CN110568176A - method for directional coupling of antibody and magnetic bead - Google Patents
method for directional coupling of antibody and magnetic bead Download PDFInfo
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- CN110568176A CN110568176A CN201910865034.XA CN201910865034A CN110568176A CN 110568176 A CN110568176 A CN 110568176A CN 201910865034 A CN201910865034 A CN 201910865034A CN 110568176 A CN110568176 A CN 110568176A
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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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Abstract
The invention relates to the field of biotechnology detection, in particular to a method for directionally coupling an antibody and magnetic beads, which comprises (1) magnetic bead pretreatment; (2) S-NHS treatment; (3) EDC treatment; (4) the antibody is added. The method of the invention can realize directional coupling of the antibody and the magnetic bead, greatly improve the yield of the effective antibody and further improve the actual sensitivity and accuracy of detection.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to a method for directionally coupling an antibody and magnetic beads.
Background
The chemiluminescence immunodiagnosis technology uses substances such as chemiluminescence agent, catalytic luminescent enzyme and the like to mark an antigen or an antibody, and when the marked antigen or antibody is combined with a corresponding antibody and antigen, a luminous substrate is acted by the substances such as the chemiluminescence agent, the catalytic enzyme and the like to generate oxidation-reduction reaction. The visible light released in the reaction is finally collected and processed by matched equipment to obtain the concentration of the measured substance, thereby achieving the purpose of measurement. Compared with the traditional immunological technology, chemiluminescence has the advantages of high automation degree, good specificity, high accuracy, wide detection range and the like.
In the development and preparation processes of magnetic particle chemiluminescence reagents, a plurality of coupling methods of antibodies and magnetic bead microspheres exist, but the coupling methods have a problem that the direction of the antibodies connected to the microspheres has great randomness and cannot be controlled. The region where the antibody binds to the antigen is located on the F (ab) fragment of the antibody, and if the F (ab) fragment of the antibody is attached to the microsphere during the coupling process, the Fc fragment that has no binding activity to the antigen is stretched outwards, which results in the coupled antibody being an ineffective antibody, and the presence of the ineffective antibody makes the utilization rate of the antibody low, and greatly reduces the final sensitivity of the reagent.
The existing directional coupling method is that a full-length antibody reduces and opens two pairs of disulfide bonds between heavy chains of an Fc segment to obtain an antibody fragment with one heavy chain and one light chain connected through the disulfide bonds, and the formed free sulfydryl is utilized for directional coupling. However, by screening the antibody reduction scheme by using different reducing agents, different reduction times and different reduction conditions, the yield of the final target product is not more than 25%, and the labeling efficiency is extremely low. In addition, the method is easy to cause the interference of Rheumatoid Factors (RF), and the sensitivity and specificity of the reagent are reduced.
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 the directional coupling of the antibody and the magnetic bead is provided, the method is simple to operate, the directional coupling of the antibody and the coating substance can be realized, and the sensitivity and the specificity of the detection reagent are greatly improved.
In order to solve the technical problems, the technical scheme of the invention is as follows:
A method for directional coupling of antibodies to magnetic beads, the method comprising the steps of:
(1) Magnetic bead pretreatment: adding magnetic bead mother liquor into MES buffer solution, oscillating uniformly, magnetically separating, discarding supernatant, adding MES buffer solution, and oscillating uniformly for later use;
(2) S-NHS treatment: adding an S-NHS solution prepared by MES buffer solution into the step (1), oscillating and uniformly mixing, and adjusting the pH value of a reaction system to be acidic;
(3) EDC treatment: adding an EDC solution prepared by MES buffer solution into the reaction system in the step (2), oscillating and mixing uniformly, adjusting the pH value of the reaction system to be acidic, placing the reaction system in a constant temperature and humidity box, rotating and mixing uniformly for reaction for a period of time, removing supernatant after magnetic separation, adding MES buffer solution, and oscillating and mixing uniformly;
(4) adding an antibody: adding an antibody into the step (3), placing a magnetic bead labeled reactant into a constant temperature and humidity box, rotating and uniformly mixing the reactant and the antibody for a period of time, removing the supernatant after magnetic separation, adding a TRIS buffer solution, uniformly oscillating the reactant and the supernatant, adding a magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly mixing the reactant and the magnetic bead labeled reactant, placing the magnetic bead labeled reactant into the constant temperature and humidity box, standing the reactant and the magnetic bead labeled reactant for a period of time, removing the supernatant after magnetic separation, adding the magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly oscillating the reactant and the supernatant, adding a magnetic bead preservation solution into the magnetic bead labeled reactant, and placing the.
