CN113030461B - IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof - Google Patents
IgG antibody marked by amino group or carboxyl group-containing substance, human IgG magnetic particle chemical detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an IgG antibody and human IgG magnetic particle chemical detection kit containing amino or carboxyl group substance markers and a preparation method thereof, wherein the preparation method of the antibody containing the amino or carboxyl group substance markers comprises the following steps: 1) dissolving the IgG monoclonal antibody in an acidic buffer solution, and then adding pepsin to carry out enzyme digestion reaction; 2) dialyzing the mixed solution after enzyme digestion by using a neutral buffer solution overnight; 3) subjecting the obtained liquid to Protein A affinity purification and DEAE cellulose anion exchange chromatography to obtain IgG-F (ab')2(ii) a 4) Subjecting the obtained liquid to Protein A affinity purification and DEAE cellulose anion exchange chromatography to obtain IgG-F (ab')2(ii) a 5) And covalently bonding the obtained antibody intermediate with a substance containing amino or carboxyl groups to obtain the IgG antibody labeled with the substance containing amino or carboxyl groups. The directional coupling technology of the invention can reduce the influence of complement on the detection reagent and reduce the interference caused by nonspecific combination.
Description
Technical Field
The invention belongs to the field of in-vitro diagnosis, and particularly relates to an IgG antibody labeled by amino or carboxyl group-containing substances, a human IgG magnetic particle chemical detection kit and a preparation method thereof.
Background
The antibody for detection is usually a monoclonal antibody or a polyclonal antibody, and is an IgG. When the antibody binds to the antigen, complement binds to the complement binding site of the Fc fragment of the antibody, which interferes with the detection reagent. In addition, the presence of the Fc fragment may also falsely interfere with the detection reagent.
In addition, covalent coupling is frequently used in current antibody coupling techniques. Since the surface of the antibody contains abundant amino and carboxyl groups, the two groups are also the most used functional groups in antibody coupling. The commonly adopted strategy is to activate carboxyl on the surface of a solid phase such as magnetic microspheres, polystyrene microspheres or enzymes by EDC/NHS, and then form stable amido bond with amino on the surface of the antibody. EDC/NHS is rarely used to activate carboxyl groups on the surface of antibodies, since the surface of antibodies itself has amino groups and is susceptible to self-crosslinking. This conjugation method is also random, since amino and carboxyl groups are ubiquitous on antibody surfaces. When labeled on the complementarity determining region of an antibody, the antibody is inactivated. Even the label is located in the Fc region, the immunological activity is reduced due to the presence of steric hindrance.
Human immunoglobulin G (Human IgG) is mainly synthesized and secreted by plasma cells in the spleen and lymph nodes, and exists in a monomeric form. Immunoglobulin G is the major antibody component in serum and represents approximately 75% of the total Ig in serum. Immunoglobulin G is the only Ig that can pass through the placenta and plays an important role in natural passive immunity. In addition, the immunoglobulin G also has the functions of opsonophagocytosis, ADCC, SPA binding and the like. Because of the above characteristics of igg, igg plays a major role in immune protection in the body, and most of the antibacterial, antiviral, and antitoxin antibodies belong to the class of igg antibodies. The artificial passive immunization can be carried out by applying the C or placental globulin of the lying-in women or normal people with immunity to measles, hepatitis A and the like, and can effectively prevent corresponding infectious diseases. Many autoantibodies such as anti-thyroglobulin antibodies, systemic lupus erythematosus LE factor (antinuclear antibodies) and causing type III allergy immune complexes in the antibody also belong to immunoglobulin G.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide an IgG antibody labeled with an amino group or a carboxyl group, a human IgG magnetic particle chemical detection kit, and a preparation method thereof.
In order to achieve the above object, the present invention provides a method for preparing an IgG antibody labeled with a substance having an amino group or a carboxyl group, comprising the steps of:
1) dissolving the IgG monoclonal antibody in an acidic buffer solution, and then adding pepsin to carry out enzyme digestion reaction;
2) dialyzing the mixed solution subjected to enzyme digestion in the step 1) by using a neutral buffer solution overnight;
3) subjecting the liquid obtained in step 2) to Protein A affinity purification and DEAE cellulose anion exchange chromatography to obtain IgG-F (ab')2；
4) Covalently coupling the IgG-F (ab') 2 obtained in step 3) with HOOC- (CHCHO) n-NH2 to form an antibody intermediate;
5) covalently bonding the antibody intermediate obtained in the step 4) with a substance containing amino or carboxyl groups to obtain the antibody marked by the substance containing amino or carboxyl groups.
