. Adsorbed antibodies

Two kinds of clenbuterol monoclonal antibodies (Abnova, MAB5578 and Novus Biologicals, NB100-2193), two kinds of salbutamol monoclonal antibodies (Abnova, MAB21953 and Novus Biologicals, NBP2-75934) and two kinds of ractopamine monoclonal antibodies (Novus Biologicals, NBP1-28847 and Fitzgerald, 10R-1875) were added to the above microsphere solutions having particle diameters of 1 μm, 2 μm and 3 μm, respectively, and stirred at 4 ℃ for 12 hours. The supernatant was removed by high speed centrifugation and the precipitated microspheres were resuspended in MES buffer. According to the method, microspheres with the sizes of 1 micron, 2 microns and 3 microns are crosslinked with different antibodies, and the three are uniformly mixed. As shown in FIG. 5, each microsphere 1 is coated with two

monoclonal antibodies

2 and 3.

4. Sealing of

BSA (bovine serum albumin) solution was added to the crosslinked microspheres, and after stirring at room temperature for 1 hour, the supernatant was centrifuged off. The pellet was resuspended in MES buffer (2- (N-morpholine) ethanesulfonic acid). Thus preparing the required reagent.

5. Detection of

0.5ml of the above reagent was taken, and the diameter and the number of particles were measured on a particle size analyzer. After mixing 0.5ml of reagent with 50ul of sample and reacting for 5 minutes, the particle diameter and number were measured again.

After the sample is added, the analyte 4 (antigen) contained therein and the

monoclonal antibody

2 or 3 corresponding to the surface of the microsphere are immunoreactive and bound to each other, and the microsphere is polymerized together by the formed antigen/antibody conjugate to form a microsphere polymer, as shown in fig. 6. The concentration of the analyte is related to the amount of polymerization forming the microspheres, resulting in a reduction in the original particle size of the microspheres. The number of particles reduced is compared in the same diameter range. Thereby calculating the concentration of the analyte.

6. Statistics of test results

The lost particle fraction for the salbutamol project was 32.7%, indicating that the sample contained salbutamol, and the other two lost particles fraction was close to 0%, indicating that the sample contained no clenbuterol or ractopamine.

7. Semi-quantitative method

Preparing salbutamol standard solutions with known concentrations (0ng/ml, 0.2ng/ml, 2ng/ml, 5ng/ml and 10ng/ml), respectively testing on a computer to obtain corresponding percent of lost particles, and drawing a standard curve by taking the concentration of the salbutamol standard solution as an X axis and the percent of lost particles as a Y axis, wherein the standard curve is shown in figure 7. According to the loss particle percentage of the sample to be tested being 32.7%, the curve is searched to obtain the corresponding concentration being 1.5ng/ml (in the range of 0.2-2 ng/ml). The concentration of the salbutamol is divided into four groups of <0.2ng/ml, 0.2-5 ng/ml, 5-10 ng/ml and 10-20 ng/ml, which are respectively defined as-, + + and + + +, and the semi-quantitative test result of the sample is "+", namely the content of the salbutamol in the sample is between 0.2-5 ng/ml.

Example 3

Myocardial three-phase (myoglobin, creatine kinase MB type isozyme and troponin I) detection reagent

1. Preparation of carboxyl modified polymethyl methacrylate microspheres

Adding alcohol-water solution of polyvinylpyrrolidone into a flask, fully stirring, adding methyl methacrylate, azodiisobutyronitrile and ethylene glycol dimethacrylate under the protection of nitrogen, introducing nitrogen for 30 minutes, heating to 70 ℃, stirring, condensing and refluxing for 12 hours. The size of the polystyrene microspheres generated by the reaction is controlled by adjusting the adding amount of methyl methacrylate and ethylene glycol dimethacrylate, and microspheres with average particle sizes of about 1 micron, 2 microns and 3 microns are prepared in sequence.

Then taking part of polymethyl methacrylate microspheres to carry out saponification reaction in 0.5mol/L sodium hydroxide solution at 80 ℃ for 40 hours, centrifuging, washing, separating, and drying in vacuum at 40 ℃ to constant weight.

2. Microsphere activation

3. Cross-linked antibodies

5ug of myoglobin polyclonal antibody (Bioss Antibodies, bs-3805R), 10ug of creatine kinase MB type isozyme polyclonal antibody (GenScript, A01456) and 5ug of creatine kinase MB type isozyme polyclonal antibody (Bioss Antibodies, bs-0799R) were added to the above activated microsphere solutions having particle diameters of 1 μm, 2 μm and 3 μm, respectively, and stirred in a water bath at 30 ℃ for 4 hours. The supernatant was removed by high speed centrifugation and the precipitated microspheres were resuspended in MES buffer. According to the method, microspheres with the sizes of 1 micron, 2 microns and 3 microns are crosslinked with different antibodies, and the three are uniformly mixed. As shown in FIG. 1, each microsphere 1 is coated with a polyclonal antibody 5.

4. Sealing of

5. Detection of

0.5ml of the above reagent was taken, and the diameter and the number of particles were measured on a particle size analyzer. After mixing 0.5ml of the reagent with 20ul of the whole blood sample for 10 minutes, the particle diameter and number were measured again. The number of particles reduced is compared in the same diameter range. Thereby calculating the concentration of the analyte.

6. Statistics of test results

The proportion of lost particles corresponding to the myoglobin item was 59.7%, the proportion of lost particles corresponding to the creatine kinase MB-type isozyme item was 26.4%, and the proportion of lost particles corresponding to the creatinine I item was 58.4%. It is shown that the sample contains myoglobin, creatine kinase MB-type isozyme, and troponin I.