As an improved technical scheme, in the step (1), the magnetic bead mother liquor is carboxyl magnetic bead mother liquor, the addition amount of the magnetic bead mother liquor is 10-15mg, the first addition amount of MES buffer is 0.5-1ml, and the second addition amount of MES buffer is 1-2 ml.
As an improved technical scheme, the concentration of the S-NHS solution in the step (2) is 10mg/mL-20mg/mL, and the addition amount of the S-NHS solution is 50 mu L.
As an improved technical scheme, the pH value of the reaction system is adjusted to be 5.0-6.5 in the step (2).
As a modified technical scheme, the concentration of the EDC solution in the step (3) is 8-12mg/mL, and the addition amount of the EDC solution is 50 mu L.
As an improved technical scheme, the pH value of the reaction system is adjusted to be 5.0-6.5 in the step (3).
As an improved technical scheme, the temperature in the constant temperature and humidity chamber in the step (3) is 26 +/-1 ℃, and the time of the rotary reaction is 20-40 min.
as an improved technical scheme, the adding amount of the antibody in the step (4) is 200-500 mug, the time of the rotary mixing reaction is 120-150min, the adding amount of the TRIS buffer solution is 1-2.5 mL, the adding amount of the magnetic bead labeling buffer solution A is 1-2.5 mL, and the time of the standing reaction is 60-90 min.
as an improved technical scheme, the formula of the magnetic bead labeling buffer solution A in the step (4) is 6.05g of Tris, 9g of NaCl, 0.5g of PC300, 5g of BSA and 0.5g of Tween-20, the components are dissolved to 1L by double distilled water, and the pH is adjusted to 7.3-7.5 by 1M HCl.
as an improved technical scheme, the formula of the magnetic bead preservation solution in the step (4) comprises the following components: 6.05g Tris, 9g NaCl, 0.5g PC300, 10g BSA, 1g Tween-20, the above components were dissolved to 1L with double distilled water, and the pH was adjusted to 7.3-7.5 with 1M HCl.
After the technical scheme is adopted, the invention has the beneficial effects that:
according to the invention, an EDC/NHS two-step coupling mode is adopted, the pH environment during EDC/NHS two-step coupling is adjusted to 5.0-6.5 by adjusting the pH, and in the pH environment, both the Fab end and the Fc end of an antibody are positively charged, so that the antibody can be attached to the surface of a PS microsphere through electrostatic adsorption. At this time, the Fab terminal amino group is in a protonated state (NH3+), the reaction priority is lowered, the Fc terminal is positively charged but is significantly lower in charge than the Fab terminal, the hydration layer is thinner, the hydrophobicity is stronger, and the reaction is more likely to be involved. In the two-step reaction, the pH of the second antibody coupling reaction is reduced, the NHS ester hydrolysis speed is slower, the amino-carboxyl reaction speed is also slower, and time is provided for the adjustment of the direction of the antibody on the carboxyl surface.
