CN115656494A - Method for coupling blue microspheres with antibody and application - Google Patents
Method for coupling blue microspheres with antibody and application Download PDFInfo
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- CN115656494A CN115656494A CN202211266834.8A CN202211266834A CN115656494A CN 115656494 A CN115656494 A CN 115656494A CN 202211266834 A CN202211266834 A CN 202211266834A CN 115656494 A CN115656494 A CN 115656494A
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Abstract
The invention discloses a method for coupling blue microspheres with an antibody and application thereof, which relate to the technical field of biological detection and comprise the following steps: step one, buffer solution is prepared; step two, removing supernatant; step three, neutralizing the final concentration; step four, adding the monoclonal antibody; step five, adding a coupling reagent; and step six, post-treatment. The method comprises the steps of preparing a phosphate buffer solution and washing microspheres, removing supernatant by using a centrifugal machine to obtain a precipitate, adding the buffer solution and water into the precipitate to neutralize the final concentration, then adding a monoclonal antibody, uniformly mixing on a shaking table to obtain a solution, adding a coupling reagent, namely propyl phosphoric anhydride into the solution, then adding a bovine serum albumin solution to seal, centrifuging again after sealing is finished to remove the supernatant, and finally performing ultrasonic treatment to complete the coupling process of the blue microspheres and the antibody, and has the advantages of simple operation, short reaction time and high coupling efficiency of the blue microspheres and the antibody; the requirements on the types and the pH of the buffer solution are wide, and the like.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for coupling blue microspheres with an antibody and application thereof.
Background
The microsphere is coupled with an antibody and can also be coupled with an antigen, the surface of a carrier should have chemical active groups, and the groups can be directly coupled with antibiotics (antibiotics) or pesticide molecules, which is the premise of preparing the antigen by chemical coupling; secondly, the carrier should have a certain capacity to couple enough molecules; the carrier should also be inert and should not interfere with the function of the coupling molecule; the carrier should have sufficient stability and should be inexpensive and readily available, and bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, human serum albumin, artificially synthesized polylysine, and the like are commonly used as carrier proteins for synthesizing artificial antigens. In these protein molecules, a part of alpha and epsilon-amino groups (isoelectric points 8 and 10), phenol groups, mercapto groups (isoelectric point 9), imidazole groups (isoelectric point 7), carboxyl groups (isoelectric points 2 to 4, mostly from beta-and gamma-carboxyl groups of aspartic acid or glutamic acid) and the like become protons under the condition of isoelectric point pH, and the other part of unprotonated nucleophilic groups have reactivity and can be combined with corresponding groups in haptens. In addition, when these proteins are solubilized with organic solvents (e.g., pyridine, dimethylformamide), the active groups remain soluble, and thus, these two proteins are the most commonly used carrier proteins. In recent years, it has been reported that the use of synthetic polypeptides (most commonly polylysine) as carriers has been widely used to increase the immunogenicity of haptens, thereby increasing the possibility of generating specific antibodies against haptens.
At present, two common methods for coupling carboxyl microspheres with amino groups are available: one is a one-step method using carbodiimide (EDC) as a cross-linking agent, and the other is a two-step method using carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linking agents.
The disadvantage of the one-step method is that the EDC reagent is sensitive to water, the preparation is required to be carried out at present, and the intermediate product is extremely unstable in aqueous solution and easy to hydrolyze, so that the coupling efficiency is low, the whole reaction is uncontrollable, and the final product may have a large amount of self-linked products of a plurality of proteins besides the target product, thereby not only wasting the proteins, but also greatly reducing the reaction efficiency.
The by-product formed by the two-step process inhibits the localization of carboxyl groups on the conjugated protein, resulting in a decrease in coupling efficiency. Meanwhile, EDC is sensitive to pH value, the optimum pH value of activated carboxyl is 3.5-4.5, but the optimum pH value of coupling an intermediate product formed after the carboxyl is activated and amino is 4.5-7.5, and in the practical application process, the operation is complicated, and the reaction efficiency is influenced. In addition, EDC reacts with phosphate groups, so that a phosphate buffer cannot be used during the reaction in which EDC participates, but the phosphate buffer is a buffer that is commonly used in the art, easily prepared, and inexpensive. Therefore, both the one-step method and the two-step method in the prior art have the defects of harsh operation conditions, complicated operation steps, long reaction time, complex reaction process, low coupling efficiency and the like.
Disclosure of Invention
The invention provides a method for coupling blue microspheres with an antibody and application thereof, which aim to solve the problems in the background technology.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for coupling blue microspheres with antibodies and application thereof comprise the following steps of preparing a buffer solution, removing a supernatant, neutralizing final concentration, adding monoclonal antibodies, adding a coupling reagent, and performing aftertreatment.
