Protein coupling reagents, protein coupling microspheres, microplates and their applications

Abstract

The present invention relates to the field of biological detection, and in particular to protein coupling reagents, protein coupling microspheres, microplates, and their applications. The present invention provides a protein coupling reagent comprising a reagent I and a reagent II; the reagent I comprises MES, EDC, and NHS; and the reagent II comprises a recombinant anti-phospholipase A2 protein. By coating the microplate with the microspheres after protein coupling, the surface area of the carrier is increased, thereby improving the sensitivity of the ELISA plate.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a protein coupling reagent, a protein coupling microsphere, a microplate and applications thereof. Background Art

[0002] The chemiluminescent enzyme immunoassay (cLEIA) is a type of enzyme immunoassay, except that the substrate for the enzyme reaction is a luminescent agent. The procedure is identical to that of an ELISA: antigens or antibodies coated on a polystyrene plate capture specific substances in the sample and combine with enzyme-labeled bioactive substances to form an immune complex. During the test, the enzyme on the immune complex catalyzes the substrate to emit light, and the chemiluminescence analyzer measures the light quanta emitted by the catalytic reaction to obtain the final test result.

[0003] The purity, concentration, and ratio of the antigens, antibodies, labeled antibodies, or antigens involved in immunological reactions; the type, concentration, and ionic strength of the buffer; the pH value; the reaction temperature; and the reaction time play a key role in the ELISA plate. Furthermore, the solid-phase polystyrene surface, acting as a carrier, also plays an important role in the adsorption of antigens, antibodies, or antigen-antibody complexes.

[0004] Latex microspheres are a new type of material developed by combining immunology and magnetic microspheres. Due to their high efficiency and low toxicity, they are currently widely used in the fields of biochemistry, medicine, and food testing.

[0005] The coupling of antibodies to latex microspheres involves coupling monoclonal antibodies to microspheres bearing functional groups. There are two methods for linking antibodies to microspheres: covalent coupling and physical absorption. Physical absorption is highly unstable and can easily fall off under certain conditions, while covalent coupling involves the covalent bonding of antibodies to groups on the microsphere surface, firmly binding the antibodies to the microspheres. Aldehyde and epoxy groups on the microspheres can directly bind to nitrogen groups on the target molecule, while microspheres containing other groups require activation before linking to the target molecule. Common activation methods include the carbodiimide method, the diazo method, the hydrocarbonation method, the cyanogen bromide method, the glutaraldehyde method, and the Woodward reagent K method.

[0006] Solid-phase supports serve as adsorbents and containers in ELISA assays and do not participate in chemical reactions. Polystyrene is the most commonly used. Coating is the process of binding proteins to the surface of the solid-phase support. Proteins bind to the polystyrene solid-phase support through physical adsorption, which relies on the interaction between hydrophobic groups in the protein's molecular structure and those on the solid-phase support's surface. This physical adsorption is nonspecific and is affected by factors such as the protein's molecular weight, isoelectric point, and concentration.

[0007] In the prior art, a coating solution is typically added to an ELISA plate during coating. Proteins in the coating solution are adsorbed onto the polystyrene plate. However, the polystyrene plate has limited space within a fixed area, so the amount of adsorbed protein is limited. Detection using such coated microplates results in low sensitivity. Summary of the Invention

[0008] In view of this, the present invention provides a protein coupling reagent, protein coupling microspheres, a microplate and their applications. The present invention enlarges the protein area on the carrier and improves the sensitivity of the ELISA plate by coating the microplate after coupling the protein to the microspheres.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a protein coupling reagent, including a reagent and reagents ; The reagent Comprising: MES, EDC and NHS; the reagent Includes: phospholipase A2 receptor PLA2R.

[0011] In some embodiments of the present invention, the MES in the above-mentioned protein coupling reagent is added in the form of an MES solution; the concentration of the MES solution is 0.05 mol / L, and the pH value is 6.0; the mass ratio of the EDC to the NHS in the above-mentioned protein coupling reagent is 1:15.

[0012] The present invention also provides the use of the protein coupling reagent in preparing protein-coupled microspheres.

