CN110596373A - Method for coupling magnetic particles and antibody - Google Patents
Method for coupling magnetic particles and antibody Download PDFInfo
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- CN110596373A CN110596373A CN201910885281.6A CN201910885281A CN110596373A CN 110596373 A CN110596373 A CN 110596373A CN 201910885281 A CN201910885281 A CN 201910885281A CN 110596373 A CN110596373 A CN 110596373A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
Abstract
The invention relates to the field of biotechnology detection, in particular to a method for coupling magnetic particles and antibodies, which comprises the following steps of (1) taking a magnetic particle solution, magnetically separating to remove supernatant, adding a buffer solution, and uniformly mixing at room temperature to obtain a magnetic particle suspension; (2) carrying out magnetic separation on the magnetic particle suspension, removing supernatant, adding phosphate buffer solution dissolved with the composite coupling reinforcing agent, and uniformly mixing for later use; (3) adding an antibody, uniformly mixing, coupling, controlling the coupling temperature and the coupling time, carrying out magnetic separation after coupling, and removing supernatant to obtain magnetic particles coupled with the antibody; (4) adding the magnetic particles coupled with the antibody in the step (3) into the sealing liquid, uniformly mixing, and sealing; (5) coupling magnetic particles with antibodies, magnetically separating to remove supernatant, adding storage solution, mixing, and storing. The method can improve the coupling efficiency of the magnetic particles and the antibody, and further improve the linear range and accuracy of the kit.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to a method for coupling magnetic particles and antibodies.
Background
The traditional immunology detection mostly uses an enzyme label plate as a solid phase carrier, but the suspended magnetic particles as the carrier have higher specific surface area, can react with a sample more fully, and have the advantages of higher sensitivity, higher detection speed, better repeatability and the like compared with the enzyme label plate carrier in addition to the flexible application of an external magnetic field, so that the traditional immunology detection is widely applied to the fields of biological and medical detection and the like. The magnetic particle chemiluminescence immunoassay technology integrates a magnetic particle carrier technology and a chemiluminescence immunoassay technology, the efficiency of the existing coupling method of magnetic particles and antibodies is generally low, raw materials are wasted, the sensitivity of reagents is reduced, and the phenomenon of missed detection of some detection items is caused. Therefore, it is highly desirable to develop a novel coupling method that allows efficient binding of an antibody to magnetic microparticles and further improves the sensitivity of a reagent.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, the method for coupling the magnetic particles and the antibodies is provided, and the method can improve the coupling efficiency of the magnetic particles and the antibodies and further improve the linear range and accuracy of the kit.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method of coupling magnetic particles to antibodies, the method comprising the steps of:
(1) taking the magnetic particle solution, performing magnetic separation to remove supernatant, adding a buffer solution, and uniformly mixing at room temperature to obtain a magnetic particle suspension;
(2) performing magnetic separation on the magnetic particle suspension obtained in the step (1), removing supernatant, adding a phosphate buffer solution dissolved with the composite coupling reinforcing agent into the magnetic particles, and uniformly mixing at room temperature for later use;
(3) adding the antibody into the step (2), uniformly mixing, placing in a constant-temperature culture shaking table for coupling, controlling the coupling temperature and the coupling time, carrying out magnetic separation after coupling, and removing supernatant to obtain magnetic particles coupled with the antibody;
(4) taking the magnetic particles coupled with the antibody in the step (3), adding a sealing liquid, uniformly mixing, and placing in a constant-temperature culture shaking table for sealing;
(5) and (4) closing the magnetic particles coupled with the antibody by the closing liquid in the step (4) for a period of time, carrying out magnetic separation to remove supernatant, adding storage liquid, uniformly mixing at room temperature, and storing in an environment at 2-8 ℃.
As an improved technical scheme, the magnetic particle solution in the step (1) is a tosyl magnetic particle solution, the addition amount of the tosyl magnetic particle solution is 1-5mL, and the concentration of the tosyl magnetic particle solution is 10-100 mg/mL.
As an improved technical scheme, the composite coupling reinforcing agent in the step (2) is composed of Na2SO4、MgSO4、(NH4)2SO4And buffer solution, wherein the addition amount of the composite coupling agent is 2-20ml, and each 1L of the composite coupling reinforcing agent contains Na2SO4、MgSO4And (NH)4)2SO4The molar concentrations of (A) and (B) are respectively 0.1-3M.
