CN110596373B - Method for coupling magnetic particles with antibody - Google Patents
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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 and removing 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, removing supernatant, adding phosphate buffer solution dissolved with the composite coupling enhancer, and uniformly mixing for later use; (3) Adding an antibody, uniformly mixing, performing coupling, controlling the coupling temperature and the coupling time, performing magnetic separation after coupling is finished, 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 sealing; (5) Magnetic particles coupled with the antibody are subjected to magnetic separation, supernatant is removed, and then storage solution is added, uniformly mixed and stored. The method can improve the coupling efficiency of the magnetic particles and the antibody, and further improve the linearity 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 with antibodies.
Background
The traditional immunological detection mostly uses an ELISA plate as a solid-phase carrier, but the suspended magnetic particles as a 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 ELISA plate carrier due to flexible application of an externally applied magnetic field. The magnetic particle chemiluminescence immunoassay technology combines a magnetic particle carrier technology and a chemiluminescence immunoassay technology, and the coupling method of magnetic particles and antibodies is generally low in efficiency, raw materials are wasted, and meanwhile, the sensitivity of reagents is reduced, so that detection omission occurs in some detection items. Therefore, there is a need to develop a novel coupling method for efficiently binding an antibody to magnetic particles, thereby improving the sensitivity of a reagent.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects of the prior art, a method for coupling magnetic particles with an antibody is provided, and the method can improve the coupling efficiency of the magnetic particles with the antibody and further improve the linear range and the 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 a magnetic particle solution, removing supernatant by magnetic separation, 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 in the step (1), removing the supernatant, and then adding a phosphate buffer solution in which a composite coupling enhancer is dissolved into the magnetic particles, and uniformly mixing at room temperature for later use;
(3) Adding the antibody into the step (2), uniformly mixing, placing the mixture into a constant-temperature culture shaking table for coupling, controlling the coupling temperature and the coupling time, magnetically separating the mixture after the coupling is finished, and removing the 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) After the magnetic particles coupled with the antibody are subjected to the sealing of the sealing liquid in the step (4) for a period of time, removing the supernatant by magnetic separation, adding the storage liquid, uniformly mixing at room temperature, and storing in an environment of 2-8 ℃.
As an improved technical scheme, the magnetic particle solution in the step (1) is a tosyl magnetic particle solution, the adding amount of the tosyl magnetic particle solution is 1-5mL, and the concentration of the tosyl magnetic particle solution is 10-100mg/mL.
As an improved technical scheme, the composite coupling enhancer in the step (2) is prepared from Na 2 SO 4 、MgSO 4 、(NH 4 ) 2 SO 4 Is prepared with buffer solution, wherein the adding amount of the composite coupling agent is 2-20ml, and each 1L of the composite coupling reinforcing agent isThe Na is 2 SO 4 、MgSO 4 (NH) 4 ) 2 SO 4 The molar concentration of (2) is 0.1-3M respectively.
As a preferable technical scheme, the Na in the composite coupling agent 2 SO 4 、MgSO 4 (NH) 4 ) 2 SO 4 The molar concentration ratio of (2) 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 pH of 7-8 or borate buffer solution with pH of 8-9.5 or carbonate buffer solution with pH 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 6-16h.
As an improved technical scheme, the blocking time in the step (4) is 1-6h, the adding amount of the blocking liquid is 4-20mL, and the blocking liquid is prepared from phosphate buffer solution and at least one of BSA, casein and glycine.
As a preferable technical scheme, the blocking solution in the step (4) is prepared from phosphate buffer solution with the pH of 7.4-8 in BSA, casein and glycine, wherein the mass concentration of the BSA, casein and glycine in the blocking solution is respectively 0.1% -1.0%.
As an improved technical scheme, the adding amount of the storage solution in the step (5) is 4-20mL, and the storage solution is prepared from at least one of BSA, casein and glycine and Tris buffer solution.