The technical scheme of the invention is further improved as follows: in the first step, 0.1M phosphate buffer solution with the pH value of 6.0-8.0 is prepared.
The technical scheme of the invention is further improved as follows: and in the second step, the microspheres are washed for 1 time by using the 0.1M phosphate buffer solution with the pH value of 6.0-8.0 obtained in the first step, the microspheres are centrifuged for 20min by using a centrifuge to keep the rotating speed at 12000rpm, and the supernatant is removed to obtain a precipitate.
The technical scheme of the invention is further improved as follows: and in the third step, adding 0.1M phosphate buffer solution with the pH value of 6.0-8.0 and water into the precipitate obtained in the second step to ensure that the final concentration of the phosphate buffer solution is 5-50 mM and the final concentration of the blue microspheres is 1% w/v to obtain the blue microsphere solution.
The technical scheme of the invention is further improved as follows: and in the fourth step, the monoclonal antibody is added into the microsphere solution obtained in the third step, the final concentration of the antibody is 5-50 ug/mL, the mixture is uniformly mixed, and the mixture is reacted on a shaking table for 5min to obtain a solution.
The technical scheme of the invention is further improved as follows: and step five, adding propyl phosphoric anhydride (T3P) into the solution obtained in step four to ensure that the final concentration of the T3P is 0.01-1%, uniformly mixing, and reacting for 40-60 min in a shaking table.
The technical scheme of the invention is further improved as follows: and step six, sealing the solution after the shaking table reaction by using a bovine serum albumin solution, keeping the final concentration of the bovine serum albumin at 0.1% -1%, continuing to react for 30min on the shaking table, centrifuging the solution at 12000rpm for 20min after the reaction is finished, removing the supernatant, washing for 2 times by using Tris-HCl buffer solution to obtain a precipitate, redissolving the precipitate by using a microsphere composite solution, carrying out ultrasonic treatment on the redissolved microsphere for 2-5 min by using an ultrasonic machine, and finishing the coupling process of the antibody-blue microsphere.
The technical scheme of the invention is further improved as follows: the LH antibody-blue microsphere conjugate is prepared into a binding pad, and is matched with an LH coated plate to prepare an LH detection test strip, when a sample to be detected contains a certain amount of antigen, the sample flows through the binding pad through the sample pad and is bound with the antibody on the binding pad to form a complex, then the complex further binds the antibody on the detection line when the sample flows through the detection line of the LH detection test strip, after a period of time, a line appears on the LH detection test strip, when no antigen exists in the detection sample, the antibody 2 on the binding pad cannot react with the antibody on the LH detection test strip to form the complex, and at the moment, the detection line is blank.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the invention provides a method for coupling blue microspheres with an antibody and application thereof, which comprises the steps of preparing a buffer solution, removing a supernatant, neutralizing the final concentration, adding a monoclonal antibody, adding a coupling reagent, carrying out post-treatment and the like, preparing a phosphate buffer solution and washing the microspheres, removing the supernatant by using a centrifuge to obtain a precipitate, adding the buffer solution and water into the precipitate to neutralize the final concentration, then adding the monoclonal antibody to the precipitate to be uniformly mixed on a shaker to obtain a solution, adding a coupling reagent of propyl phosphoric anhydride into the solution, then adding a bovine serum albumin solution to seal, carrying out centrifugation again after the sealing is finished to carry out supernatant removal treatment, and finally completing the coupling process of the antibody and the blue microspheres by ultrasound. The method has the advantages of simple operation, short reaction time and high coupling efficiency of the blue microspheres and the antibody; the requirements on the types and the pH of the buffer solution are wide, and the like.