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
The kit for detecting VEGF prepared by the directional coupling method comprises the following components: the kit comprises a magnetic microsphere suspension coated with a VEGF antibody (the working concentration of the magnetic microsphere is 0.1-0.35 mg/mL, the concentration of the VEGF antibody coated on the surface of the magnetic microsphere is 10-50ug/mg), a VEGF reference product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF reference product are respectively 0pg/mL, 30pg/mL, 125pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL), a VEGF quality control product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF quality control product are 125pg/mL and 1000pg/mL), a VEGF enzyme conjugate (the VEGF enzyme conjugate is a horseradish peroxidase-labeled VEGF monoclonal antibody, and the concentration of the VEGF enzyme conjugate is diluted according to the ratio of the VEGF enzyme conjugate diluent to 3000), a concentrated washing solution (0.04-0.06 mol, 0.06mol and the concentration of the VEGF enzyme conjugate is 0., Tris-HCl buffer solution with pH7.4-pH7.6, and Tween-80 in 0.04-0.06% v/v and Proclin-300 in 0.7-0.8% v/v in every 1L of buffer solution. ) And luminescence substrate liquid (every 250ml of the luminescence substrate liquid A contains 2.3-2.6g of N, O-bis trimethylsilyl acetamide, 1.3-1.6g of trihydroxymethyl aminomethane, 0.4-0.6g of 3-aminophthalic hydrazide, 0.007-0.008g of para-iodophenol and 4-40g of sodium hydroxide, and the pH value of the working solution is 10-13; the luminescent substrate liquid B comprises 2.3-2.6g of N, O-bis (trimethylsilyl) acetamide, 1.3-1.6g of tris (hydroxymethyl) aminomethane and 0.1-0.3ml of hydrogen peroxide per 250ml of solution. ) The reference substance diluent is Tris-HCl buffer solution with the pH value of 7.5, and each 1L of the buffer solution also contains 18-22g of BSA, 14-15g of EDTA, 3.8-4g of 2-mercaptoethanol and 0.3-0.5mL of Proclin-300. ) The enzyme conjugate diluent is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 8-11g of bovine serum albumin, 5-6g of calcium chloride, 3-6% v/v of calf serum, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
The method for directionally coupling the antibody and the magnetic bead (namely preparing the magnetic microsphere suspension coated with the VEGF antibody) comprises the following steps:
(1) Magnetic bead pretreatment: adding 10mg of magnetic bead mother liquor (carboxyl magnetic bead mother liquor) into 0.5-1ml of MES buffer solution, oscillating for 1min, magnetically separating for 1min, discarding supernatant, repeating for 3 times, adding 1ml of MES buffer solution, and oscillating for 1min for later use;
(2) S-NHS treatment: adding 50 mu L of S-NHS solution with the concentration of 10mg/mL prepared by MES buffer solution into the step (1), uniformly mixing for 1min by oscillation, and adjusting the pH value of the reaction system to 5.0;
(3) EDC treatment: adding 50 mu L of EDC solution with the concentration of 10mg/mL prepared by MES buffer solution into the reaction system in the step (2), oscillating and uniformly mixing for 1min, adjusting the pH value of the reaction system to 5.0, placing the reaction system in a constant temperature and humidity box (26 +/-1 ℃), rotating and uniformly mixing for 30min, magnetically separating for 1min, then discarding supernatant, adding MES buffer solution, and oscillating and uniformly for 1-3 min;
(4) Adding an antibody: adding 350 mu g of antibody into the step (3), placing a magnetic bead labeled reactant into a constant temperature and humidity box (26 +/-1 ℃), uniformly mixing and reacting for 120min in a rotating mode, removing the supernatant after 1min of magnetic separation, adding 1ml of TRIS buffer solution, oscillating uniformly for 1min, repeating for 3 times, removing the supernatant, adding 1ml of magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly mixing, placing the magnetic bead labeled reactant into the constant temperature and humidity box (26 +/-1 ℃), standing and reacting for 60min, removing the supernatant after 1min of magnetic separation, adding 1ml of magnetic bead labeled buffer solution A, oscillating uniformly for 1min, removing the supernatant, repeating for 3 times, finally adding a magnetic bead preservation solution, and placing the mixture in an environment at 4 ℃ for storage.
The MES buffer solution formula in the step (1), the step (3) and the step (4) comprises 9.76g of MES, 9g of NaCl and 0.5g of PC300, the components are subjected to volume fixing to 1L by double distilled water, and the pH value is adjusted to 5.8 by 3M NaOH.