As a preferable technical scheme, the storage solution in the step (4) is prepared from Tris buffer solution with pH of 7.4-8 in BSA, casein and glycine, wherein the mass concentration of BSA, casein and glycine in the storage solution is respectively 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 prepared from Na 2 SO 4 、MgSO 4 、(NH 4 ) 2 SO 4 Is prepared with buffer solution and Na 2 SO 4 、MgSO 4 、(NH 4 ) 2 SO 4 The concentration in the composite coupling enhancer is 0.1-3M, the coupling temperature is controlled to be 35-40 ℃ and the coupling time is controlled to be 6-16h when the antibody is coupled with the magnetic particles, the use of the composite coupling enhancer can provide proper salt concentration for the antibody and the magnetic particles, the three are synergistic, 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 coupling of the antibody and the magnetic particles is finished, adding a blocking solution for blocking for 1-6 hours, wherein the blocking solution is prepared from BSA, casein and glycine and a phosphate buffer solution, the mass concentration of the BSA, casein and glycine in the blocking solution is respectively 0.1% -1.0%, the pH value of the phosphate buffer solution is 7.4-8, and the blocking solution adopting the components can block unbound sites, reduce non-specific adsorption and prevent interference.
(3) After the magnetic particles coupled with the antibody are blocked by a blocking solution, a storage solution is prepared from BSA, casein and glycine and a Tris buffer solution, wherein the mass concentration of the BSA, casein and glycine in the storage solution is 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 bound on the magnetic particles through storage of the storage solution.
In summary, the invention adds the composite coupling reinforcing agent when the antibody is coupled with the magnetic particles, and controls the coupling temperature and the coupling time, and the use of the composite coupling reinforcing agent can increase 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 then put into the storage liquid for storage for standby, the interference can be prevented through the operation, the antibodies combined on the magnetic particles are stabilized, and the linear range and the accuracy of the kit are further improved.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A method of magnetic particle coupled alpha fetoprotein antibody molecule (AFP), comprising the steps of:
(1) Taking 1mL of 10mg/mL magnetic particle solution, adding 1mL of phosphate buffer solution with pH of 7.8 after removing supernatant by magnetic separation, and uniformly mixing by vortex for 10s at room temperature to obtain magnetic particle suspension;
(2) Taking the magnetic particle suspension in the step (1), removing the supernatant by magnetic separation, and adding 2mL of a composite coupling enhancer (prepared from Na 2 SO 4 、Mg SO 4 、(NH 4 ) 2 SO 4 Is prepared with phosphate buffer solution with pH of 7.8, wherein Na in the composite coupling reinforcing agent 2 SO 4 、Mg SO 4 、(NH 4 ) 2 SO 4 The molar concentration ratio of (2) is 1:1:1), and vortex for 10s at room temperature to mix uniformly;
(3) Adding 0.1mg of AFP antibody into the step (2), vortexing for 10s at room temperature, and then placing into a constant temperature culture shaking table at 37 ℃ for coupling for 6h;
(4) After magnetically separating the supernatant, adding 4mL of blocking solution (prepared by BSA, casein and glycine and phosphate buffer solution with pH7.5, wherein the mass concentration of BSA, casein and glycine in the blocking solution is respectively 0.1%, 0.5% and 0.3%), swirling at room temperature for 10 hours, and then placing in a constant temperature culture shaking table at 37 ℃ for blocking for 1 hour;
(5) After magnetic separation of the supernatant, stock solution (prepared from BSA, casein and glycine in combination with Tris buffer pH7.5, wherein the mass concentrations of BSA, casein and glycine in the stock solution are 0.01%, 0.02% and 0.003%, respectively) was added, vortexed for 10s at room temperature, and stored at 2-8deg.C.
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 method are used as components of a kit for testing. A comparative example was also established, in which the kit components were identical to the present invention, the only difference being that the magnetic particles to which the antibodies were coupled were only added with ammonium sulfate during the coupling.