In a specific embodiment of the invention, in step 1), the mass ratio of the IgG antibody, pepsin, and acidic buffer to the acidic buffer is (0.5-1): (0.5-1): 10000, preferably 1: 10000. the IgG antibody is a monoclonal antibody capable of specifically binding to an antigen, can be cleaved into F (ab') 2 fragments by pepsin, and retains the activity of binding to the antigen.
In a specific embodiment of the invention, in step 1), the temperature of the enzyme digestion reaction is 37 ℃ to 55 ℃, preferably 37 ℃, which means that the activity of the enzyme is better at 37 ℃; the time of the enzyme digestion reaction is 0.5 to 2 hours. The preferable time is 30min, the reaction degree reaches the highest under the preferable time, the time tends to be stable again, and the reaction can be fully performed within 30min, and the test time can be saved.
In a specific embodiment of the invention, in step 1), the acidic buffer is a buffer with a pH value of 1-7, preferably pH3.0, and preferably enables pepsin to retain the enzyme digestion activity, including but not limited to a citric acid buffer, an acetic acid buffer, a phosphate buffer, a Tris-phosphate buffer, an organic acid buffer, an amino acid buffer, and a MES buffer, preferably a citric acid buffer, so that the pepsin retains the enzyme digestion activity to the maximum after the selection.
In one embodiment of the present invention, in step 2), the neutral dialysis buffer is a buffer with a pH between 6 and 8, including but not limited to phosphate buffer, Tris-hydrochloric acid buffer, barbiturate sodium-hydrochloric acid buffer, boric acid-borax buffer, MES buffer, preferably 0.1M phosphate buffer pH7.0, so that the activity of the dialysis substance can be preferably maintained.
In one embodiment of the present invention, wherein step 2), the cut-off molecular weight of the dialysis is 7000-50000D.
In one embodiment of the invention, in the step 4), n in the HOOC- (CHCHO) n-NH2 is between 3 and 100000, preferably HOOC- (CH CH O)12-NH2, so that the coupling efficiency can be higher after preference.
In one embodiment of the present invention, in the step 4), the mass ratio of IgG-F (ab') 2 to HOOC- (CHCHO) n-NH2 is (0.5-1): 1. the preferred ratio is 1:1, preferably, the antibody can fully react with HOOC- (CHCHO) n-NH2, and the reaction degree is optimal; not only does not waste raw materials, but also can achieve the best combination effect.
In one embodiment of the present invention, in step 5), the mass ratio of the antibody intermediate to the amino or carboxyl group-containing substance is 1: (5-10), preferably 1:6, and when reaching 1:6, the reaction reaches the highest point and tends to be smooth backwards, so that the raw materials can be saved and the reaction degree can reach the peak at 1: 6.
In a specific embodiment of the invention, in step 5), the substance containing amino or carboxyl groups is protein, antibody, antigen, enzyme, amino acid, polystyrene microsphere, quantum dot, magnetic particle, metal particle or colloidal gold, and is not limited by choice but not limited by the amount of raw materials, and preferably metal particle, so that the obtained labeled substance has better dispersibility and separation capability.
In order to achieve the above object, the present invention also provides an antibody labeled with a substance having an amino group or a carboxyl group, which is produced by the above method; the substance containing amino or carboxyl groups is protein, antibody, antigen, enzyme (such as alkaline phosphatase), amino acid, polystyrene microsphere, quantum dot, magnetic particle, metal particle, and colloidal gold.
In order to achieve the above object, the present invention further provides a human IgG magnetic particle chemical detection kit, which comprises biotin-labeled IgG antibody, streptavidin-labeled magnetic bead, and the above IgG antibody labeled with an amino group-or carboxyl group-containing substance, wherein the weight ratio of the biotin-labeled IgG antibody to the streptavidin-labeled magnetic bead to the amino group-or carboxyl group-containing substance is (0.5-1): (0.5-1), preferably 1:1:1, and thus, preferably, the detection target substance can be better bound.