Drawings
Fig. 1-2 are schematic flow diagrams of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples:
example 1
As shown in FIGS. 1-2, the invention provides a method for coupling blue microspheres with antibodies and application thereof, comprising the following steps of preparing a buffer solution in the first step, removing a supernatant in the second step, neutralizing the final concentration in the third step, step four, adding a monoclonal antibody, step five, adding a coupling reagent, step six, post-processing, preparing 0.1M phosphate buffer solution with pH of 6.0-8.0 in the step one, washing the microspheres for 1 time by using the 0.1M phosphate buffer solution with pH of 6.0-8.0 obtained in the step one in the step two, centrifuging at 12000rpm by using a centrifuge at a rotating speed for 20min, removing supernatant to obtain a precipitate, adding 0.1M phosphate buffer solution with pH of 6.0-8.0 and water into the precipitate obtained in the step two in the step three to ensure that the final concentration of the phosphate buffer solution is 5-50 mM and the final concentration of the blue microspheres is 1% w/v to obtain a blue microsphere solution, in the fourth step, the monoclonal antibody is added into the microsphere solution obtained in the third step, the concentration of the monoclonal antibody is 5-50 ug/mL, the mixture is evenly mixed, reacting on a shaking table for 5min to obtain a solution, adding propylphosphoric anhydride (T3P) into the solution obtained in the step four in the step five to ensure that the final concentration of the T3P is 0.01-1%, uniformly mixing, reacting on the shaking table for 40-60 min, in the sixth step, the solution after the table shaking reaction is closed by bovine serum albumin solution, the final concentration of the bovine serum albumin is 0.1% -1%, the table shaking reaction is continued for 30min, the solution is centrifuged at 12000rpm for 20min after the reaction is finished, the supernatant is removed, and then washing the precipitate for 2 times by using Tris-HCl buffer solution to obtain a precipitate, re-dissolving the precipitate by using a microsphere complex solution, carrying out ultrasonic treatment on the re-dissolved microsphere for 2-5 min by using an ultrasonic machine, and finishing the coupling process of the antibody-blue microsphere.
In this example, 1ml of phosphate buffer (ph 7.4) was added to the centrifuge tube, 20ul of blue microspheres (solid content 5%) were added to the centrifuge tube, after shaking, the supernatant was centrifuged, 50ug of LH monoclonal antibody was added, mixing was performed, shaking was performed for 5min, T3P was added to the centrifuge tube, the final concentration of T3P was 0.1%, reaction was performed for 40min, after the reaction was completed, the supernatant was centrifuged, the protein concentration in the supernatant was measured with a BCA kit to calculate the labeling efficiency, labeling was performed again, the above steps were repeated, BSA bovine serum albumin was added to each centrifuge tube to seal, the final concentration of BSA was 0.1%, sealing was performed for 30min to prevent non-specific adsorption, after the reaction was completed, the solution was centrifuged at 12000rpm for 20min, the supernatant was removed, then Tris-Hcl buffer was used to wash for 2 times, the pellet microsphere complex solution was redissolved, the redissolved microspheres were subjected to ultrasonic LH 2-5 min, the LH antibody-blue microsphere coupling process was completed, the antibody-blue microsphere conjugate was made into a conjugate pad, and was prepared into LH test strip.
Example 2
As shown in fig. 1-2, in this example, on the basis of example 1, an LH test strip was prepared in the same manner as in example 1, the reaction buffer was changed to MES (ph 6.0), the coupling agent was EDC, and the final concentration was 0.1%; an LH test strip was prepared in the same manner as in example 1, the reaction buffer was changed to MES (ph 6.0), the coupling agents were EDC and NHS, the final concentration of EDC was 0.1%, and the final concentration of NHS was 0.01%.
The results of coupling efficiency and test paper performance of 3 different methods are shown in tables 1 and 2;
the batch differences of the LH test strips prepared by coupling 3 different methods are shown in Table 3;
the batch differences of the LH test strips prepared by coupling 3 different methods are shown in Table 4.
TABLE 1
TABLE 2
In-batch variation of LH test paper prepared by different cross-linking agents
TABLE 3
Batch-to-batch difference of LH test paper prepared by different cross-linking agents
TABLE 4
Example 3
As shown in fig. 1-2, based on example 1, in this embodiment, an application of a blue microsphere coupled antibody is proposed, where an LH antibody-blue microsphere conjugate is made into a conjugate pad, and is matched with an LH coated plate to make an LH detection test strip, when a sample to be detected contains a certain amount of antigen, the sample flows through the conjugate pad via the sample pad, and is bound with the antibody on the conjugate pad to form a complex, and then flows through a detection line of the LH detection test strip, the antibody on the detection line is further bound, after a certain period of time, a line appears on the LH detection test strip, and when no antigen exists in the detection sample, the antibody 2 on the conjugate pad cannot react with the antibody on the LH detection test strip to form a complex, and the detection line is blank.
The following is a specific description of the method for coupling the blue microsphere with the antibody and the working principle of the application.
