CN114034865A - Method for coating magnetic beads with alpha-fetoprotein antibodies, coupled magnetic beads and kit - Google Patents
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- 102000013529 alpha-Fetoproteins Human genes 0.000 title claims abstract description 101
- 108010026331 alpha-Fetoproteins Proteins 0.000 title claims abstract description 101
- 239000011324 bead Substances 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000011248 coating agent Substances 0.000 title claims abstract description 15
- 238000000576 coating method Methods 0.000 title claims abstract description 15
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 37
- 230000008878 coupling Effects 0.000 claims abstract description 28
- 238000010168 coupling process Methods 0.000 claims abstract description 28
- 238000005859 coupling reaction Methods 0.000 claims abstract description 28
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 15
- 239000000243 solution Substances 0.000 claims description 40
- 239000007987 MES buffer Substances 0.000 claims description 20
- 238000007789 sealing Methods 0.000 claims description 19
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000007983 Tris buffer Substances 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 20
- 210000002966 serum Anatomy 0.000 abstract description 20
- 238000003018 immunoassay Methods 0.000 abstract description 11
- 239000006249 magnetic particle Substances 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 description 23
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 3
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
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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
- G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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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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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
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- 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
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- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
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Abstract
The invention relates to a method for coating a magnetic bead with an alpha fetoprotein antibody, a coupled magnetic bead and a kit. Before the alpha-fetoprotein antibody is coupled with the carboxyl magnetic beads, the alpha-fetoprotein antibody is pretreated by the sodium dodecyl sulfate, so that the coupling efficiency of the alpha-fetoprotein antibody and the magnetic beads can be obviously improved, and the sensitivity and the signal-to-noise ratio of detecting the alpha-fetoprotein in human serum by using a chemiluminescence magnetic particle immunoassay method in the prior art are improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for coating a magnetic bead with an alpha fetoprotein antibody, a coupled magnetic bead and a kit.
Background
Alpha-fetoprotein (AFP) is a glycoprotein belonging to the albumin family, is mainly synthesized by fetal liver cells and yolk sac, and has a molecular weight of 70000 daltons. The alpha-fetoprotein has higher concentration in the blood circulation of the fetus, is reduced after birth, and is replaced by leucocytes in 2-3 months after birth, so the content in the serum of an adult is extremely low and the alpha-fetoprotein is difficult to detect.
AFP has important physiological functions including transport function, growth factor regulation, T lymphocyte-induced apoptosis, immunosuppression, and the like. The AFP is closely related to the occurrence and development of chronic viral hepatitis, liver cirrhosis and various tumors, can show high concentration in various tumors, and can be used as a positive detection index of various tumors. At present, the AFP can be clinically used for the auxiliary diagnosis of primary liver cancer, and can also be used for the joint detection of Human Chorionic Gonadotropin (HCG) and the condition monitoring of patients with non-seminiferous cell type testicular cancer.
The conventional method for detecting alpha-fetoprotein is enzyme-linked immunosorbent assay, but the method has a lot of defects, such as complex operation, low detection sensitivity, narrow detection linear range, poor reproducibility and the like. In recent years, chemiluminescence immunoassay methods, which have the advantages of high sensitivity, wide detection range, easy operation, and the like, have become the most desirable detection methods. For example, patent application publication No. CN112763719A discloses a method for detecting alpha-fetoprotein by using aptamer magnetic beads, in which an alpha-fetoprotein aptamer is mixed with magnetic beads, and then the magnetic beads are separated for detection.
Disclosure of Invention
The invention aims to provide a method for coating a magnetic bead with an alpha-fetoprotein antibody, a coupled magnetic bead and a kit, wherein the alpha-fetoprotein antibody is pretreated by using sodium dodecyl sulfate, so that the coupling efficiency of the alpha-fetoprotein antibody and the magnetic bead is improved, and the technical problems of insufficient sensitivity and signal-to-noise ratio of the detection of the alpha-fetoprotein in human serum by using a chemiluminescence magnetic particle immunoassay in the prior art are finally improved.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for coating magnetic beads with alpha fetoprotein antibodies comprises the steps of adding pretreatment liquid into the alpha fetoprotein antibodies for pretreatment, and then coupling the pretreated alpha fetoprotein antibodies with carboxyl magnetic beads to obtain alpha fetoprotein antibody coupled magnetic beads, wherein the pretreatment liquid comprises a sodium dodecyl sulfate solution.
