CN112698025B - Method for coating magnetic particles with antigen or antibody, application and kit - Google Patents
Method for coating magnetic particles with antigen or antibody, application and kit Download PDFInfo
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Abstract
The invention discloses a method for coating magnetic particles with antigen or antibody, application and a kit, comprising the following steps: s1, taking magnetic particles, and resuspending the magnetic particles by PBS; s2, magnetic particle coating: mixing the short-arm biotin labeled antigen and the long-arm biotin labeled antigen in a certain ratio, or mixing the short-arm biotin labeled antibody and the long-arm biotin labeled antibody in a certain ratio, adding the mixture into the resuspended magnetic particles obtained in the step S1, uniformly mixing and reacting; s3, magnetic particle sealing: and (3) adding the confining liquid into the solution obtained in the step (S2), uniformly mixing and reacting. The invention simultaneously uses short-arm biotin and long-arm biotin labeled antigen or antibody to coat the magnetic particles, can greatly reduce steric hindrance and improve the reaction efficiency of the antigen and the antibody, and can obviously improve the signal and the sensitivity of the whole system when the magnetic particles A and B are mixed for use.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method for coating magnetic particles with an antigen or an antibody, application and a kit.
Background
The chemiluminescence immunoassay method is to connect antigen or antibody with carrier or enzyme by using biotin and avidin. Due to the high affinity of biotin, avidin, their binding is rapid, specific, stable and has a multi-step amplification effect. It can be coupled with macromolecular bioactive substances such as antigen and antibody, and can also be labeled by materials such as fluorescein and enzyme. The chemiluminescence immunoassay method has the characteristics of high sensitivity, wide linear range, strong specificity, good stability, simple operation and automation.
Biotin has a relatively low molecular weight and, after reacting with an antigen or an antibody to form a biotin-labeled conjugate, can interfere with the binding of biotin to avidin due to steric hindrance of the macromolecular protein.
Disclosure of Invention
The invention aims to provide a method for coating magnetic particles with an antigen or an antibody, which can reduce steric hindrance effect, reduce interference of the steric hindrance effect on the combination of biotin and avidin and further improve the reaction efficiency of the antigen or the antibody.
In addition, the invention also provides the magnetic particles prepared by the method, application and a kit.
The invention is realized by the following technical scheme:
a method for coating magnetic particles with an antigen or antibody, comprising the steps of:
s1, taking magnetic particles, and resuspending the magnetic particles by PBS;
s2, magnetic particle coating: mixing a short-arm biotin labeled antigen and a long-arm biotin labeled antigen according to a certain proportion, or mixing a short-arm biotin labeled antibody and a long-arm biotin labeled antibody according to a certain proportion, adding the mixture into the heavy suspension magnetic particles obtained in the step S1, uniformly mixing and reacting;
s3, magnetic particle sealing: and (3) adding the confining liquid into the solution obtained in the step (S2), uniformly mixing and reacting.
Steric hindrance of macromolecular proteins can interfere with the binding of biotin to avidin. A certain number of groups can be connected on a side chain of a biotin molecule to form a connecting arm, so that the distance between biotin and a marked macromolecule is increased, for example, the long-arm biotin can reduce the steric effect. The invention utilizes the characteristic that the short-arm biotin and the long-arm biotin are combined to reduce the steric hindrance effect to the maximum extent, thereby improving the reaction efficiency of the antigen and the antibody.
The short-arm biotin adopted by the invention is biotin-N-hydroxysuccinimide, sigma-Aldrich, cat: h1759; the long-armed biotin is N-hydroxysuccinimide ester of biotin amide hexanoate, sigma-Aldrich, cat: b2643; the short-arm biotin labeled antigen and the long-arm biotin labeled antigen are respectively obtained by labeling the short-arm biotin or the long-arm biotin; the short-arm biotin labeled antibody and the long-arm biotin labeled antibody are obtained by labeling the short-arm biotin or the long-arm biotin respectively.