The formulation of the magnetic bead labeling buffer solution A in the step (4) is 6.05g of Tris, 9g of NaCl, 0.5g of PC300, 5g of BSA and 0.5g of Tween-20, the components are dissolved to 1L by double distilled water, and the pH value is adjusted to 7.3-7.5 by 1M HCl.
the formula of the magnetic bead preservation solution in the step (4) comprises the following components: 6.05g Tris, 9g NaCl, 0.5g PC300, 10g BSA, 1g Tween-20, the above components were dissolved to 1L with double distilled water, and the pH was adjusted to 7.3-7.5 with 1M HCl.
example 2
The kit for detecting VEGF prepared by the directional coupling method comprises the following components: the kit comprises a magnetic microsphere suspension coated with a VEGF antibody (the working concentration of the magnetic microsphere is 0.1-0.35 mg/mL, the concentration of the VEGF antibody coated on the surface of the magnetic microsphere is 10-50ug/mg), a VEGF reference product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF reference product are respectively 0pg/mL, 30pg/mL, 125pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL), a VEGF quality control product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF quality control product are 125pg/mL and 1000pg/mL), a VEGF enzyme conjugate (the VEGF enzyme conjugate is a horseradish peroxidase-labeled VEGF monoclonal antibody, and the concentration of the VEGF enzyme conjugate is diluted according to the ratio of the VEGF enzyme conjugate diluent to 3000), a concentrated washing solution (0.04-0.06 mol, 0.06mol and the concentration of the VEGF enzyme conjugate is 0., Tris-HCl buffer solution with pH7.4-pH7.6, and Tween-80 in 0.04-0.06% v/v and Proclin-300 in 0.7-0.8% v/v in every 1L of buffer solution. ) And luminescence substrate liquid (every 250ml of the luminescence substrate liquid A contains 2.3-2.6g of N, O-bis trimethylsilyl acetamide, 1.3-1.6g of trihydroxymethyl aminomethane, 0.4-0.6g of 3-aminophthalic hydrazide, 0.007-0.008g of para-iodophenol and 4-40g of sodium hydroxide, and the pH value of the working solution is 10-13; the luminescent substrate liquid B comprises 2.3-2.6g of N, O-bis (trimethylsilyl) acetamide, 1.3-1.6g of tris (hydroxymethyl) aminomethane and 0.1-0.3ml of hydrogen peroxide per 250ml of solution. ) The reference substance diluent is Tris-HCl buffer solution with the pH value of 7.5, and each 1L of the buffer solution also contains 18-22g of BSA, 14-15g of EDTA, 3.8-4g of 2-mercaptoethanol and 0.3-0.5mL of Proclin-300. ) The enzyme conjugate diluent is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 8-11g of bovine serum albumin, 5-6g of calcium chloride, 3-6% v/v of calf serum, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
the method for directionally coupling the antibody and the magnetic bead (namely preparing the magnetic microsphere suspension coated with the VEGF antibody) comprises the following steps:
(1) Magnetic bead pretreatment: taking 1ml of MES buffer solution, adding 10mg of magnetic bead mother liquor (carboxyl magnetic bead mother liquor), oscillating for 1min, magnetically separating for 1min, discarding supernatant, repeating for 3 times, adding 2ml of MES buffer solution, oscillating for 1min for later use;
(2) S-NHS treatment: adding 50 mu L of S-NHS solution prepared by MES buffer solution and having the concentration of 15mg/mL into the step (1), uniformly mixing for 1min by oscillation, and adjusting the pH value of a reaction system to 6.0;
(3) EDC treatment: adding 50 mu L of EDC solution with the concentration of 10mg/mL prepared by MES buffer solution into the reaction system in the step (2), oscillating and uniformly mixing for 1min, adjusting the pH value of the reaction system to be 6.0, placing the reaction system in a constant temperature and humidity box (26 +/-1 ℃), rotating and uniformly mixing for 30min, magnetically separating for 3min, then discarding supernatant, adding MES buffer solution, and oscillating and uniformly mixing for 3 min;
(4) Adding an antibody: adding 500 mu g of antibody into the step (3), placing a magnetic bead labeled reactant into a constant temperature and humidity box (26 +/-1 ℃), uniformly mixing and reacting for 150min in a rotating mode, removing the supernatant after 1min of magnetic separation, adding 2.5ml of TRIS buffer solution, oscillating uniformly for 1min, repeating for 3 times, removing the supernatant, adding 2.5ml of magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly mixing, placing the magnetic bead labeled reactant into the constant temperature and humidity box (26 +/-1 ℃), standing and reacting for 60min, removing the supernatant after 1min of magnetic separation, adding 1ml of magnetic bead labeled buffer solution A, oscillating uniformly for 2min, removing the supernatant, repeating for 3 times, finally adding a magnetic bead preservation solution, and placing in an environment at 4 ℃ for storage.