As a preferred technical scheme, the Na in the composite coupling agent2SO4、MgSO4And (NH)4)2SO4The molar concentration ratio of (A) to (B) is 1-2:1: 1.
As an improved technical scheme, the buffer solution in the step (2) and the step (1) is phosphate buffer solution with the pH value of 7-8 or borate buffer solution with the pH value of 8-9.5 or carbonate buffer solution with the pH value of 8-9.5.
As an improved technical scheme, the coupling temperature in the step (3) is controlled to be 35-40 ℃, and the coupling time is controlled to be 6-16 h.
As an improved technical scheme, in the step (4), the blocking time is 1-6h, the adding amount of the blocking solution is 4-20mL, and the blocking solution is prepared from phosphate buffer solution and at least one of BSA, casein and glycine.
As a preferable technical scheme, in the step (4), the blocking solution is prepared by a phosphate buffer solution with the pH value of 7.4-8 in BSA, casein and glycine, wherein the mass concentration of the BSA, the casein and the glycine in the blocking solution is 0.1-1.0% respectively.
As an improved technical scheme, the adding amount of the stock solution in the step (5) is 4-20mL, and the stock solution is prepared by at least one of BSA, casein and glycine and a Tris buffer solution.
As a preferable technical scheme, the stock solution in the step (4) is prepared by mixing a Tris buffer solution with the pH value of 7.4-8 in BSA, casein and glycine, wherein the mass concentration of the BSA, the casein and the glycine in the stock solution is 0.01-0.05%.
After the technical scheme is adopted, the invention has the beneficial effects that:
(1) the invention adds a composite coupling reinforcing agent when the antibody is coupled with the tosyl magnetic particles, wherein the composite coupling reinforcing agent is Na2SO4、MgSO4、(NH4)2SO4Is prepared from Na and buffer solution2SO4、MgSO4、(NH4)2SO4The concentration in the composite coupling reinforcing agent is 0.1-3M, the coupling temperature is controlled to be 35-40 ℃ when the antibody and the magnetic particles are coupled, the coupling time is 6-16h, the composite coupling reinforcing agent can provide proper salt concentration for the antibody and the magnetic particles, the three components have synergistic effect, the deposition speed of the antibody on the surface of the magnetic particles is promoted, the exposure of hydrophobic groups of the antibody is accelerated, the magnetic particles and the antibody can be coupled more quickly, the coupling is more sufficient and firmer, the coupling efficiency of the antibody and the magnetic particles is greatly improved, and the sensitivity of the kit is further improved.
(2) After the antibody and the magnetic particles are coupled, adding a blocking solution for blocking for 1-6h, wherein the blocking solution is prepared from BSA, casein and glycine and a phosphate buffer solution, the mass concentrations of the BSA, the casein and the glycine in the blocking solution are respectively 0.1-1.0%, and the pH value of the phosphate buffer solution is 7.4-8.
(3) After the magnetic particles coupled with the antibody are sealed by a sealing solution, a storage solution is prepared by mixing BSA, casein and glycine with a Tris buffer solution, wherein the mass concentrations of the BSA, the casein and the glycine in the storage solution are respectively 0.01-0.05%, and the pH value of a phosphate buffer solution is 7.4-8. The magnetic particles coupled with the antibody can stabilize the antibody combined on the magnetic particles through storage of the storage solution.