Further preferably, the concentration of the sodium dodecyl sulfate solution is 0.2-0.3%, the pretreatment time is 5-10 min, and the pretreatment temperature is 20-25 ℃.
More preferably, the concentration of the sodium dodecyl sulfate solution is 0.2%, the pretreatment time is 10min, and the pretreatment temperature is 25 ℃.
More preferably, the pretreatment solution further comprises a MES buffer solution with a concentration of 10 mM-80 mM.
More preferably, the carboxyl magnetic beads are mixed and activated with a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide before coupling of the alpha-fetoprotein antibody, wherein the concentrations of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide are both 8-12 mg/ml; the activation temperature is 23-28 ℃, and the activation time is 10-20 min.
More preferably, the activated carboxyl magnetic beads are washed in MES buffer solution for 2-3 times, wherein the concentration of the MES buffer solution is 10 mM-80 mM, the pH value is 5.5-7.5, and then the temperature for mixing and coupling the pretreated alpha-fetoprotein antibody and the washed carboxyl magnetic beads is 22-28 ℃, and the mixing and coupling time is 1-5 h.
Preferably, the coupled magnetic beads are placed into MES buffer solution containing 0.1% BSA for sealing treatment, wherein the sealing time is 5-8 h, the sealing temperature is 22-28 ℃, and then the coupled magnetic beads subjected to sealing treatment are added into Tris buffer solution for resuspension and preservation, and the preservation temperature is 2-8 ℃.
Further preferably, the amount of the alpha fetoprotein antibody coating per milligram of magnetic beads is 10-30 mu g.
Coupled magnetic beads prepared by the method for coating the magnetic beads with the alpha fetoprotein antibody.
A kit comprising coupled magnetic beads prepared by the method for coating magnetic beads with alpha fetoprotein antibodies as described in any one of the above.
Has the advantages that: in general, hydrophilic amino acid residues of the alpha-fetoprotein antibody are positioned on the surface of molecules, and hydrophobic amino acid residues are buried in proteins, so that the protein space structure of the recombinant alpha-fetoprotein antibody can be changed by pretreating the alpha-fetoprotein antibody with sodium dodecyl sulfate before the alpha-fetoprotein antibody is coupled with magnetic beads, binding sites of the alpha-fetoprotein antibody are effectively released, non-essential sites are covered, the coupling efficiency of the alpha-fetoprotein antibody and the carboxyl magnetic beads is greatly improved, and in addition, when the alpha-fetoprotein in human serum is detected by using a chemiluminescence magnetic particle immunoassay method, the detection is carried out by using the alpha-fetoprotein antibody coupled magnetic beads prepared by the method, and the sensitivity and the signal-to-noise ratio of the detection are obviously improved.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below.
Example 1:
a method for coating a magnetic bead with an alpha fetoprotein antibody, a coupled magnetic bead and a kit comprise the following steps:
1. pretreatment of alpha-fetoprotein antibody: preparing 0.1 mass percent of dodecyl sulfuric acid and 10 mM-80 mM of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, and mixing to prepare a pretreatment solution; and then adding the alpha fetoprotein antibody into the pretreatment solution, and placing the mixture into a shaking table for shaking and mixing, wherein the rotation speed of the shaking table is 50r/min, the treatment time is 10min, and the treatment temperature is 25 ℃.
2. Magnetic bead activation: preparing a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), wherein the concentration of EDC and NHS is 8-12 mg/L. And then adding 0.05-1.0 mu m of carboxyl magnetic beads into the mixed solution, and mixing in a constant temperature box by using a rotary mixing machine to fully activate the carboxyl magnetic beads, wherein the temperature of the constant temperature box is 25 ℃, and the mixing time is 10 min.
3. Washing with carboxyl magnetic beads: and (3) washing the activated carboxyl magnetic beads in the step (2) for 2-3 times by using an MES buffer solution, wherein the concentration of the MES buffer solution is 50mM, and the pH value is 6.5.
4. Coupling: and (3) adding the washed carboxyl magnetic beads in the step (3) into the alpha fetoprotein antibody pretreated in the step (1), and oscillating and mixing in a shaking table, wherein the rotating speed of the shaking table is 60r/min, the coupling time is 1-5 h, and the coupling temperature is 25 ℃.