the MES buffer solution formula in the step (1), the step (3) and the step (4) comprises 9.76g of MES, 9g of NaCl and 0.5g of PC300, the components are subjected to volume fixing to 1L by double distilled water, and the pH value is adjusted to 6.0 by 3M NaOH.
the formulation of the magnetic bead labeling buffer solution A in the step (4) is 6.05g of Tris, 9g of NaCl, 0.5g of PC300, 5g of BSA and 0.5g of Tween-20, the components are dissolved to 1L by double distilled water, and the pH value is adjusted to 7.3-7.5 by 1M HCl.
the formula of the magnetic bead preservation solution in the step (4) comprises the following components: 6.05g Tris, 9g NaCl, 0.5g PC300, 10g BSA, 1g Tween-20, the above components were dissolved to 1L with double distilled water, and the pH was adjusted to 7.3-7.5 with 1M HCl.
Example 3
The kit for detecting VEGF prepared by the directional coupling method comprises the following components: the kit comprises a magnetic microsphere suspension coated with a VEGF antibody (the working concentration of the magnetic microsphere is 0.1-0.35 mg/mL, the concentration of the VEGF antibody coated on the surface of the magnetic microsphere is 10-50ug/mg), a VEGF reference product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF reference product are respectively 0pg/mL, 30pg/mL, 125pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL), a VEGF quality control product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF quality control product are 125pg/mL and 1000pg/mL), a VEGF enzyme conjugate (the VEGF enzyme conjugate is a horseradish peroxidase-labeled VEGF monoclonal antibody, and the concentration of the VEGF enzyme conjugate is diluted according to the ratio of the VEGF enzyme conjugate diluent to 3000), a concentrated washing solution (0.04-0.06 mol, 0.06mol and the concentration of the VEGF enzyme conjugate is 0., Tris-HCl buffer solution with pH7.4-pH7.6, and Tween-80 in 0.04-0.06% v/v and Proclin-300 in 0.7-0.8% v/v in every 1L of buffer solution. ) And luminescence substrate liquid (every 250ml of the luminescence substrate liquid A contains 2.3-2.6g of N, O-bis trimethylsilyl acetamide, 1.3-1.6g of trihydroxymethyl aminomethane, 0.4-0.6g of 3-aminophthalic hydrazide, 0.007-0.008g of para-iodophenol and 4-40g of sodium hydroxide, and the pH value of the working solution is 10-13; the luminescent substrate liquid B comprises 2.3-2.6g of N, O-bis (trimethylsilyl) acetamide, 1.3-1.6g of tris (hydroxymethyl) aminomethane and 0.1-0.3ml of hydrogen peroxide per 250ml of solution. ) The reference substance diluent is Tris-HCl buffer solution with the pH value of 7.5, and each 1L of the buffer solution also contains 18-22g of BSA, 14-15g of EDTA, 3.8-4g of 2-mercaptoethanol and 0.3-0.5mL of Proclin-300. ) The enzyme conjugate diluent is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 8-11g of bovine serum albumin, 5-6g of calcium chloride, 3-6% v/v of calf serum, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
The method for directionally coupling the antibody and the magnetic bead (namely preparing the magnetic microsphere suspension coated with the VEGF antibody) comprises the following steps:
(1) Magnetic bead pretreatment: taking 1ml of MES buffer solution, adding 15mg of magnetic bead mother liquor (carboxyl magnetic bead mother liquor), oscillating for 1min, magnetically separating for 1min, discarding supernatant, repeating for 3 times, adding 2ml of MES buffer solution, oscillating for 1min for later use;
(2) S-NHS treatment: adding 50 mu L of S-NHS solution prepared by MES buffer solution and having the concentration of 20mg/mL into the step (1), uniformly mixing for 1min by oscillation, and adjusting the pH value of a reaction system to 6.5;
(3) EDC treatment: adding 50 mu L of EDC solution with the concentration of 10mg/mL prepared by MES buffer solution into the reaction system in the step (2), oscillating and uniformly mixing for 1min, adjusting the pH value of the reaction system to 5.0, placing the reaction system in a constant temperature and humidity box (26 +/-1 ℃), rotating and uniformly mixing for 30min, magnetically separating for 1min, then discarding supernatant, adding MES buffer solution, and oscillating and uniformly for 1-3 min;
(4) adding an antibody: adding 200-500 mug of antibody into the step (3), placing a magnetic bead labeled reactant into a constant temperature and humidity box (26 +/-1 ℃), uniformly mixing and reacting for 120min in a rotating mode, removing the supernatant after 1min of magnetic separation, adding 1ml of TRIS buffer solution, uniformly oscillating for 1min, repeating for 3 times, removing the supernatant, adding 2.5ml of magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly mixing, placing the magnetic bead labeled reactant into the constant temperature and humidity box (26 +/-1 ℃), standing and reacting for 60min, removing the supernatant after 1min of magnetic separation, adding 2ml of magnetic bead labeled buffer solution A, uniformly oscillating for 1min, removing the supernatant, repeating for 3 times, finally adding a magnetic bead preservation solution, and placing the mixture in an environment at 4 ℃ for storage.