[0013] The present invention also provides a method for preparing protein-coupled microspheres, comprising the following steps:

[0014] S1: After activation of the biomagnetic beads, activated microspheres are obtained;

[0015] S2: taking the activated microspheres and coupling them to obtain the protein-coupled microspheres;

[0016] The activation described in S1 and the coupling described in S2 employed the above-mentioned protein coupling reagents.

[0017] In some embodiments of the present invention, the biomagnetic beads in the above preparation method include carboxyl microspheres.

[0018] In some embodiments of the present invention, the activation in the preparation method S1 is performed using the reagent ; The volume ratio of the biomagnetic beads to the MES is 1:2500.

[0019] In some embodiments of the present invention, the coupling in the preparation method S2 uses reagents ; The mass ratio of the volume of the activated microspheres to the phospholipase A2 receptor PLA2R is 1:1~5.

[0020] In some embodiments of the present invention, the concentration of the MES solution in the above preparation method is 0.05 mol / L and the pH value is 6.0.

[0021] In some embodiments of the present invention, the volume of the MES solution in the above preparation method is 500 μL.

[0022] In some embodiments of the present invention, the volume of the biomagnetic beads in the above preparation method is 0.2 μL.

[0023] In some embodiments of the present invention, the mass ratio of the EDC to the NHS in the above preparation method is 1:15.

[0024] In some embodiments of the present invention, the mass of the EDC in the above preparation method is 0.03 mg.

[0025] In some embodiments of the present invention, the mass of NHS in the above preparation method is 0.045 mg.

[0026] In some embodiments of the present invention, the mass of the phospholipase A2 receptor PLA2R in the above preparation method is 20-100 μg.

[0027] In some embodiments of the present invention, the mass of the phospholipase A2 receptor PLA2R in the above preparation method is 20 μg, 50 μg or 100 μg.

[0028] In some embodiments of the present invention, the activation time in the above preparation method S1 is 30 minutes and the temperature is 20-25°C.

[0029] In some embodiments of the present invention, the coupling time in the above preparation method S2 is 20 minutes and the temperature is 20-25°C.

[0030] The present invention also provides the use of the protein coupling reagent or the protein coupling microspheres obtained by the preparation method in preparing a microplate.

[0031] The present invention also provides a method for preparing a microporous plate, comprising the steps of blocking the protein-coupled microspheres, centrifuging, removing the supernatant, ultrasonicating, diluting, and then plating the microporous plate to obtain the microporous plate.

[0032] In some embodiments of the present invention, the sealing time in the above preparation method is 30 minutes and the temperature is 20-25°C.

[0033] In some embodiments of the present invention, the centrifugation speed in the above preparation method is 16000 rpm and the time is 30-60 min.

[0034] In some embodiments of the present invention, the centrifugation in the above preparation method includes a first centrifugation and a second centrifugation; the first centrifugation has a rotation speed of 16000 rpm and a time of 30 min; the second centrifugation has a rotation speed of 16000 rpm and a time of 30 min.

[0035] In some embodiments of the present invention, the second centrifugation in the above preparation method includes a step of mixing with 50 mm TB buffer.

[0036] In some embodiments of the present invention, the ultrasonic parameters in the above preparation method are 10%, 3 / 3, and the time is 2 minutes.

[0037] In some embodiments of the present invention, the preparation method further comprises the steps of coating, sealing and drying after plating.

[0038] In some embodiments of the present invention, the coating time in the above preparation method is 18 hours and the temperature is 2-8°C.

[0039] In some embodiments of the present invention, the blocking time in the above preparation method is 1 hour and the temperature is 20-25°C.

[0040] In some embodiments of the present invention, the drying time in the above preparation method is 2 hours and the temperature is 37°C.

[0041] In some embodiments of the present invention, the dilution in the above preparation method uses CB buffer; the concentration after the dilution is 0.1-0.2 μg / mL.

[0042] In some embodiments of the present invention, the concentration after dilution in the above preparation method is 0.1 μg / mL, 0.15 μg / mL or 0.2 μg / mL.

[0043] The present invention also provides a microporous plate obtained by the above preparation method.

[0044] The present invention also provides the use of the protein coupling reagent, the protein coupling microspheres obtained by the preparation method, and the microporous plate in protein or nucleic acid detection.