5. And (3) sealing: and (3) adding the coupled carboxyl magnetic beads obtained in the step (4) into MES buffer solution containing 0.1% BSA for sealing treatment, wherein the sealing treatment time is 5-8 h, and the sealing temperature is 25 ℃.
6. And (4) resuspension preservation: and (3) adding the carboxyl magnetic beads subjected to the blocking treatment in the step (5) into a Tris buffer solution, and resuspending and storing at 2-8 ℃ to obtain the alpha fetoprotein antibody coupled magnetic beads.
In other embodiments, the concentrations of sodium dodecyl sulfate were set to 0.2%, 0.3%, 0.4%, and 0.5%, respectively, and other parameters were unchanged to make different alpha-fetoprotein antibody-coupled magnetic beads.
The alpha fetoprotein antibody coupling magnetic beads obtained in the embodiment are placed in a reagent kit to prepare five groups of reagent kits, the alpha fetoprotein in human serum is detected by using a chemiluminescence magnetic particle immunoassay method respectively, and meanwhile, a group of blank groups are arranged, namely the existing reagent kit for detecting the alpha fetoprotein in the serum. S1-S6 are the detection sensitivity results of six groups of kits corresponding to different detection samples, wherein S1 corresponds to the detection sensitivity result of a negative sample, namely, the detection object is a serum sample containing no or little alpha-fetoprotein. The sensitivity and signal-to-noise ratio data for each set of assays are shown in table 1 below:
TABLE 1
As can be seen from table 1, when the concentration of sodium dodecyl sulfate is 0.2% to 0.3% in the process of pretreating the alpha-fetoprotein antibody, the alpha-fetoprotein antibody shows higher sensitivity and signal-to-noise ratio in the process of detecting the alpha-fetoprotein in human serum by using a chemiluminescence magnetic particle immunoassay, and the coupling efficiency of the alpha-fetoprotein antibody and the carboxyl magnetic beads under the treatment condition is also obviously improved; especially, when the concentration is 0.2%, the sensitivity and the signal-to-noise ratio of the alpha fetoprotein in human serum can be obviously improved.
Example 2
1. Pretreatment of alpha-fetoprotein antibody: preparing 0.2 mass percent of dodecyl sulfuric acid and 10 mM-80 mM of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, and mixing to prepare a pretreatment solution; and then adding the alpha fetoprotein antibody into the pretreatment solution, and placing the mixture into a shaking table for shaking and mixing, wherein the rotation speed of the shaking table is 50r/min, the treatment time is 5min, and the treatment temperature is 25 ℃.
2. Magnetic bead activation: preparing a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), wherein the concentration of EDC and NHS is 8-12 mg/L. And then adding 0.05-1.0 mu m of carboxyl magnetic beads into the mixed solution, and mixing in a constant temperature box by using a rotary mixing machine to fully activate the carboxyl magnetic beads, wherein the temperature of the constant temperature box is 25 ℃, and the mixing time is 10 min.
3. Washing with carboxyl magnetic beads: and (3) washing the activated carboxyl magnetic beads in the step (2) for 2-3 times by using an MES buffer solution, wherein the concentration of the MES buffer solution is 50mM, and the pH value is 6.5.
4. Coupling: and (3) adding the washed carboxyl magnetic beads in the step (3) into the alpha fetoprotein antibody pretreated in the step (1), and oscillating and mixing in a shaking table, wherein the rotating speed of the shaking table is 60r/min, the coupling time is 1-5 h, and the coupling temperature is 25 ℃.
5. And (3) sealing: and (3) adding the coupled carboxyl magnetic beads obtained in the step (4) into MES buffer solution containing 0.1% BSA for sealing treatment, wherein the sealing treatment time is 5-8 h, and the sealing temperature is 25 ℃.
6. And (4) resuspension preservation: and (3) adding the carboxyl magnetic beads subjected to the blocking treatment in the step (5) into a Tris buffer solution, and resuspending and storing at 2-8 ℃ to obtain the alpha fetoprotein antibody coupled magnetic beads.
In other embodiments, the pretreatment time of the alpha-fetoprotein antibody is set to 5min, 15min and 20min respectively, and other parameters are unchanged, so that the alpha-fetoprotein antibody coupled magnetic beads at different pretreatment times are prepared.