In conclusion, the composite coupling reinforcing agent is added when the antibody is coupled with the magnetic particles, the coupling temperature and the coupling time are controlled, and the use of the composite coupling reinforcing agent can improve the coupling efficiency of the antibody and the magnetic particles; and then the magnetic particles coupled with the antibodies are sealed by the sealing liquid and are placed in a storage liquid for storage and standby, the operation can prevent interference, the antibodies combined on the magnetic particles are stabilized, and the linear range and the accuracy of the kit are further improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A method of magnetic particle coupling of alpha fetoprotein antibody molecules (AFPs) comprising the steps of:
(1) taking 1mL of 10mg/mL magnetic particle solution, removing supernatant through magnetic separation, adding 1mL of phosphate buffer solution with pH of 7.8, and uniformly mixing by swirling for 10s at room temperature to obtain magnetic particle suspension;
(2) taking the magnetic particle suspension in the step (1), carrying out magnetic separation to remove supernatant, and adding 2mL of composite coupling enhancer (prepared from Na)2SO4、Mg SO4、(NH4)2SO4Is prepared by mixing with phosphate buffer solution with pH of 7.8, wherein Na in the composite coupling reinforcing agent2SO4、Mg SO4、(NH4)2SO4The molar concentration ratio of 1:1:1), and uniformly mixing by swirling for 10s at room temperature;
(3) adding 0.1mg of AFP antibody into the step (2), swirling for 10s at room temperature, and then placing the mixture in a constant-temperature culture shaking table at 37 ℃ for coupling for 6 h;
(4) after magnetically separating the supernatant, adding 4mL of blocking solution (prepared by adding BSA, casein and glycine and a phosphate buffer solution with pH7.5, wherein the mass concentrations of the BSA, the casein and the glycine in the blocking solution are respectively 0.1%, 0.5% and 0.3%), performing vortex 10 at room temperature, and then placing in a constant-temperature culture shaker at 37 ℃ for blocking for 1 h;
(5) after magnetic separation of the supernatant, a stock solution (prepared from BSA, casein and glycine and a Tris buffer solution with pH of 7.5, wherein the mass concentrations of the BSA, the casein and the glycine in the stock solution are respectively 0.01%, 0.02% and 0.003%) is added, vortexed at room temperature for 10s and mixed uniformly, and the mixture is stored at 2-8 ℃.
In order to prove that the magnetic particles coupled with the antibody obtained by the coupling method have higher coupling effect, the magnetic particles coupled with the antibody in the coupling method are used as components of a kit for testing. A comparative example was also established in which the kit components were the same as in the present invention, the only difference being that only ammonium sulfate was added to the antibody-conjugated magnetic microparticles at the time of conjugation.
The specific detection method comprises the following steps: 50uL of magnetic particle coupling reagent +50uLAFP standard (SO-S5 concentrations of 0ng/mL,2.5ng/mL,5ng/mL,10ng/mL,20ng/mL,40ng/mL) +50uL of enzyme labeling reagent (acridinium ester label), 30min incubation at 37 ℃, 3 washes of 0.1M Tris wash containing 0.1% BSA by mass at pH8 +100uL of pre-excitation solution (containing 1.32% w/v hydrogen peroxide in acidic solution, available from Yapei trade (Shanghai) Co., Ltd.) were tested, and the results are shown in Table 1:
TABLE 1
The experimental results in table 1 show that, at the same concentration point, when the magnetic particles coupled with the antibody are coupled, three coupling reinforcing agents are added, compared with the case where one coupling reinforcing agent is added, the magnetic particle coupling reagents added with the three coupling reinforcing agents are higher in numerical value measured as the kit components under the same operation, which indicates that the coupling efficiency of the antibody and the magnetic particles is higher, and the linear range of the kit is further improved.
Example 2
A method of magnetic particle coupling to a thyrotropin antibody molecule (TSH) comprising the steps of:
(1) taking 1mL of 50mg/mL magnetic particle solution, carrying out magnetic separation to remove supernatant, adding 5mL of phosphate buffer (pH is 8), and uniformly mixing by swirling for 10s at room temperature to obtain magnetic particle suspension;
(2) taking the magnetic particle suspension in the step (1), carrying out magnetic separation to remove supernatant, and adding 2mL of composite coupling enhancer (prepared from Na)2SO4、Mg SO4、(NH4)2SO4Is prepared by mixing with phosphate buffer solution with pH of 8, wherein Na in the composite coupling reinforcing agent2SO4、Mg SO4、(NH4)2SO4The molar concentration ratio of 1:0.5:0.5), and uniformly mixing by swirling for 10s at room temperature;
(3) adding 0.3mg of TSH antibody into the step (2), swirling for 10s at room temperature, and then placing in a constant-temperature culture shaking table at 37 ℃ for coupling for 6 h;
(4) after magnetically separating the supernatant, adding 10mL of confining liquid (prepared by glycine and phosphate buffer solution with pH of 8, the mass concentration of the glycine is 0.5%), swirling for 10s at room temperature, and sealing in a constant-temperature culture shaking table at 37 ℃ for 3 h;
(5) after magnetic separation of the supernatant, 5mL of stock solution (prepared from glycine and Tris with pH8, the mass concentration of glycine is 0.1%) was added, vortexed at room temperature for 10s, and stored at 2-8 ℃.