The space position can be reduced by using the long-arm biotin label compared with that of the short-arm biotin label, and the applicant finds that the signal is higher when the long-arm biotin and the short-arm biotin are used in a mixed mode through experiments compared with the signal when the long-arm biotin is used alone.
The steric hindrance problem can be reflected by the detection signal result, because in the case of consistent biotin antibody and definite amount of biotin antibody, the signal is increased due to the decrease of steric hindrance, as can be seen from the data in tables 1-3 of the present application: the long-arm biotin and the short-arm biotin are mixed for use, so that the steric hindrance can be reduced, and compared with the single use of the long-arm biotin, the signal is higher.
In conclusion, the invention uses two different biotin-labeled antigens or antibodies to coat the magnetic particles; the steric hindrance can be greatly reduced, and the reaction efficiency of the antigen antibody is improved; the signal and the sensitivity of the system are improved.
Further, the ratio of the short-arm biotin labeled antigen to the long-arm biotin labeled antigen or the ratio of the arm biotin labeled antibody to the long-arm biotin labeled antibody is 1.
Furthermore, the short-arm biotin labeled antigen, the long-arm biotin labeled antigen, the short-arm biotin labeled antibody and the long-arm biotin labeled antibody are all products of biotin labeling based on macromolecular protein.
Further, the macromolecular protein includes BNP antibody, TG antigen.
Further, the concentration of the resuspended magnetic microparticles in step S1 was 2mg/mL.
Further, the magnetic particles are a mixture of the magnetic particles a and the magnetic particles B.
Magnetic particle A (Dynabeads) TM MyOne TM Streptavidin T1), magnetic particles B (Dynabeads) TM M-280Streptavidin)：
The detection signal of the magnetic particle A is high, and the background signal is high. The background signal of the magnetic particle B is low, but the coating amount is low relative to the magnetic particle A. The method mixes the magnetic particles A and the magnetic particles B for use, achieves the purposes of reducing background signals, improving the sensitivity of the system and simultaneously better meeting the linear range of the whole system.
Further, the mass ratio of the magnetic particles a to the magnetic particles B is 7.
The magnetic particles are prepared by adopting the method for coating the magnetic particles with the antigen or the antibody.
The method for coating the magnetic particles with the antigen or the antibody is applied to chemiluminescence immunization.
A kit comprising magnetic particles as described above.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention uses two different biotin-labeled antigens or antibodies to coat the magnetic particles; the steric hindrance can be greatly reduced, and the reaction efficiency of the antigen antibody is improved; the signal and the sensitivity of the system are improved.
2. The invention mixes the magnetic particles A and B for use, thereby achieving the purposes of reducing background signals, improving the sensitivity of the system and simultaneously better meeting the linear range of the whole system.
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 below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limiting the present invention.
Example 1:
a method for coating magnetic particles with an antigen or antibody, comprising the steps of:
s1, taking a certain amount of magnetic particles, washing the magnetic particles 3 times by using 1% BSA, and then re-suspending the magnetic particles by using 1% BSA to fix the volume to a concentration of 2mg/mL;
s2, magnetic particle coating: taking the prepared short-arm biotin labeled BNP antibody and long-arm biotin labeled BNP antibody, adding the short-arm biotin labeled BNP antibody or the long-arm biotin labeled BNP antibody into the resuspended magnetic particles according to the coating amount of 10ug/mg (namely adding 5ug/mg of the short-arm biotin labeled BNP antibody and 5ug/mg of the long-arm biotin labeled BNP antibody into the resuspended magnetic particles, wherein the total coating amount is 10 ug/mg), and performing vortex mixing and rolling reaction at room temperature for 30min;
s3, magnetic particle sealing: adding 10mM vitamin H (50 ul of vitamin H confining liquid added to 1mg magnetic beads) into the solution obtained in the step S2, uniformly mixing by vortex, and carrying out rolling reaction at room temperature for 30min;
s4, cleaning: BNP magnetic particle coating was completed after washing the above magnetic particles three times with 1% bsa solution.