The specific detection method comprises the following steps: 50uL magnetic particle coupling reagent+50 uLAFP standard (SO-S5 concentration of 0ng/mL,2.5ng/mL,5ng/mL,10ng/mL,20ng/mL,40 ng/mL) +50uL enzyme labeling reagent (acridine ester labeling), 30min incubation at 37 ℃,3 times +100uL pre-excitation solution (containing 1.32% w/v hydrogen peroxide acid solution, purchased from Atlantic trade (Shanghai) Inc.) of Tris wash containing 0.1% BSA by mass concentration at 0.1M and pH8, and test results are shown in Table 1:
TABLE 1
As can be seen from the experimental results in Table 1, at the same concentration point, when the magnetic particles coupled with the antibody are added with three coupling enhancers during the coupling operation, compared with the addition of one coupling enhancer, the magnetic particle coupling reagents added with three coupling enhancers are used as kit components, and the measured values under the same operation are higher, so 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 coupled thyrotropin antibody molecules (TSH) comprising the steps of:
(1) Taking 1mL of 50mg/mL magnetic particle solution, adding 5mL phosphate buffer (pH 8) after removing supernatant by magnetic separation, and uniformly mixing by vortex for 10s at room temperature to obtain magnetic particle suspension;
(2) Taking the magnetic particle suspension in the step (1), removing the supernatant by magnetic separation, and adding 2mL of a composite coupling enhancer (prepared from Na 2 SO 4 、Mg SO 4 、(NH 4 ) 2 SO 4 Is prepared with phosphate buffer solution with pH of 8, wherein Na in the composite coupling reinforcing agent 2 SO 4 、Mg SO 4 、(NH 4 ) 2 SO 4 The molar concentration ratio of (2) is 1:0.5:0.5), and vortex for 10s at room temperature for uniform mixing;
(3) Adding 0.3mg of TSH antibody into the step (2), vortexing for 10s at room temperature, and then placing the mixture in a constant temperature culture shaking table at 37 ℃ for coupling for 6h;
(4) After magnetically separating the supernatant, adding 10mL of sealing solution (prepared by glycine and phosphate buffer solution with pH of 8, wherein the mass concentration of glycine is 0.5%), swirling for 10s at room temperature, and sealing in a constant temperature culture shaking table at 37 ℃ for 3h;
(5) After magnetic separation of the supernatant, 5mL of stock solution (prepared from glycine and Tris with pH of 8, mass concentration of glycine of 0.1%) was added, vortexed at room temperature for 10s, mixed well and stored at 2-8deg.C.
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 method are used as components of a kit for testing. A comparative example was also established, in which the kit components were identical to the present invention, the only difference being that the magnetic particles to which the antibodies were coupled were only added with ammonium sulfate during the coupling.
The specific detection method comprises the following steps: 50uL magnetic particle coupling reagent+50 uL TSH standard (S0-S5 concentration of 0uIU/mL,0.2uIU/mL,1uIU/mL,5uIU/mL,20uIU/mL,60 uIU/mL) +50uL enzyme-labeled reagent (acridine ester labeled), 30min incubation at 37 ℃,3 times of 0.1M and pH8 Tris wash containing 0.1% BSA by mass concentration +100uL pre-excitation solution (containing 1.32% w/v hydrogen peroxide acid solution, available from Atlantic trade (Shanghai) Co., ltd.) test, the test results are shown in Table 2:
TABLE 2
As can be seen from the experimental results in Table 2, at the same concentration point, when the magnetic particles coupled with the antibody are added with three coupling enhancers during the coupling operation, compared with the addition of one coupling enhancer, the magnetic particle coupling reagents added with three coupling enhancers are used as kit components, and the measured values under the same operation are higher, so 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 particle-coupled carcinoembryonic antigen antibody molecule (CEA), comprising the steps of:
(1) Taking 0.1mL of 50mg/mL magnetic particle solution, magnetically separating supernatant, adding 1mL of phosphate buffer solution (pH 9), and vortex for 10s at room temperature to mix uniformly to obtain magnetic particle suspension;
(2) Taking the magnetic particle suspension in the step (1), removing the supernatant by magnetic separation, adding 0.8mL of a composite coupling enhancer (prepared from 1M Na 2 SO 4 、0.5M Mg SO 4 、0.5M(NH 4 ) 2 SO 4 Is prepared with phosphate buffer solution with pH of 9, wherein Na in the composite coupling reinforcing agent 2 SO 4 、Mg SO 4 、(NH 4 ) 2 SO 4 The molar concentration ratio of (2) is 1:1:1), and vortex for 10s at room temperature to mix uniformly;
(3) Adding 0.08mg CEA antibody, vortexing for 10s at room temperature, and coupling for 6h in a constant temperature culture shaker at 37 ℃;
(4) After magnetic separation of the supernatant, 2mL of blocking solution (prepared from BSA, casein and phosphate buffer solution with pH8, wherein the mass concentration of BSA is 0.5% and the mass concentration of casein is 0.1%) with pH8 is added, vortexed for 10s at room temperature and then placed in a constant temperature culture shaking table at 37 ℃ for 6h of blocking;
(5) After magnetic separation of the supernatant, 1mL of a stock solution containing 0.1% BSA at pH8 was added, vortexed at room temperature for 10s, and stored at 2-8deg.C.