In a specific embodiment of the invention, the human IgG magnetic particle chemical detection kit comprises an incubation solution, a label reagent, a magnetic bead reagent, a substrate, a washing solution, a calibrator and a quality control product, wherein the volume ratio of the incubation solution to the label reagent is (0.5-1): (0.5-1): (0.5-1): (2-4): (2-4): (0.1-0.2): (0.1-0.2), preferably 0.5: 0.5: 0.5: 2: 2: 0.1: 0.1. the optimal post-reaction effect is optimal, the combination is most sufficient, the raw materials are used least, and the cost is saved to the greatest extent.
In a specific embodiment of the invention, the incubation solution is 0.05mol/L Tris buffer solution with biotin-labeled human IgG coated antibody and 0.5 wt% bovine serum albumin, and the pH value is 7.0; the weight ratio of the biotin-labeled human IgG coating antibody to the Tris buffer solution is 1: (1000-3000), preferably 1: 1000. preferably, the biotin-labeled human IgG-coated antibody is sufficiently soluble in the buffer, and the insolubility of the labeled antibody does not affect the subsequent test results.
In a specific embodiment of the invention, wherein the marker reagent is Tris buffer solution with 0.05mol/L, pH7.0, containing IgG antibody marked by substances containing amino or carboxyl groups and 0.5 wt% of bovine serum albumin; the weight ratio of the IgG antibody marked by the substance containing amino or carboxyl groups to the Tris buffer solution is 1: (1000-3000), preferably 1: 1000. preferably, the IgG antibody labeled with the substance containing amino or carboxyl groups can be sufficiently dissolved in the buffer, and the situation that the labeled antibody is insoluble and the subsequent test results are not influenced does not occur.
In one embodiment of the invention, the magnetic bead reagent is Tris buffer solution with 0.05mol/L and pH7.0 containing streptavidin-labeled magnetic beads and 0.5 wt% of bovine serum albumin, and the ratio of the streptavidin-labeled magnetic beads to the Tris buffer solution is 0.5-1mg/ml, preferably 0.5mg/ml, so that the kit linearity can be better by cooperating with other parameters after optimization.
In a specific embodiment of the invention, the substrate is 0.05mol/L Tris buffer with 0.2 wt% of lucigenin and pH 7.0.
In a specific embodiment of the present invention, wherein the washing solution is Tris buffer containing 0.1 wt% Tween-20 and 8 wt% NaCl at 0.05mol/L and pH 7.0.
In a specific embodiment of the invention, the calibrator is 0.05mol/L Tris buffer solution with human IgG antigen and 0.5 wt% bovine serum albumin, and the pH value of the Tris buffer solution is 7.0, and the calibrator comprises five liquid calibrators with human IgG antigen concentrations of 10ng/mL, 100ng/mL, 500ng/mL, 2000ng/mL and 10000ng/mL respectively.
In a specific embodiment of the invention, the quality control substance is a Tris buffer solution with 0.05mol/L and pH7.0 containing human IgG antigen and 0.5 wt% of bovine serum albumin, and the quality control substance comprises two liquid quality control substances with human IgG antigen concentrations of 100ng/ml and 2000ng/ml respectively.
In a specific embodiment of the invention, the linear range of the human IgG magnetic particle chemical detection kit is 10-10000 ng/ml.
In order to achieve the above object, the present invention also provides a preparation method of the human IgG magnetic particle chemical detection kit, comprising the following steps:
1a) preparation of incubation solution: dissolving a biotin-labeled human IgG coating antibody in 0.05mol/L Tris buffer solution containing 0.5 wt% of bovine serum albumin and having the pH value of 7.0 to obtain an incubation solution;
2a) preparation of marker reagents: dissolving the IgG antibody marked by the substance containing the amino group or the carboxyl group in Tris buffer solution which contains 0.5wt percent of bovine serum albumin and has the pH value of 7.0 to obtain a marker reagent;
3a) preparing a magnetic bead reagent: dissolving streptavidin-labeled magnetic beads in Tris buffer solution which contains 0.5 wt% of bovine serum albumin and has the concentration of 0.05mol/L and the pH value of 7.0 to obtain a magnetic bead reagent;
4a) preparation of the substrate: preparing 0.05mol/L Tris buffer solution with pH7.0 and containing 0.2 wt% of lucigenin;
5a) preparation of a washing solution: preparing a Tris buffer solution containing 0.1 wt% of Tween-20 and 8 wt% of NaCl and having a concentration of 0.05mol/L and a pH value of 7.0;
6a) preparing a calibrator and a quality control product: human lgG antigen is respectively dissolved in 0.05mol/L Tris buffer solution with pH7.0 of 0.5 wt% bovine serum albumin according to certain concentration, and calibration substances and quality control substances with the concentration of 10, 100, 500, 2000, 10000ng/mL, 100ng/mL and 2000ng/mL are prepared in sequence.