[0045] The present invention provides a protein coupling reagent, including a reagent and reagents ; The reagent Comprising: MES, EDC and NHS; the reagent Includes: phospholipase A2 receptor PLA2R.

[0046] The invention enlarges the protein area on the carrier and improves the sensitivity of the enzyme labeling plate by coating the microplate after the microspheres are coupled with proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0048] Figure 1 The figure shows the results of quality screening of protein coupling by activated microspheres;

[0049] Figure 2 The figure shows the results of protein coating concentration screening;

[0050] Figure 3 Graph showing the experimental results of the embodiment and the control group in the verification example. DETAILED DESCRIPTION

[0051] The invention discloses a protein coupling reagent, a protein coupling microsphere, a microporous plate and applications thereof.

[0052] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0053] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0054] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0055] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0056] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0057] The invention provides a preparation method of a polystyrene microplate for improving its sensitivity, comprising the steps of protein coupling, enzyme labeling plate coating, blocking, specimen incubation, enzyme conjugate incubation, substrate color development, and termination.

[0058] In Examples 1 to 9 and Effect Examples 1 to 3 of the present invention, phospholipase A2 receptor PLA2R (source: origene, batch number: WX03364F), CE210 solution (source: Beijing Borma, 2502LB-02), rabbit anti-PLA2R antibody (source: abcam, batch number: GR3266954-4),

[0059] In Examples 1 to 9 and Effect Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0060] The present invention will be further described below in conjunction with the embodiments:

[0061] Example 1 Preparation of coating buffer

[0062] (1) Prepare 100 mL of CB buffer: Weigh 0.159 g of Na2CO3 and 0.293 g of NaHCO3. Pour 55 mL of purified water into a beaker using a graduated cylinder. Place a stirring rotor in the beaker and stir on a magnetic stirrer until completely dissolved. Measure the pH of the solution with a calibrated pH meter. Adjust the pH to 9.60 ± 0.20 with 2 M HCl or 2 M NaOH and dilute to 100 mL.

[0063] (2) Prepare 100 mL of MES solution: Weigh MES and use a graduated cylinder to measure 90 mL of purified water into a beaker. Place the beaker in a stirring rotor and stir on a magnetic stirrer until completely dissolved. Measure the pH of the solution with a calibrated pH meter and adjust the pH to 6.00 ± 0.20 with 2 M HCl or 2 M NaOH. Dose to 100 mL.

[0064] (3) 100 mL of 10× concentrated washing solution: Weigh 2.422 g of NaCl and 8.766 g of Tris and dissolve them in purified water. Add 110 μL of Proclin 300 and 50 μL of Tween 20, mix well, adjust the pH to 7.2 ± 0.2, and make up to 100 mL with purified water.

[0065] (4) 1× washing solution: dilute the 10× concentrated washing solution obtained in step (3) 10 times.

[0066] (5) Sample diluent: Weigh 8.766 g of NaCl and 2.422 g of Tris, dissolve them in purified water, add 100 μL of ProClin 300, 1 g of BSA, and 50 μL of Tween 20, mix well, adjust the pH to 7.2 ± 0.2, and dilute to 100 mL with purified water.

[0067] (6) Basic blocking solution: Weigh 25 g of trehalose and dissolve it in purified water. Add 500 μL of ProClin 300 and 10 g of BSA, mix well, adjust the pH to 7.2 ± 0.2, and dilute to 500 mL with purified water.

[0068] (7) Coupling storage solution: Weigh 1.5 g of BSA and 0.03 g of PG20000, add 0.3 mL of 0.2 M boric acid, and add 29.7 mL of purified water to fully dissolve and mix.

[0069] Example 2 Coupling

[0070] (1) Activated microspheres

[0071] The MES solution obtained in Example 1 was used as the activation buffer solution. 0.2 μL of carboxyl microspheres was placed in a 2 ml centrifuge tube, 500 μL of activation buffer solution was added, and the mixture was mixed evenly on a vortex oscillator. 0.03 mg of EDC and 0.045 mg of N-hydroxysuccinimide (NHS) were added, respectively, and the mixture was mixed evenly on a vortex oscillator. The carboxyl groups on the surface of the magnetic beads were activated at 20-25° C. for 30 min.