the MES buffer solution formula in the step (1), the step (3) and the step (4) comprises 9.76g of MES, 9g of NaCl and 0.5g of PC300, the components are subjected to volume fixing to 1L by double distilled water, and the pH value is adjusted to 6.5 by 3M NaOH.
The formulation of the magnetic bead labeling buffer solution A in the step (4) is 6.05g of Tris, 9g of NaCl, 0.5g of PC300, 5g of BSA and 0.5g of Tween-20, the components are dissolved to 1L by double distilled water, and the pH value is adjusted to 7.3-7.5 by 1M HCl.
The formula of the magnetic bead preservation solution in the step (4) comprises the following components: 6.05g Tris, 9g NaCl, 0.5g PC300, 10g BSA, 1g Tween-20, the above components were dissolved to 1L with double distilled water, and the pH was adjusted to 7.3-7.5 with 1M HCl.
The formulations of TRIS buffer in example 1, example 2 and example 3 are given in table 1 below:
TABLE 1
| Manufacturer of the product | Dosage of | |
| Tris | Sigma | 6.05g |
| Tris-HCI | Sigma | 6.59g |
| NaCl | Chemical reagent for Chinese medicine | 9.00g |
| PC300 | Sigma | 0.5g |
| 1MHCI | Chemical reagent for Chinese medicine | the pH of the mixture was adjusted to 7.4. + -. 0.1 |
| ddH2O | / | Constant volume is 1L |
In order to better prove that the kit for detecting VEGF prepared by the directional coupling method has better detection effect compared with the existing unconjugated kit, two comparative examples are given below.
Comparative example 1
The Jianping jinxing product is sold in the market.
Comparative example 2
the same kit components as in example 3, the only difference is the antibody-magnetic bead coupling method:
The kit comprises a magnetic microsphere suspension coated with a VEGF antibody (the working concentration of the magnetic microsphere is 0.1-0.35 mg/mL, the concentration of the VEGF antibody coated on the surface of the magnetic microsphere is 10-50ug/mg), a VEGF reference product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF reference product are respectively 0pg/mL, 30pg/mL, 125pg/mL, 500pg/mL, 1000pg/mL and 2000 pg/mL), a VEGF quality control product (the VEGF reference product is prepared from a VEGF standard product and a reference product diluent, the concentrations of the VEGF quality control product are 125pg/mL and 1000pg/mL), a VEGF enzyme conjugate (the VEGF enzyme conjugate is a horseradish peroxidase-labeled VEGF monoclonal antibody, and the concentration of the VEGF enzyme conjugate is diluted according to the ratio of the VEGF enzyme conjugate diluent to 3000), a concentrated washing solution (0.04-0.06 mol, 0.06mol and the concentration of the VEGF enzyme conjugate is 0., Tris-HCl buffer solution with pH7.4-pH7.6, and Tween-80 in 0.04-0.06% v/v and Proclin-300 in 0.7-0.8% v/v in every 1L of buffer solution. ) And luminescence substrate liquid (every 250ml of the luminescence substrate liquid A contains 2.3-2.6g of N, O-bis trimethylsilyl acetamide, 1.3-1.6g of trihydroxymethyl aminomethane, 0.4-0.6g of 3-aminophthalic hydrazide, 0.007-0.008g of para-iodophenol and 4-40g of sodium hydroxide, and the pH value of the working solution is 10-13; the luminescent substrate liquid B comprises 2.3-2.6g of N, O-bis (trimethylsilyl) acetamide, 1.3-1.6g of tris (hydroxymethyl) aminomethane and 0.1-0.3ml of hydrogen peroxide per 250ml of solution. ) The reference substance diluent is Tris-HCl buffer solution with the pH value of 7.5, and each 1L of the buffer solution also contains 18-22g of BSA, 14-15g of EDTA, 3.8-4g of 2-mercaptoethanol and 0.3-0.5mL of Proclin-300. ) The enzyme conjugate diluent is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 8-11g of bovine serum albumin, 5-6g of calcium chloride, 3-6% v/v of calf serum, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