In a specific embodiment of the present invention, wherein in step 1a), the weight ratio of the biotin-labeled human IgG-coated antibody to Tris buffer is 1: 1000-3000; in the step 2a), the weight ratio of the IgG antibody marked by the substance containing amino or carboxyl groups to the Tris buffer solution is 1: 1000-3000; in the step 3a), the ratio of the streptavidin-labeled magnetic beads to the Tris buffer solution is 0.5-1 mg/ml.
In order to achieve the above object, the present invention also provides a method for using the human IgG magnetic particle chemical detection kit, wherein the sample used in the method is selected from human serum and plasma.
The antibody marked by the substance containing amino or carboxyl groups, the human IgG magnetic particle chemical detection kit containing the antibody and the preparation method thereof have the following beneficial effects:
1. the invention can reduce the influence of complement on the detection reagent and reduce the interference caused by nonspecific combination through directional coupling.
2. The invention can reduce the steric hindrance of the labeled antibody and improve the activity of the labeled antibody by the directional coupling technology.
3. The invention can improve the detection sensitivity and specificity of the original antibody through directional coupling.
4. The invention can reduce the antibody dosage and save the cost through the directional coupling.
Drawings
FIG. 1 is a graph of a standard human IgG sample of the present invention;
FIG. 2 is a ROC curve interface of the kit obtained in example 2 of the present invention for performing human IgG detection on 40 different samples (20 normal persons and 20 positive patients);
FIG. 3 is a ROC curve interface of the kit obtained in comparative example 1 according to the present invention for human IgG detection of 40 different samples (20 normal persons and 20 positive patients).
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The following materials or reagents, unless otherwise specified, are commercially available.
Example 1:
1. preparation of human IgG magnetic particle chemical detection reagent
1) Preparation of IgG-F (ab') 2
10mg of human IgG monoclonal antibody (purchased from Nanjing Keotai) was diluted to 2mg/ml with 0.1M citric acid buffer pH 3-5. Then, the mixture was dialyzed for 6 hours against 0.1M citric acid buffer (cut-off 7000D) at pH3-5 under stirring and then placed in a vial.
0.5mg of pepsin is added into a penicillin bottle, stirred and mixed evenly, and then incubated for 3 hours at 37 ℃. It was then dialyzed against PBS (molecular weight cut-off 5 kilomillion D) at 0.1M, pH8.0 for 24 hours.
The Protein A affinity column was equilibrated with 0.1M PB (pH7.0) buffer at a flow rate of 1ml/min, a volume of >20ml, to an OD280< 0.01. The dialyzed sample was loaded at a flow rate of 1ml/min, and the flow-through was collected and the volume was recorded. And washing the loaded chromatographic column with 0.1M PB (pH7.0) buffer solution at the flow rate of 2ml/min and the volume of 50ml until OD280 is less than 0.01, and reserving liquid for testing. Eluting the loaded chromatographic column with 0.1M citric acid (pH3.0) buffer solution at a flow rate of 1ml/min and an unlimited volume, collecting the eluate in tubes while adjusting the pH to about 7.0, eluting until the OD280 is less than 0.01, retaining the eluate, finally measuring the OD280 with a spectrophotometer to calculate the primary yield, placing the obtained sample in a dialysis bag, dialyzing with 0.01M PBS (pH7.0-7.5) for 24h, fully dialyzing, changing the solution for 2-3 times, and collecting the dialyzed sample. Weighing the DEAE with the required amount according to the proportion of 100mg/100mg, soaking the DEAE in distilled water overnight and changing water for 3 times to remove small particles, then pumping out, soaking in 0.5mL/L NaOH for 1-2 hours, pumping out, washing with double distilled water for 2-3 times, adjusting the pH to about 8.0, soaking in 0.5mL/L HCl for 1-2 hours, pumping out, washing with double distilled water for 2-3 times, and adjusting the pH to about 6.0. Soaking the treated DEAE-cellulose in 0.01mol/L phosphate buffer solution with pH of 6 for 1-3 hr, changing the solution for 1-2 times, and stirring to fill into the chromatographic column until the height of the column is about 17 cm. After the bed was prepared, the column was washed with 0.01mol/L phosphate buffer by air washing until the pH of the effluent was completely the same as the pH of the phosphate buffer. After the balance is finished, closing the lower opening, sucking liquid in the column, which is more than the lower opening of the column, controlling the flow rate to enable the sample to slowly enter the column, and adding a layer of phosphate buffer solution with the pH value of 6 and 0.1mol/L on the column bed after the sample completely enters the column. And opening the lower opening of the column for elution after the sample loading is finished, controlling the flow rate to be 0.5ml/min, collecting eluent according to 10 drops of each tube, taking 1 drop of the collected liquid from each tube, just putting the collected liquid into a black color comparison hole, adding 1 drop of 20 wt% yellow salicylic acid to check whether white precipitates are generated, wherein the white precipitates firstly appear in the collected liquid under the condition that the collected liquid is purified lgG until no white precipitates appear in the collected liquid.