7. Semi-quantitative method

Myoglobin standard solutions (0ng/ml, 100ng/ml, 200ng/ml, 400ng/ml, 800ng/ml), creatine kinase MB type isozyme standard solutions (0U/L, 24U/L, 100U/L, 200U/L, 500U/L), troponin I standard solutions (0ng/ml, 0.1ng/ml, 0.5ng/ml, 25ng/ml, 100ng/ml) were prepared at known concentrations.

And respectively testing on a computer to obtain corresponding lost particle percentages, and drawing a standard curve by taking the concentration of the myoglobin standard solution, the concentration of the creatine kinase MB type isozyme standard solution and the concentration of the troponin I standard solution as an X axis and the lost particle percentages as a Y axis, wherein the standard curve is shown in figures 8, 9 and 10.

From the 59.7% percent of particles lost from the sample to be tested, a curve was developed giving a myoglobin concentration of 200ng/ml (in the range of 100ng/ml to 200 ng/ml). The concentration of myoglobin is divided into four groups of <100ng/ml, 100-200 ng/ml, 200-400 ng/ml and 400-800 ng/ml, which are respectively defined as-, + + + + and + + +, and the sample semi-quantitative test result is "+".

According to the percent of lost particles of the sample to be tested being 26.4 percent, the concentration of the creatine kinase MB isozyme is 28U/L (in the range of 24U/L-100U/L) by curve checking. The concentration of creatine kinase MB isozyme is divided into four groups of <24U/L, 24-100U/L, 100-200U/L and 200-500U/L, which are respectively defined as-, + + + + + and + + +, and the semi-quantitative test result of the sample is "+".

According to the percent of lost particles of the sample to be tested, which is 58.4%, the curve is searched to obtain the concentration of the troponin I, which is 30ng/ml (in the range of 25ng/ml to 100 ng/ml). The concentration of troponin I is divided into four groups of <0.1ng/ml, 0.1-0.5 ng/ml, 0.5-25 ng/ml and 25-100 ng/ml, which are respectively defined as-, + + and + + +, and the sample is in a semi-quantitative test result of "+ + +".

Namely, the content of myoglobin in the sample is 100-200 ng/ml, the content of creatine kinase MB type isozyme is 24-100U/L, and the content of troponin I is 25-100 ng/ml.

Claims

1. A high-throughput and rapid semi-quantitative immunoassay method is characterized in that the semi-quantitative immunoassay method comprises the steps of coating one polyclonal antibody or at least two monoclonal antibodies of an object to be detected on the surfaces of microspheres and then carrying out immunoassay on the object to be detected, and estimating the concentration of the object to be detected in a sample by the reduction amount or reduction ratio of the number of the microspheres with the primary particle size.

2. The semi-quantitative immunoassay method according to claim 1, wherein the concentration of the analyte is estimated by:

3. The semi-quantitative immunoassay method according to claim 2, wherein the semi-quantitative immunoassay method comprises preparing microspheres with different particle sizes, coating the surface of the microspheres with different particle sizes with one polyclonal antibody or at least two monoclonal antibodies of different analytes, and then performing immunoassay of multiple analytes simultaneously, and estimating the concentration of the analytes in the sample by the reduction amount or reduction ratio of the number of the microspheres with different particle sizes.

4. The semi-quantitative immunoassay method according to any one of claims 1 to 3, wherein the microsphere is a polymer microsphere.

5. The semi-quantitative immunoassay method according to claim 4, wherein the microsphere is a polystyrene microsphere, a polystyrene/polymethyl methacrylate microsphere, a polymethyl methacrylate microsphere, or a polystyrene/polyacrylic acid microsphere.

6. The semi-quantitative immunoassay method according to any one of claims 1 to 3, wherein the antibody is coated on the surface of the microsphere by carboxyl covalent crosslinking and physical adsorption.

7. The semi-quantitative immunoassay method according to claim 5, wherein the microspheres are polystyrene microspheres, and surface antibody coating is performed by the following method: firstly, preparing carboxyl modified polystyrene microspheres, then activating the microspheres, then performing antibody crosslinking, and finally sealing.

8. A high-throughput and rapid semi-quantitative immunoassay reagent is characterized in that the semi-quantitative immunoassay reagent is prepared by the following method:

9. The semi-quantitative immunoassay reagent of claim 8, wherein when the semi-quantitative immunoassay reagent can simultaneously perform immunoassay of a plurality of analytes, a plurality of microspheres with different particle sizes corresponding to the analytes are respectively selected in step (1), the microspheres with different particle sizes sequentially pass through the subsequent steps (1), (2), (3) and (4) to respectively perform cross-linking and sealing of different antibodies corresponding to the analytes, and finally, all microsphere solutions after cross-linking of the antibodies are mixed to obtain the semi-quantitative immunoassay reagent.

10. The semi-quantitative immunoassay reagent of claim 9, wherein when the semi-quantitative immunoassay reagent can simultaneously perform immunoassay of three analytes, corresponding microspheres with three different particle sizes are respectively selected in step (1), the three particle sizes are respectively controlled to be 1 μm, 2 μm and 3 μm, the microspheres with different particle sizes sequentially pass through the subsequent steps (1), (2), (3) and (4) to respectively perform cross-linking closure of different antibodies corresponding to the analytes, and finally, all microsphere solutions after cross-linking of the antibodies are mixed to obtain the semi-quantitative immunoassay reagent.