[0072] (2) Microsphere-coupled protein

[0073] Add 20 μg of phospholipase A2 receptor PLA2R to the activated microspheres obtained in step (1), mix evenly on a vortex shaker, rotate and shake slowly during the process to avoid microsphere precipitation, and couple at 20-25°C for 20 minutes.

[0074] (3) Microsphere sealing

[0075] After step (2), add 50 μL of CE210 solution and block at 20-25°C for 30 min. After blocking, centrifuge at 16,000 rpm for 30 min and remove the supernatant.

[0076] (4) Centrifugal washing

[0077] Add 500 μL of 50 mm TB to the centrifuge tube in step (3), centrifuge at 16,000 rpm for 30 min, and remove the supernatant.

[0078] (5) Storage

[0079] Add 500 μL of coupling storage solution to the centrifuge tube in step (4), and ultrasonicate for 2 min at ultrasonic parameters of 10%, 3 / 3 to obtain protein-coupled microspheres.

[0080] Example 3 Coupling

[0081] (1) Activated microspheres

[0082] The MES solution obtained in Example 1 was used as the activation buffer solution. 0.2 μL of carboxyl microspheres was placed in a 2 ml centrifuge tube, 500 μL of activation buffer solution was added, and the mixture was mixed evenly on a vortex oscillator. 0.03 mg of EDC and 0.045 mg of N-hydroxysuccinimide (NHS) were added, respectively, and the mixture was mixed evenly on a vortex oscillator. The carboxyl groups on the surface of the magnetic beads were activated at 20-25° C. for 30 min.

[0083] (2) Microsphere-coupled protein

[0084] Add 50 μg of phospholipase A2 receptor PLA2R to the activated microspheres obtained in step (1), mix evenly on a vortex shaker, rotate and shake slowly during the process to avoid microsphere precipitation, and couple at 20-25°C for 20 minutes.

[0085] (3) Microsphere sealing

[0086] After step (2), add 50 μL of CE210 solution and block at 20-25°C for 30 min. After blocking, centrifuge at 16,000 rpm for 30 min and remove the supernatant.

[0087] (4) Centrifugal washing

[0088] Add 500 μL of 50 mm TB to the centrifuge tube in step (3), centrifuge at 16,000 rpm for 30 min, and remove the supernatant.

[0089] (5) Storage

[0090] Add 500 μL of coupling storage solution to the centrifuge tube in step (4), and ultrasonicate for 2 min at ultrasonic parameters of 10%, 3 / 3 to obtain protein-coupled microspheres.

[0091] Example 4 Coupling

[0092] (1) Activated microspheres

[0093] The MES solution obtained in Example 1 was used as the activation buffer solution. 0.2 μL of carboxyl microspheres was placed in a 2 ml centrifuge tube, 500 μL of activation buffer solution was added, and the mixture was mixed evenly on a vortex oscillator. 0.03 mg of EDC and 0.045 mg of N-hydroxysuccinimide (NHS) were added, respectively, and the mixture was mixed evenly on a vortex oscillator. The carboxyl groups on the surface of the magnetic beads were activated at 20-25° C. for 30 min.

[0094] (2) Microsphere-coupled protein

[0095] Add 100 μg of phospholipase A2 receptor PLA2R to the activated microspheres obtained in step (1), mix evenly on a vortex shaker, and rotate and shake slowly during the process to avoid microsphere precipitation. Couple at 20-25°C for 20 minutes.

[0096] (3) Microsphere sealing

[0097] After step (2), add 50 μL of CE210 solution and block at 20-25°C for 30 min. After blocking, centrifuge at 16,000 rpm for 30 min and remove the supernatant.

[0098] (4) Centrifugal washing

[0099] Add 500 μL of 50 mm TB to the centrifuge tube in step (3), centrifuge at 16,000 rpm for 30 min, and remove the supernatant.

[0100] (5) Storage

[0101] Add 500 μL of coupling storage solution to the centrifuge tube in step (4), and ultrasonicate for 2 min at ultrasonic parameters of 10%, 3 / 3 to obtain protein-coupled microspheres.