The alpha fetoprotein antibody coupling magnetic beads obtained in the above embodiment are placed in a reagent kit to prepare four groups of reagent kits, the chemiluminescence magnetic particle immunoassay method is respectively utilized to detect the alpha fetoprotein in the human serum, and meanwhile, a group of blank groups is arranged, namely the existing reagent kit for detecting the alpha fetoprotein in the serum. S1-S6 are detection sensitivity results of five groups of kits corresponding to different detection samples, wherein S1 corresponds to the detection sensitivity result of a negative sample, namely, a detection object is a serum sample containing no or little alpha-fetoprotein. The sensitivity and signal-to-noise ratio data for each set of assays are shown in table 1 below:
TABLE 2
As can be seen from Table 2, in the process of pretreating the alpha-fetoprotein antibody, 0.2% of sodium dodecyl sulfate is adopted to pretreat the alpha-fetoprotein antibody at 25 ℃, after pretreatment for 10-15 min, in the process of finally detecting the alpha-fetoprotein in human serum by using a chemiluminescence magnetic particle immunoassay method, higher sensitivity and signal-to-noise ratio can be shown compared with a blank group, and the coupling efficiency of the alpha-fetoprotein antibody and the carboxyl magnetic beads under the treatment condition is also obviously improved. Especially, when the processing time is 10min, the sensitivity and the signal-to-noise ratio of the alpha fetoprotein in the human serum can be obviously improved.
Example 3:
1. pretreatment of alpha-fetoprotein antibody: preparing 0.2 mass percent of dodecyl sulfuric acid and 10 mM-80 mM of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, and mixing to prepare a pretreatment solution; and then adding the alpha fetoprotein antibody into the pretreatment solution, and placing the mixture into a shaking table for shaking and mixing, wherein the shaking speed of the shaking table is 50r/min, the treatment time is 10min, and the treatment temperature is 25 ℃.
2. Magnetic bead activation: preparing a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), wherein the concentration of EDC and NHS is 8-12 mg/L. And then adding 0.05-1.0 mu m of carboxyl magnetic beads into the mixed solution, and mixing in a constant temperature box by using a rotary mixing machine to fully activate the carboxyl magnetic beads, wherein the temperature of the constant temperature box is 25 ℃, and the mixing time is 10 min.
3. Washing with carboxyl magnetic beads: and (3) washing the activated carboxyl magnetic beads in the step (2) for 2-3 times by using an MES buffer solution, wherein the concentration of the MES buffer solution is 50mM, and the pH value is 6.5.
4. Coupling: and (3) adding the washed carboxyl magnetic beads in the step (3) into the alpha fetoprotein antibody pretreated in the step (1), and oscillating and mixing in a shaking table, wherein the rotating speed of the shaking table is 60r/min, the coupling time is 1-5 h, and the coupling temperature is 25 ℃.
5. And (3) sealing: and (3) adding the coupled carboxyl magnetic beads obtained in the step (4) into MES buffer solution containing 0.1% BSA for sealing treatment, wherein the sealing treatment time is 5-8 h, and the sealing temperature is 25 ℃.
6. And (4) resuspension preservation: and (3) adding the carboxyl magnetic beads subjected to the blocking treatment in the step (5) into a Tris buffer solution, and resuspending and storing at 2-8 ℃ to obtain the alpha fetoprotein antibody coupled magnetic beads.
In other embodiments, the pretreatment temperatures of the alpha-fetoprotein antibody are set to 4 ℃, 16 ℃, 20 ℃ and 37 ℃ respectively, and other parameters are unchanged, so that the alpha-fetoprotein antibody coupled magnetic beads at different pretreatment temperatures are prepared.
The alpha fetoprotein antibody coupling magnetic beads obtained in the embodiment are placed in a reagent kit to prepare five groups of reagent kits, the alpha fetoprotein in human serum is detected by using a chemiluminescence magnetic particle immunoassay method respectively, and meanwhile, a group of blank groups are arranged, namely the existing reagent kit for detecting the alpha fetoprotein in the serum. S1-S6 are the detection sensitivity results of six groups of kits corresponding to different detection samples, wherein S1 corresponds to the detection sensitivity result of a negative sample, namely, the detection object is a serum sample containing no or little alpha-fetoprotein. The sensitivity and signal-to-noise ratio data for each set of assays are shown in table 3 below:
TABLE 3
From table 3, it can be known that, in the process of pretreating the alpha-fetoprotein antibody, 0.2% of sodium dodecyl sulfate is adopted to pretreat the alpha-fetoprotein antibody at the temperature of 20-25 ℃ for 10min, and in the process of finally detecting the alpha-fetoprotein in human serum by using a chemiluminescence magnetic particle immunoassay, higher sensitivity and signal-to-noise ratio can be shown compared with a blank experiment, and the coupling efficiency of the alpha-fetoprotein antibody and the carboxyl magnetic beads under the treatment condition is also obviously improved. Especially at 25 ℃, the sensitivity and the signal-to-noise ratio of detecting the alpha fetoprotein in human serum can be obviously improved.