As shown in fig. 1-2, 1ml of phosphate buffer (ph 7.4) is added into a centrifuge tube, 20ul of blue microspheres (solid content is 5%) is added into the centrifuge tube, after shaking, supernatant is centrifugally removed, 50ug of LH monoclonal antibody is added, mixing is performed, shaking is performed for 5min, T3P is added into the centrifuge tube, the final concentration of T3P is 0.1%, reaction is performed for 40min, after the reaction is finished, supernatant is centrifugally removed, the protein concentration in the supernatant is measured by using a BCA kit to calculate the labeling efficiency, labeling is performed again, the above steps are repeated, BSA bovine serum albumin is added into each centrifuge tube to perform sealing, the final concentration of BSA is 0.1%, sealing is 30min, after the reaction is finished, the solution is 12000rpm, centrifuged for 20min, the supernatant is removed, then Tris-Hcl buffer is used for washing for 2 times, a precipitation microsphere complex solution is redissolved, the ultrasound antibody-blue microsphere coupling process is completed, an antibody-blue microsphere conjugate is made into a conjugate pad, and is matched with a coated plate to prepare a test strip, so that the problem of low coupling efficiency in the existing technology is solved. And the problem of large batch-to-batch difference caused by complicated coupling process and poor controllability, and has the advantages of simple operation, short reaction time, high coupling efficiency of the blue microspheres and the antibody, wide requirements on the types and pH values of the buffer solution and the like.
The present invention has been described in general terms in the foregoing, but it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Therefore, modifications or improvements are within the scope of the invention without departing from the spirit of the inventive concept.
Claims (8)
1. A method for coupling blue microspheres with antibodies is characterized by comprising the following steps:
step one, buffer solution is prepared;
step two, removing supernatant;
step three, neutralizing the final concentration;
step four, adding the monoclonal antibody;
step five, adding a coupling reagent;
and step six, post-treatment.
2. The method and the application of the blue microsphere coupled antibody according to claim 1, wherein the method comprises the following steps: in the first step, 0.1M phosphate buffer solution with the pH value of 6.0-8.0 is prepared.
3. The method and the application of the blue microsphere coupled antibody according to claim 2, wherein the method comprises the following steps: and in the second step, the microspheres are washed for 1 time by using the 0.1M phosphate buffer solution with the pH value of 6.0-8.0 obtained in the first step, the microspheres are centrifuged for 20min by using a centrifuge to keep the rotating speed at 12000rpm, and the supernatant is removed to obtain a precipitate.
4. The method and the application of the blue microsphere coupled antibody according to claim 3, wherein the method comprises the following steps: and in the third step, adding 0.1M phosphate buffer solution with the pH value of 6.0-8.0 and water into the precipitate obtained in the second step to ensure that the final concentration of the phosphate buffer solution is 5-50 mM and the final concentration of the blue microspheres is 1% w/v to obtain a blue microsphere solution.
5. The method and the application of the blue microsphere coupled antibody according to claim 4, wherein the method comprises the following steps: and in the fourth step, the monoclonal antibody is added into the microsphere solution obtained in the third step, the final concentration of the antibody is 5-50 ug/mL, the mixture is uniformly mixed, and the mixture is reacted on a shaking table for 5min to obtain a solution.
6. The method and the application of the blue microsphere coupled antibody according to claim 4, wherein the method comprises the following steps: and step five, adding propyl phosphoric anhydride (T3P) into the solution obtained in step four to ensure that the final concentration of the T3P is 0.01-1%, uniformly mixing, and reacting in a shaking table for 40-60 min.
7. The method and the application of the blue microsphere coupled antibody according to claim 6, wherein the method comprises the following steps: and in the sixth step, the solution obtained after the shaking table reaction in the fifth step is sealed by a bovine serum albumin solution, the final concentration of the bovine serum albumin is 0.1% -1%, the shaking table reaction is continued for 30min, the solution is centrifuged at 12000rpm for 20min after the reaction is finished, the supernatant is removed, then Tris-HCl buffer solution is used for washing for 2 times to obtain a precipitate, the precipitate is redissolved by a microsphere composite solution, the redissolved microspheres are subjected to ultrasonic treatment for 2-5 min by an ultrasonic machine, and the coupling process of the antibody-blue microspheres is finished.
8. The method for coupling blue microspheres with an antibody according to any one of claims 1 to 7, wherein the LH antibody-blue microsphere conjugate is prepared as a conjugate pad, and is combined with an LH-coated plate to prepare an LH detection strip, when a sample to be detected contains a certain amount of antigen, the LH antibody-blue microsphere conjugate flows through the conjugate pad via the sample pad, and is combined with the antibody on the conjugate pad to form a complex, and then flows through a detection line of the LH detection strip, the LH detection strip is further combined with the antibody on the detection line, after a certain period of time, a line appears on the LH detection strip, when no antigen exists in the detection sample, the antibody 2 on the conjugate pad cannot react with the antibody on the LH detection strip to form a complex, and then the detection line is blank.
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| CN117471110A (en) * | 2023-12-27 | 2024-01-30 | 天津德祥生物技术股份有限公司 | Blocking liquid and blocking method for blocking antigen-microsphere indication system |
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