In order to prove that the magnetic particles coupled with the antibody obtained by the coupling method have higher coupling effect, the magnetic particles coupled with the antibody in the coupling method are used as components of a kit for testing. A comparative example was also established in which the kit components were the same as in the present invention, the only difference being that only ammonium sulfate was added to the antibody-conjugated magnetic microparticles at the time of conjugation.
The specific detection method comprises the following steps: 50uL of magnetic microparticle coupling reagent +50uL of TSH standard (S0-S5 concentrations of 0uIU/mL,0.2uIU/mL,1uIU/mL,5uIU/mL,20uIU/mL,60uIU/mL, respectively) +50uL of enzyme-labeled reagent (acridinium ester label), Tris wash containing 0.1% BSA by mass concentration at 37 ℃ for 30min, 0.1M and pH8 +100uL of pre-excitation solution (containing 1.32% w/v hydrogen peroxide in acidic solution, available from Yapei trade (Shanghai) Co., Ltd.) were tested and the results are shown in Table 2:
TABLE 2
The experimental results in table 2 show that, at the same concentration point, when the magnetic particles coupled with the antibody are coupled, three coupling reinforcing agents are added, compared with the case where one coupling reinforcing agent is added, the magnetic particle coupling reagents added with the three coupling reinforcing agents are higher in measured value as the kit components under the same operation, which indicates that the coupling efficiency of the antibody and the magnetic particles is higher, and the linear range of the kit is further improved.
Example 3
A method of magnetic microparticle coupling a carcinoembryonic antigen antibody molecule (CEA) comprising the steps of:
(1) taking 0.1mL of 50mg/mL magnetic particle solution, magnetically separating supernate, adding 1mL of phosphate buffer (pH is 9), and uniformly mixing by swirling for 10s at room temperature to obtain magnetic particle suspension;
(2) taking the magnetic particle suspension in the step (1), carrying out magnetic separation to remove supernatant, and adding 0.8mL of composite coupling enhancer (1M Na)2SO4、0.5M Mg SO4、0.5M(NH4)2SO4Is prepared by mixing with phosphate buffer solution with pH of 9, wherein Na in the composite coupling reinforcing agent2SO4、Mg SO4、(NH4)2SO4The molar concentration ratio of 1:1:1), and uniformly mixing by swirling for 10s at room temperature;
(3) adding 0.08mg CEA antibody, vortexing at room temperature for 10s, and coupling in a constant-temperature culture shaker at 37 deg.C for 6 h;
(4) after magnetically separating the supernatant, adding 2mL of a pH 8-containing blocking solution (prepared from BSA, casein and a pH8 phosphate buffer solution, wherein the mass concentration of BSA is 0.5%, and the mass concentration of casein is 0.1%), swirling at room temperature for 10s, and then placing in a constant-temperature culture shaker at 37 ℃ for blocking for 6 h;
(5) after magnetic separation of the supernatant, 1mL of stock solution containing 0.1% BSA at pH8 was added, vortexed at room temperature for 10s and mixed, and stored at 2-8 ℃.
In order to prove that the magnetic particles coupled with the antibody obtained by the coupling method have higher coupling effect, the magnetic particles coupled with the antibody in the coupling method are used as components of a kit for testing. A comparative example was also established in which the kit components were the same as in the present invention, the only difference being that only ammonium sulfate was added to the antibody-conjugated magnetic microparticles at the time of conjugation.