[0102] Example 5 Preparation of ELISA Plate

[0103] (1) Pre-coating

[0104] Rabbit anti-PLA2R antibody was diluted to 0.15 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to a white microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0105] (2) Coating

[0106] The phospholipase A2 receptor PLA2R was diluted to 0.15 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to the pre-coated microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0107] (2) Closed

[0108] The wells were blocked with basic blocking solution. 180 μL of the basic blocking solution obtained in Example 1 was added to each well. The wells were blocked at 20-25° C. for 1 h, and the liquid was discarded.

[0109] (3) Drying and bagging

[0110] Place the plate stabilized in step (2) in an oven and dry at 37°C for 2 hours. Remove the plate and place it in an aluminum foil bag, add desiccant, and seal the bag for later use.

[0111] Example 6 Preparation of ELISA Plate

[0112] (1) Pre-coating

[0113] Rabbit anti-PLA2R antibody was diluted to 0.15 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to a white microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0114] (2) Coating

[0115] The protein-coupled microspheres obtained in Example 4 were diluted to 0.1 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to the pre-coated microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0116] (3) Closed

[0117] The wells were blocked with basic blocking solution. 180 μL of the basic blocking solution obtained in Example 1 was added to each well. The wells were blocked at 20-25° C. for 1 h, and the liquid was discarded.

[0118] (4) Drying and bagging

[0119] Place the plate stabilized in step (2) in an oven and dry at 37°C for 2 hours. Remove the plate and place it in an aluminum foil bag, add desiccant, and seal the bag for later use.

[0120] Example 7 Preparation of ELISA Plate

[0121] (1) Pre-coating

[0122] Rabbit anti-PLA2R antibody was diluted to 0.15 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to a white microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0123] (2) Coating

[0124] The protein-coupled microspheres obtained in Example 4 were diluted to 0.15 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to the pre-coated microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0125] (3) Closed

[0126] The wells were blocked with basic blocking solution. 180 μL of the basic blocking solution obtained in Example 1 was added to each well. The wells were blocked at 20-25° C. for 1 h, and the liquid was discarded.

[0127] (4) Drying and bagging

[0128] Place the plate stabilized in step (2) in an oven and dry at 37°C for 2 hours. Remove the plate and place it in an aluminum foil bag, add desiccant, and seal the bag for later use.

[0129] Example 8 Preparation of ELISA Plate

[0130] (1) Pre-coating

[0131] Rabbit anti-PLA2R antibody was diluted to 0.15 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to a white microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0132] (2) Coating

[0133] The protein-coupled microspheres obtained in Example 4 were diluted to 0.2 μg / mL with the CB buffer obtained in Example 1. The diluted coating solution was added to the pre-coated microplate at 100 μL / well. The coated plate was placed in a refrigerator (at 2-8°C) for 18 h. The plate was washed twice with the 1× washing solution obtained in Example 1 and patted dry after washing.

[0134] (2) Closed

[0135] Block with basic blocking solution. Add 180 μL of basic blocking solution to each well, block at 20-25°C for 1 h, and discard the liquid.

[0136] (3) Drying and bagging

[0137] Place the plate stabilized in step (2) in an oven and dry at 37°C for 2 hours. Remove the plate and place it in an aluminum foil bag, add desiccant, and seal the bag for later use.

[0138] Example 9 Detection Method

[0139] (1) Preparation of enzyme conjugate

[0140] Dilute goat anti-human IgG-HRP 6000 times and mix well.

[0141] (2) Sample incubation

[0142] PLA2R-positive and -negative samples of varying concentrations were diluted 100-fold with sample diluent, or diluted to 1024-fold using high-concentration samples. The samples were then added to the ELISA plates obtained in Examples 5 to 8, equilibrated to 20-25°C, at a rate of 100 μL / well. A blank control was also added, with 100 μL / well of sample diluent added directly. The plates were incubated at 20-25°C for 30 min. The plates were then washed three times with 300 μL of the 1× wash buffer obtained in Example 1, and any remaining liquid was patted dry.