In the present embodiment, the magnetic particles are magnetic particles A (Dynabeads) TM MyOne TM Streptavidin T1)。
Comparative example 1:
this comparative example is based on example 1 and differs from example 1 in that:
and step S2, adding 10ug/mg of short-arm biotin labeled BNP antibody into the resuspended magnetic particles to perform magnetic particle coating.
Comparative example 2:
this comparative example is based on example 1 and differs from example 1 in that: in step S2, adding 10ug/mg of long-arm biotin labeled BNP antibody into the resuspended magnetic particles to perform magnetic particle coating.
The BNP magnetic particles coated in example 1, comparative example 1 and comparative example 2 were subjected to signal value-to-signal ratio detection (chemiluminescence immunoassay), and the results are shown in table 1:
TABLE 1
From the data in table 1 it can be seen that:
the signal of the magnetic particles coated with the short-arm biotin labeled BNP antibody (comparative example 1) is about 60% of that of example 1, and the signal of the magnetic particles coated with the long-arm biotin labeled antibody (comparative example 2) is about 70% of that of example 1. The signal of the example 1 is 30-40% higher than that of the original method, and the background signal has no obvious change.
Example 2:
a method for coating magnetic particles with an antigen or antibody, comprising the steps of:
s1, taking a certain amount of magnetic particles, washing the magnetic particles 3 times by 5% BSA, re-suspending the magnetic particles by 5% BSA, and fixing the volume to a concentration of 2mg/mL;
s2, coating magnetic particles: taking the prepared short-arm biotin-marked 25-OH VD antibody and long-arm biotin-marked 25-OH VD antibody, adding the short-arm biotin-marked 25-OH VD antibody and the long-arm biotin-marked 25-OH VD antibody according to the coating amount of 1ug/mg (namely adding 0.5ug/mg of the short-arm biotin-marked 25-OH VD antibody and 0.5ug/mg of the long-arm biotin-marked 25-OH VD antibody into the resuspended magnetic particles, wherein the total coating amount is 1 ug/mg), uniformly mixing by vortex, and carrying out rolling reaction at room temperature for 30min;
s3, magnetic particle sealing: adding 10mM vitamin H (50 ul of vitamin H confining liquid added to 1mg magnetic beads) into the solution obtained in the step S2, uniformly mixing by vortex, and carrying out rolling reaction at room temperature for 30min;
s4, cleaning: after washing the above magnetic fine particles three times with 5% by weight of BSA solution, the 25-OH VD magnetic fine particle coating was completed.
In the present embodiment, the magnetic particles are magnetic particles A (Dynabeads) TM MyOne TM Streptavidin T1)。
Comparative example 3:
this comparative example is based on example 2, with the difference from example 2 that:
and step S2, adding 1ug/mg of short-arm biotin labeled 25-OH VD antibody into the resuspended magnetic particles to coat the magnetic particles.
Comparative example 4:
this comparative example is based on example 2, differing from example 2 in that:
in the step S2, 1ug/mg of the long-arm biotin labeled 25-OH VD antibody is added into the resuspended magnetic particles for magnetic particle coating.
The 25-OH VD magnetic particles coated in example 2, comparative example 3 and comparative example 4 were subjected to signal value to signal ratio detection (chemiluminescence immunoassay), and the results are shown in Table 2:
TABLE 2
Shown in table 2:
and the 25-OH VD is a small molecule, so that the steric hindrance is small. Compared with the signal of the method, the signal of the magnetic particle coated by the short-arm biotin-labeled 25-OH VD antibody and the signal of the magnetic particle coated by the long-arm biotin-labeled antibody are about 10 percent lower, and the difference is not obvious.