The method for directionally coupling the antibody and the magnetic bead (namely the magnetic microsphere suspension coated with the VEGF antibody) comprises the following steps:
(1) magnetic bead pretreatment: taking 1ml of PBS buffer solution, adding 15mg of magnetic bead mother solution, oscillating for 1min, magnetically separating for 1min, discarding supernatant, repeating for 3 times, adding 2ml of MES buffer solution, oscillating for 1min for later use;
(2) S-NHS treatment: adding 50 mu L of S-NHS solution prepared by MES buffer solution and having the concentration of 20mg/mL into the step (1), uniformly mixing for 1min by oscillation, and adjusting the pH value of a reaction system to 7.8;
(3) EDC treatment: adding 50 mu L of EDC solution with the concentration of 10mg/mL prepared by MES buffer solution into the reaction system in the step (2), oscillating and uniformly mixing for 1min, adjusting the pH value of the reaction system to 7.8, placing the reaction system in a constant temperature and humidity box (26 +/-1 ℃), rotating and uniformly mixing for reaction for 30min, magnetically separating for 1min, then removing supernatant, adding MES buffer solution, oscillating and uniformly mixing for 3 min;
(4) Adding an antibody: adding 500 mu g of antibody into the step (3), placing a magnetic bead labeled reactant into a constant temperature and humidity box (26 +/-1 ℃), uniformly mixing and reacting for 120min in a rotating mode, removing the supernatant after 1min of magnetic separation, adding 1ml of TRIS buffer solution, oscillating uniformly for 1min, repeating for 3 times, removing the supernatant, adding 2.5ml of magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly mixing, placing the magnetic bead labeled reactant into the constant temperature and humidity box (26 +/-1 ℃), standing and reacting for 90min, removing the supernatant after 1min of magnetic separation, adding 2ml of magnetic bead labeled buffer solution A, oscillating uniformly for 1min, removing the supernatant, repeating for 3 times, finally adding a magnetic bead preservation solution, and placing in an environment at 4 ℃ for storage.
the formulation of PBS buffer is shown in Table 2 below.
TABLE 2
| Manufacturer of the product | Dosage of | |
| KH2PO4 | Chinese medicine | 0.24g |
| Na2HPO4 | chinese medicine | 1.44 |
| NaCl | Chemical reagent for Chinese medicine | 9.00g |
| PC300 | Sigma | 0.5g |
| 3MNaOH | Chemical reagent for Chinese medicine | Modulation of pH7.8 |
| ddH2O | / | Constant volume is 1L |
The kit prepared by the directional coupling technology (examples 1-3) and the kits in comparative example 1 and comparative example 2 are used for VEGF detection of 50 tumor patient samples, and the specific detection results are shown in Table 3.
TABLE 3
From the data in table 3, it can be seen that the detection rate of the kit prepared by using the directional coupling technique is 20% higher than that of the commercially available Jianping aventurine product and the kit in comparative example 2, and the detection rate of the embodiment not using the directional coupling technique is consistent with that of the commercially available Jianping aventurine reagent.