The sample was affinity-purified by a Protein A/G antibody purification column (10 column volumes) with 1mL of sample per time, and the penetrated sample was collected and purified by DEAE cellulose anion exchange chromatography to obtain IgG-F (ab') 2. The obtained IgG-F (ab ') 2 sample is analyzed by 3 wt% gel electrophoresis, the molecular weight of the obtained IgG-F (ab') 2 sample is about 110KD, and the purity is more than or equal to 90%.
2) Preparation of antibody intermediates
25mg of HOOC- (CHCCHO)12-NH2 was added to 1ml of DMSO and mixed uniformly, then 21.42. mu.l of the previously obtained mixed solution was added to 1ml of 1mg/ml of the above IgG-F (ab') 2, and 5mg of carbodiimide was added thereto, and the mixture was stirred at room temperature for 1 hour, and finally dialyzed against 0.01M of PBS (pH 7.2) for 24 hours (molecular weight cut-off: 7000D) to obtain 1mg/ml of an antibody intermediate solution.
3) Preparation of alkaline phosphatase-labeled IgG antibody
1ml of alkaline phosphatase (1 mg/ml) was added to 1mg/ml of carbodiimide (EDC), 1mg of N-hydroxysuccinimide (NHS) and stirred at room temperature for 3 hours at 30r/min, then added to the above 1ml of 1mg/ml antibody intermediate solution and stirred at room temperature for 1 hour at 30r/min, followed by blocking with 10mg of BSA and stirring continued for 1 hour.
Finally dialyzing in 0.01M PBS (pH 7.2) for 24 hours (molecular weight cut-off: 10 WanD), placing in a penicillin bottle, adding 5 μ L Proclin300, adding 1 wt% BSA and 1ml glycerol, mixing, and storing at-20 deg.C.
4) Preparation of Biotin-labeled IgG-coated antibody
Taking 1mg of human IgG-coated antibody, adding 0.01M PBS (0.01M PBS) labeled buffer solution of pH7.2, and dialyzing for 30 min; dissolving esterified biotin 1mg in 0.1mL of purified water; the two were mixed and reacted at room temperature for 1 hour.
The solution after 1 hour of reaction was transferred to a dialysis bag (cut-off of 7000D), and dialyzed with 0.01M, pH7.2PBS buffer for 24 hours with 1-time intermediate exchange.
Transferring the dialyzed solution into a bottle, adding 1 wt% BSA and 1ml glycerol, mixing, and storing at-20 deg.C.
5) Preparation of streptavidin-labeled magnetic microparticles
Taking 300mg of streptavidin, adding 30mL of purified water, and preparing into 10 mg/mL; 300mg of 10mg/mL magnetic microparticles purchased from Saimerfei were washed repeatedly 5 times with 0.02M PBS buffer, and then diluted to 100mg/mL with 3mL of 0.02M PBS buffer; finally, the two are mixed, 30mg of carbodiimide is added, and the mixture is uniformly mixed again and reacts for 1 hour at room temperature.
After the reaction, the reaction mixture was washed 5 times with 0.02M PBS buffer, and finally diluted again to 100mg/mL with 1 wt% bovine serum albumin, pH7.0, 0.02M Tris buffer.