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 method are used as components of a kit for testing. A comparative example was also established, in which the kit components were identical to the present invention, the only difference being that the magnetic particles to which the antibodies were coupled were only added with ammonium sulfate during the coupling.
The specific detection method comprises the following steps: 50uL magnetic particle coupling reagent+50 uLCEA standard (S0-S5 concentration of 0ng/mL,5ng/mL,10ng/mL,20ng/mL,40ng/mL,80 ng/mL) +50uL enzyme-labeled reagent (acridinium ester labeled), 30min incubation at 37 ℃,3 times +100uL pre-excitation solution (containing 1.32% w/v hydrogen peroxide acid solution, purchased from Atlantic trade (Shanghai) Co., ltd.) of Tris wash containing 0.1% BSA by mass concentration at 0.1M and pH8, and test results are shown in Table 3:
TABLE 3 Table 3
As can be seen from the experimental results in Table 3, at the same concentration point, when the magnetic particles coupled with the antibody are added with three coupling enhancers during the coupling operation, compared with the addition of one coupling enhancer, the magnetic particle coupling reagents added with three coupling enhancers are higher in value measured under the same operation as the components of the kit, 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 foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (9)
1. A method of coupling magnetic particles to antibodies, said method comprising the steps of:
(1) Taking a magnetic particle solution, removing supernatant by magnetic separation, 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 in the step (1), removing the supernatant, and then adding a phosphate buffer solution in which a composite coupling enhancer is dissolved into the magnetic particles, and uniformly mixing at room temperature for later use; the composite coupling reinforcing agent is prepared from Na 2 SO 4 、MgSO 4 、(NH 4 ) 2 SO 4 Is prepared with buffer solution, wherein the adding amount of the composite coupling reinforcing agent is 2-20ml, and the Na in each 1L of the composite coupling reinforcing agent 2 SO 4 、MgSO 4 (NH) 4 ) 2 SO 4 The molar concentration of (2) is 0.1-3M respectively;
(3) Adding the antibody into the step (2), uniformly mixing, placing the mixture into a constant-temperature culture shaking table for coupling, controlling the coupling temperature and the coupling time, magnetically separating the mixture after the coupling is finished, and removing the 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) After the magnetic particles coupled with the antibody are subjected to the sealing of the sealing liquid in the step (4) for a period of time, removing the supernatant by magnetic separation, adding the storage liquid, uniformly mixing at room temperature, and storing in an environment of 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 adding amount of the tosyl magnetic particle solution is 1-5mL, and the concentration of the tosyl magnetic particle solution is 10-100mg/mL.
3. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the Na in the composite coupling reinforcing agent 2 SO 4 、MgSO 4 (NH) 4 ) 2 SO 4 The molar concentration ratio of (2) is 1-2:1:1.
4. 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 pH7-8 or borate buffer solution with pH 8-9.5 or carbonate buffer solution with pH 8-9.5.
5. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the coupling temperature in the step (3) is controlled to be 35-40 ℃, and the coupling time is controlled to be 6-16h.
6. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the blocking time in the step (4) is 1-6h, the adding amount of the blocking liquid is 4-20mL, and the blocking liquid is prepared from phosphate buffer solution and at least one of BSA, casein and glycine.
7. A method of coupling magnetic particles to antibodies according to claim 6, wherein: 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 concentration of BSA, casein and glycine in the blocking solution is 0.1% -1.0% respectively.
8. A method of coupling magnetic particles to antibodies according to claim 1, wherein: the addition amount of the storage solution in the step (5) is 4-20mL, and the storage solution is prepared from at least one of BSA, casein and glycine and Tris buffer solution.
9. A method of coupling magnetic particles to antibodies according to claim 8, wherein: the storage solution in the step (5) is prepared from Tris buffer solution with pH of 7.4-8 in BSA, casein and glycine, wherein the mass concentration of the BSA, casein and glycine in the storage solution is 0.01% -0.05% respectively.
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Address after: 261023 No. 699, Yue He road, Weifang Economic Development Zone, Shandong Applicant after: Shandong Kanghua Biomedical Technology Co.,Ltd. Address before: 261023 No. 699, Yue He road, Weifang Economic Development Zone, Shandong Applicant before: WEIFANG KANGHUA BIOTECH Co.,Ltd. |
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