The kits of examples 1 to 3 of the present invention and comparative example 2, which are to be described herein, have the same components except that the components of the kit, namely the magnetic microsphere suspension coated with the VEGF antibody (i.e., the antibody-magnetic bead directional coupling method), are different.
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 for directional coupling of antibodies and magnetic beads, characterized in that the method comprises the following steps:
(1) Magnetic bead pretreatment: adding magnetic bead mother liquor into MES buffer solution, oscillating uniformly, magnetically separating, discarding supernatant, adding MES buffer solution, and oscillating uniformly for later use;
(2) S-NHS treatment: adding an S-NHS solution prepared by MES buffer solution into the step (1), oscillating and uniformly mixing, and adjusting the pH value of a reaction system to be acidic;
(3) EDC treatment: adding an EDC solution prepared by MES buffer solution into the reaction system in the step (2), oscillating and mixing uniformly, adjusting the pH value of the reaction system to be acidic, placing the reaction system in a constant temperature and humidity box, rotating and mixing uniformly for reaction for a period of time, removing supernatant after magnetic separation, adding MES buffer solution, and oscillating and mixing uniformly;
(4) adding an antibody: adding an antibody into the step (3), placing a magnetic bead labeled reactant into a constant temperature and humidity box, rotating and uniformly mixing the reactant and the antibody for a period of time, removing the supernatant after magnetic separation, adding a TRIS buffer solution, uniformly oscillating the reactant and the supernatant, adding a magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly mixing the reactant and the magnetic bead labeled reactant, placing the magnetic bead labeled reactant into the constant temperature and humidity box, standing the reactant and the magnetic bead labeled reactant for a period of time, removing the supernatant after magnetic separation, adding the magnetic bead labeled buffer solution A into the magnetic bead labeled reactant, uniformly oscillating the reactant and the supernatant, adding a magnetic bead preservation solution into the magnetic bead labeled reactant, and placing the.
2. The method of claim 1, wherein the method comprises the following steps: in the step (1), the magnetic bead mother liquor is carboxyl magnetic bead mother liquor, the addition amount of the magnetic bead mother liquor is 10-15mg, the first addition amount of MES buffer solution is 0.5-1ml, and the second addition amount of MES buffer solution is 1-2 ml.
3. The method of claim 1, wherein the method comprises the following steps: the concentration of the S-NHS solution in the step (2) is 10mg/mL-20mg/mL, and the adding amount of the S-NHS solution is 50 mu L.
4. the method of claim 1, wherein the method comprises the following steps: and (3) adjusting the pH value of the reaction system to 5.0-6.5 in the step (2).
5. The method of claim 1, wherein the method comprises the following steps: the concentration of the EDC solution in the step (3) is 8-12mg/mL, and the addition amount of the EDC solution is 50 mu L.
6. The method of claim 1, wherein the method comprises the following steps: and (3) adjusting the pH value of the reaction system to 5.0-6.5.
7. the method of claim 1, wherein the method comprises the following steps: in the step (3), the temperature in the constant temperature and humidity box is 26 +/-1 ℃, and the time of the rotary reaction is 20-40 min.
8. the method of claim 1, wherein the method comprises the following steps: in the step (4), the adding amount of the antibody is 200-500 mug, the time of the rotary mixing reaction is 120-150min, the adding amount of the TRIS buffer solution is 1-2.5 mL, the adding amount of the magnetic bead labeling buffer solution A is 1-2.5 mL, and the time of the standing reaction is 60-90 min.
9. The method of claim 1, wherein the method comprises the following steps: the formulation of the magnetic bead labeling buffer solution A in the step (4) is 6.05g of Tris, 9g of NaCl, 0.5g of PC300, 5g of BSA and 0.5g of Tween-20, the components are dissolved to 1L by double distilled water, and the pH value is adjusted to 7.3-7.5 by 1M HCl.
10. The method of claim 1, wherein the method comprises the following steps: the formula of the magnetic bead preservation solution in the step (4) comprises the following components: 6.05g Tris, 9g NaCl, 0.5g PC300, 10g BSA, 1g Tween-20, the above components were dissolved to 1L with double distilled water, and the pH was adjusted to 7.3-7.5 with 1M HCl.
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