The specific detection method comprises the following steps: 50uL of magnetic particle coupling reagent +50uLCEA standard (S0-S5 concentrations of 0ng/mL,5ng/mL,10ng/mL,20ng/mL,40ng/mL,80ng/mL) +50uL of enzyme-labeled reagent (acridinium ester label), 30min incubation at 37 ℃, 3 washes of 0.1M Tris wash containing 0.1% BSA by mass at pH8 +100uL of pre-excitation solution (containing 1.32% w/v hydrogen peroxide in acidic solution, available from Yapei trade (Shanghai) Co., Ltd.) were tested, and the results are shown in Table 3:
TABLE 3
The experimental results in table 3 show that, at the same concentration point, when the magnetic particles coupled with the antibody are coupled, three coupling reinforcing agents are added, compared with the case where one coupling reinforcing agent is added, the magnetic particle coupling reagents added with the three coupling reinforcing agents are higher in numerical value measured as the kit components under the same operation, which indicates that the coupling efficiency of the antibody and the magnetic particles is higher, and the linear range of the kit is further improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method of coupling magnetic particles to antibodies, the method comprising the steps of:
(1) taking the magnetic particle solution, performing magnetic separation to remove supernatant, adding a buffer solution, and uniformly mixing at room temperature to obtain a magnetic particle suspension;
(2) performing magnetic separation on the magnetic particle suspension obtained in the step (1), removing supernatant, adding a phosphate buffer solution dissolved with the composite coupling reinforcing agent into the magnetic particles, and uniformly mixing at room temperature for later use;
(3) adding the antibody into the step (2), uniformly mixing, placing in a constant-temperature culture shaking table for coupling, controlling the coupling temperature and the coupling time, carrying out magnetic separation after coupling, and removing supernatant to obtain magnetic particles coupled with the antibody;
(4) taking the magnetic particles coupled with the antibody in the step (3), adding a sealing liquid, uniformly mixing, and placing in a constant-temperature culture shaking table for sealing;
(5) and (4) closing the magnetic particles coupled with the antibody by the closing liquid in the step (4) for a period of time, carrying out magnetic separation to remove supernatant, adding storage liquid, uniformly mixing at room temperature, and storing in an environment at 2-8 ℃.
2. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the magnetic particle solution in the step (1) is a tosyl magnetic particle solution, the addition amount of the tosyl magnetic particle solution is 1-5mL, and the concentration of the tosyl magnetic particle solution is 10-100 mg/mL.
3. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the composite coupling reinforcing agent in the step (2) is composed of Na2SO4、MgSO4、(NH4)2SO4And buffer solution, wherein the addition amount of the composite coupling agent is 2-20ml, and each 1L of the composite coupling reinforcing agent contains Na2SO4、MgSO4And (NH)4)2SO4The molar concentrations of (A) and (B) are respectively 0.1-3M.
4. A method of coupling magnetic particles to antibodies according to claim 3, wherein: the Na in the composite coupling agent2SO4、MgSO4And (NH)4)2SO4The molar concentration ratio of (A) to (B) is 1-2:1: 1.
5. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the buffer solution in the step (2) and the step (1) is phosphate buffer solution with the pH value of 7-8 or borate buffer solution with the pH value of 8-9.5 or carbonate buffer solution with the pH value of 8-9.5.
6. A method of coupling magnetic particles to antibodies according to claim 1, wherein: in the step (3), the coupling temperature is controlled to be 35-40 ℃, and the coupling time is controlled to be 6-16 h.
7. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the blocking time in the step (4) is 1-6h, the addition amount of the blocking solution is 4-20mL, and the blocking solution is prepared from at least one of BSA, casein and glycine and a phosphate buffer solution.
8. The method of claim 7, wherein the magnetic particle is coupled to an antibody by: the blocking solution in the step (4) is prepared from phosphate buffer solution with pH of 7.4-8 in BSA, casein and glycine, wherein the mass concentrations of the BSA, the casein and the glycine in the blocking solution are respectively 0.1-1.0%.
9. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the adding amount of the stock solution in the step (5) is 4-20mL, and the stock solution is prepared from at least one of BSA, casein and glycine and a Tris buffer solution.
10. A method of coupling magnetic particles to antibodies according to claim 9, wherein: the stock solution in the step (4) is prepared from Tris buffer solution with the pH value of 7.4-8 in BSA, casein and glycine, wherein the mass concentrations of the BSA, the casein and the glycine in the stock solution are respectively 0.01-0.05%.
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| CN106226522A (en) * | 2016-07-04 | 2016-12-14 | 福建广生堂药业股份有限公司 | The detection method of a kind of GP73, detectable and detection kit |
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