Example 3:
a method for coating magnetic particles with an antigen or antibody, comprising the steps of:
s1, washing the magnetic particles for 3 times by using 0.02M PBS, then re-suspending the magnetic particles by using 0.02M PBS, and fixing the volume until the concentration is 2mg/mL;
s2, coating magnetic particles: taking the prepared short-arm biotin labeled TG antigen and long-arm biotin labeled TG antigen, adding 5ug/mg of short-arm biotin labeled TG antigen and 5ug/mg of long-arm biotin labeled TG antigen into the resuspended magnetic particles, coating the total amount to be 10ug/mg, uniformly mixing by vortex, and carrying out rolling reaction at room temperature for 30min;
s3, magnetic particle sealing: adding 10mM vitamin H (50 ul of vitamin H confining liquid added to 1mg of magnetic beads) into the solution obtained in the step S2, uniformly mixing by vortex, and carrying out rolling reaction at room temperature for 30min;
s4, cleaning: and washing the magnetic particles with 0.02M PBS solution for three times to finish the ANTI-TG project magnetic particle coating.
In the present embodiment, the magnetic particles are magnetic particles a (Dynabeads) TM MyOne TM Streptavidin T1)。
Comparative example 5:
this comparative example is based on example 3, differing from example 3 in that:
and step S2, adding 10ug/mg of short-arm biotin labeled TG antigen into the resuspended magnetic particles to perform magnetic particle coating.
Comparative example 6:
this comparative example is based on example 3, differing from example 3 in that:
and step S2, adding 10ug/mg of long-arm biotin labeled TG antigen into the resuspended magnetic particles to perform magnetic particle coating.
The TG magnetic particles after coating in example 3, comparative example 5 and comparative example 6 were subjected to signal value-to-signal ratio detection (chemiluminescence immunoassay), and the results are shown in table 3:
TABLE 3
From the data in table 3 it can be seen that:
the signal of the short-arm biotin-labeled TG antigen-coated magnetic particles is about 70% of that of the method, and the signal of the long-arm biotin-labeled antibody-coated magnetic particles is about 80% of that of the method. Compared with the original method, the method has the advantages that the signal is 20-30% higher, and the background signal is not obviously changed.
From the data in tables 1-3, it can be seen that:
the invention mainly has great effect on macromolecular items and has slight effect on micromolecules. Meanwhile, the short-arm biotin-labeled antigen or antibody and the long-arm biotin-labeled antigen or antibody are used for coating the magnetic beads, so that the steric hindrance of the macromolecular antigen or antibody can be greatly reduced, the coating amount of the antigen or antibody is increased, and the signal and sensitivity of the system are improved.
As can be seen from the data in tables 1-3 of the present application: the long-arm biotin and the short-arm biotin are mixed for use, so that the steric hindrance can be reduced, and compared with the single use of the long-arm biotin, the signal is higher.
Example 4:
this example is based on example 1, and differs from example 1 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 7.
Example 5:
this example is based on example 1, and differs from example 1 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 6.
Example 6:
this example is based on example 1, and differs from example 1 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 5.
Example 4-example 6 the signal values versus signal ratios for the magnetic particles prepared are shown in table 4:
TABLE 4
From the data in table 4, it can be seen that: when the magnetic particles A and the magnetic particles B are mixed for use, the background signal of the BNP item can be obviously improved, and the high-value BNP signal is reduced slightly. When the ratio of the magnetic particles A to the magnetic particles B is 6: the S/NSB of the system is most preferred at 4.
Example 7:
this example is based on example 2, and differs from example 2 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 7.
Example 8:
this example is based on example 2, and differs from example 2 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 6.
Example 9:
the present example is based on example 2, and differs from example 2 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 5.
The signal values versus signals for the magnetic particles prepared in examples 7-9 are shown in table 5:
TABLE 5
From the data in table 5, it can be seen that: when the magnetic particles A and the magnetic particles B are mixed for use, the sensitivity of the system is obviously improved, and when the ratio of the magnetic particles A to the magnetic particles B is 6:4 and 5: the S/NSB of the system is most preferred at 5.