The alpha fetoprotein antibody was pretreated according to the experimental data obtained in examples 1 to 3, and the pretreatment temperature was 25 ℃, the treatment time was 10min, and the concentration of sodium lauryl sulfate was 0.2%, as the test object. Meanwhile, the alpha-fetoprotein antibody which is not pretreated by sodium dodecyl sulfate is used as a control group, and is respectively coupled with activated carboxyl magnetic beads, and then a positive sample of the alpha-fetoprotein antibody is detected, wherein the detection results are shown in table 4:
TABLE 4
It can be seen from the data of P1-P4 in table 4 that, in the process of detecting alpha fetoprotein in human serum by chemiluminescence magnetic particle immunoassay, the coating rate of the alpha fetoprotein antibody can be significantly increased compared with the prior art by performing pretreatment on the alpha fetoprotein antibody with a concentration of 0.2% for 10min at 25 ℃ before coupling the alpha fetoprotein antibody with the carboxyl magnetic beads.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.
Claims (10)
1. A method for coating magnetic beads with alpha fetoprotein antibodies is characterized by comprising the following steps: adding a pretreatment solution into the alpha fetoprotein antibody for pretreatment, and then coupling the pretreated alpha fetoprotein antibody with carboxyl magnetic beads to obtain alpha fetoprotein antibody coupled magnetic beads, wherein the pretreatment solution comprises a sodium dodecyl sulfate solution.
2. A method of claim 1, wherein the method comprises the steps of: the concentration of the sodium dodecyl sulfate solution is 0.2-0.3%, the pretreatment time is 5-10 min, and the pretreatment temperature is 20-25 ℃.
3. A method of claim 2, wherein the method comprises the steps of: the concentration of the sodium dodecyl sulfate solution is 0.2%, the pretreatment time is 10min, and the pretreatment temperature is 25 ℃.
4. A method of claim 1, wherein the method comprises the steps of: the pretreatment solution also comprises MES buffer solution with the concentration of 10 mM-80 mM.
5. A method of claim 1, wherein the method comprises the steps of: before coupling alpha fetoprotein antibody, mixing and activating the carboxyl magnetic beads and a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the concentrations of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide are both 8-12 mg/ml; the activation temperature is 23-28 ℃, and the activation time is 10-20 min.
6. A method of claim 5, wherein the method comprises the steps of: and washing the activated carboxyl magnetic beads in an MES buffer solution for 2-3 times, wherein the concentration of the MES buffer solution is 10-80 mM, the pH is 5.5-7.5, then carrying out mixing coupling on the pretreated alpha-fetoprotein antibody and the washed carboxyl magnetic beads at the temperature of 22-28 ℃, and the mixing coupling time is 1-5 h.
7. A method of claim 6, wherein the method comprises the steps of: and (3) putting the coupled magnetic beads into MES buffer solution containing 0.1% BSA for sealing treatment, wherein the sealing time is 5-8 h, the sealing temperature is 22-28 ℃, and then adding the sealed coupled magnetic beads into the Tris buffer solution for resuspension and preservation, wherein the preservation temperature is 2-8 ℃.
8. A method of claim 1, wherein the method comprises the steps of: the quantity of the alpha fetoprotein antibody coating per milligram of the carboxyl magnetic beads is 10-30 mu g.
9. A coupled magnetic bead, comprising: coupled magnetic beads prepared by the method for coating magnetic beads with alpha-fetoprotein antibodies as claimed in any one of claims 1 to 8.
10. A kit, characterized in that: the kit comprises the alpha-fetoprotein antibody coupling magnetic beads prepared by the method for coating the magnetic beads with the alpha-fetoprotein antibodies of any one of claims 1-8.
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