[0143] (3) Enzyme conjugate incubation

[0144] 100 μL of enzyme conjugate was added to each well, and the mixture was reacted at 20-25° C. for 30 min. The 1× washing solution obtained in the example was used three times, 300 μL each time, and the residual liquid was patted dry.

[0145] (4) Color reading

[0146] Add chemiluminescent substrate to each well, mix A and B solutions in equal proportions, 100 μL / well, and read the luminescence value using a microplate reader.

[0147] Effect Example 1

[0148] The protein coupling quality of the activated microspheres of Examples 2 to 4 was screened according to the coating steps of Example 5 and the detection method of Example 9. The detection results are shown in Tables 1 and Figure 1 As shown;

[0149] Table 1

[0150]

[0151] The experimental results are as follows Figure 1As shown in Table 1, the activated microspheres were coupled with proteins of different masses. The results showed that different protein amounts affected the coupling effect. In Example 3, adding 50 μg of phospholipase A2 receptor PLA2R to the activated microspheres was the optimal choice.

[0152] Effect Example 2

[0153] The proteins of Examples 6 to 8 coated with different concentrations were screened according to the detection method of Example 9. The detection results are shown in Tables 2 and Figure 2 As shown;

[0154] Table 2

[0155]

[0156] The experimental results are as follows Figure 2 As shown in Table 2, by coating with different concentrations of coupled microglobulin, it can be concluded that 0.15 μg / mL is the optimal concentration for coating in Example 6.

[0157] Effect Example 3 Sensitivity detection of the microplate of the present invention

[0158] Control microplate: The microplate obtained in Example 5, except that the phospholipase A2 receptor PLA2R does not need to be coupled, and the protein is directly diluted with CB buffer and then coated.

[0159] Experimental microplate: the microplate obtained in Example 7.

[0160] (1) The results of the luminescence value detection are shown in Table 3;

[0161] Table 3

[0162]

[0163] (2) The mean values of the test results of the control group and the experimental group were subjected to one-way ANOVA, and the results are shown in Tables 4 and 5;

[0164] Table 4 Analysis of variance: One-way analysis of variance

[0165]

[0166] Table 5 Analysis of variance

[0167]

[0168] As shown in Table 5, if the P value is less than 0.05, the data of the experimental group and the control group are significantly different.

[0169] (3) The average values of the control group and the experimental group are analyzed by line graph, such as Figure 3As shown in the figure, it can be seen that the absorbance value of the experimental group is greater than the luminescence value of the control group, and the change trends of the two groups of data are roughly the same; when the luminescence value of the control group's detection results approaches 0, the experimental group can still detect lower concentrations.

[0170] (4) Conclusion

[0171] 1) The absorbance value increased after the activated carboxyl microspheres were coupled with the phospholipase A2 receptor PLA2R.

[0172] 2) The use of activated carboxyl microspheres coupled to the phospholipase A2 receptor PLA2R can detect lower concentrations of antibodies and improve sensitivity.

[0173] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a microplate, characterized in that: The protein-coupled microspheres were blocked, centrifuged, the supernatant was removed, sonicated, diluted to 0.15 μg / ml, and then plated and coated for 18 hours to obtain the microplate; The preparation method of the protein-coupled microspheres, The following steps are involved: S1: After activation of the biomagnetic beads, activated microspheres are obtained; S2: taking the activated microspheres and coupling them to obtain the protein-coupled microspheres; The activation described in S1 and the coupling described in S2 use protein coupling reagents; The protein coupling reagent includes reagent I and reagent II; The reagent I includes: MES, EDC and NHS; the reagent II includes: phospholipase A2 receptor PLA2R; The MES was added in the form of an MES solution; the concentration of the MES solution was 0.05 mol / L, and the pH value was 6.0; the mass ratio of the EDC to the NHS was 1:15; The activation in S1 uses the reagent I; the volume ratio of the biomagnetic beads to the MES is 1:2500; The coupling in S2 uses reagent II; the mass ratio of the activated microspheres to the phospholipase A2 receptor PLA2R is 1:1~5.

2. The microplate obtained by the preparation method according to claim 1.

3. Use of the microplate as claimed in claim 2 in protein or nucleic acid detection.

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