Example 10:
the present example is based on example 3, and differs from example 3 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 7.
Example 11:
the present example is based on example 3, and differs from example 3 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 6.
Example 12:
the present example is based on example 3, and differs from example 3 in that:
the magnetic particles are a mixture of magnetic particles a and magnetic particles B, and the mass ratio of the magnetic particles a to the magnetic particles B is 5.
The signal value versus signal ratio for the magnetic particles prepared in examples 10-12 are shown in table 6:
TABLE 6
From the data in table 6, it can be seen that:
when the magnetic particles A and the magnetic particles B are mixed for use, the background signal of an ANTI-TG project can be obviously improved, and the decrease of the high-value signal of the ANTI-TG is small. When the ratio of the magnetic particles A to the magnetic particles B is 5: the S/NSB of the system is most preferred at 5.
From the data of examples 4-12 it can be seen that:
the magnetic particle A and the magnetic particle B are combined with two magnetic beads, so that the magnetic particle A and the magnetic particle B have the advantages of being capable of combining more biotinylated antigens or antibodies, small in interference and low in background signal, and the sensitivity of the whole system is best when the mass ratio of the magnetic particle A to the magnetic particle B is 6.
In conclusion, the short-arm biotin and the long-arm biotin labeled antigen or antibody are used for coating the magnetic particles simultaneously, so that the steric hindrance can be greatly reduced, the reaction efficiency of the antigen and the antibody can be improved, and when the magnetic particles A and the magnetic particles B are mixed for use, the signal and the sensitivity of the whole system can be obviously improved.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for coating magnetic particles with antigen or antibody, which is characterized by comprising the following steps:
s1, taking magnetic particles, and resuspending the magnetic particles by PBS;
s2, coating magnetic particles: mixing a short-arm biotin labeled antigen and a long-arm biotin labeled antigen according to a certain proportion, or mixing a short-arm biotin labeled antibody and a long-arm biotin labeled antibody according to a certain proportion, adding the mixture into the heavy suspension magnetic particles obtained in the step S1, uniformly mixing and reacting;
s3, magnetic particle sealing: and (3) adding the confining liquid into the solution obtained in the step (S2), uniformly mixing and reacting.
2. The method for coating magnetic particles with antigen or antibody according to claim 1, wherein the ratio of short-arm biotin labeled antigen to long-arm biotin labeled antigen or the ratio of short-arm biotin labeled antibody to long-arm biotin labeled antibody is 1.
3. The method for coating magnetic particles with antigen or antibody according to claim 1, wherein the short-arm biotin-labeled antigen, the long-arm biotin-labeled antigen, the short-arm biotin-labeled antibody and the long-arm biotin-labeled antibody are all products of biotin labeling based on macromolecular proteins.
4. The method for coating magnetic particles with antigen or antibody according to claim 3, wherein the macromolecular protein comprises BNP antibody or TG antigen.
5. The method for coating magnetic particles with antigen or antibody according to claim 1, wherein the concentration of the resuspended magnetic particles in step S1 is 2mg/mL.
6. The method for coating magnetic particles with an antigen or antibody according to claim 1, wherein the magnetic particles are a mixture of magnetic particles A and magnetic particles B; magnetic particles A are Dynabeads TM MyOne TM Streptavidin T1, magnetic particles B are Dynabeads TM M-280 Streptavidin。
7. The method for coating magnetic particles with an antigen or an antibody according to claim 6, wherein the mass ratio of the magnetic particles A to the magnetic particles B is 7.
8. Magnetic particles prepared by the method for coating magnetic particles with the antigen or antibody according to any one of claims 1 to 7.
9. A method for coating magnetic particles with an antigen or antibody according to any one of claims 1 to 7 or the use of magnetic particles according to claim 8 in chemiluminescence immunization.
10. A kit comprising magnetic particles according to claim 8.
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