Example 2:
1. preparation of human IgG magnetic particle chemical detection kit
1) Preparation of IgG-F (ab') 2
10mg of human IgG monoclonal antibody (purchased from Nanjing Keotai) was diluted to 2mg/ml with 0.1M citric acid buffer pH 3-5. Then, the solution was dialyzed for 6 hours in 0.1M citric acid buffer (pH 3-5) with stirring (molecular weight cutoff 7000D) and then taken out and placed in a vial.
0.5mg of pepsin is added into a penicillin bottle, stirred and mixed evenly, and then incubated for 3 hours at 37 ℃. Then, it was dialyzed against 0.1M, pH8.0PBS for 24 hours (molecular weight cut-off: 5 ten thousand D).
The Protein A affinity column was equilibrated with 0.1M PB (pH7.0) buffer at a flow rate of 1ml/min, a volume of >20ml, to an OD280< 0.01. The dialyzed sample was loaded at a flow rate of 1ml/min, and the flow-through was collected and the volume was recorded. And washing the loaded chromatographic column with 0.1M PB (PH7.0) buffer solution at the flow rate of 2ml/min and the volume of more than 50ml until OD280 is less than 0.01, and reserving liquid for testing. Eluting the loaded chromatographic column with 0.1M citric acid (pH3.0) buffer solution at a flow rate of 1ml/min and an unlimited volume, collecting the eluate in tubes while adjusting pH to about 7.0, eluting until OD280 is less than 0.01, retaining the eluate, finally measuring OD280 with a spectrophotometer, calculating the primary yield, placing the obtained sample in a dialysis bag, dialyzing with 0.01M PBS (pH7.0) for 20-24 hours, changing the solution for 2-3 times, and collecting the dialyzed sample. Weighing the DEAE with the required amount according to the proportion of 75-122mg/100mg, soaking the sample and the DEAE in distilled water overnight, changing water for 2-3 times to remove small particles, then pumping out, soaking in 0.5mL/L NaOH for 1-2 hours, pumping out, washing with double distilled water for 2-3 times, adjusting the pH value to about 8.0, soaking in 0.5mL/L HCl for 1-2 hours, pumping out, washing with double distilled water for 3 times, and adjusting the pH value to about 6.0. Soaking the treated DEAE-cellulose in 0.01mol/L phosphate buffer solution with pH of 7 for 1-3 hr, changing the solution for 1-2 times, and stirring to fill into the chromatographic column until the height of the column is about 17 cm. After the bed was prepared, the column was washed with 0.01mol/L phosphate buffer by air washing until the pH of the effluent was completely the same as that of the phosphate buffer. After the balance is finished, closing the lower opening, sucking liquid in the column, which is more than the lower opening of the column, controlling the flow rate to enable the sample to slowly enter the column, and adding a layer of phosphate buffer solution with the pH value of 7 and the concentration of 0.1mol/L on the column bed after the sample completely enters the column. And opening the lower opening of the column for elution after the sample loading is finished, controlling the flow rate to be 0.5ml/min, collecting eluent according to 10 drops of each tube, taking 1 drop of the collected liquid from each tube, just putting the collected liquid into a black color comparison hole, adding 1 drop of 20 wt% yellow salicylic acid to check whether white precipitates are generated, wherein the white precipitates firstly appear in the collected liquid under the condition that the collected liquid is purified lgG until no white precipitates appear in the collected liquid.
The sample was affinity-purified by a Protein A/G antibody purification column (10 column volumes) with 1mL of sample per time, and the penetrated sample was collected and purified by DEAE cellulose anion exchange chromatography to obtain IgG-F (ab') 2. The obtained sample is analyzed by 5 wt% gel electrophoresis, the molecular weight of the obtained sample is about 110KD, and the purity is more than or equal to 90%.
2) Preparation of antibody intermediates
25mg of HOOC- (CHCCHO)16-NH2 was added to 1ml of DMSO and mixed uniformly, then 21.42. mu.l of the above mixed solution was added to 1ml of 1mg/ml IgG-F (ab') 2, and 5mg of carbodiimide was added thereto, and the mixture was stirred at room temperature for 1 hour, and finally dialyzed against 0.01M PBS (pH 7.2) for 24 hours (molecular weight cut-off 7000D), to obtain an antibody intermediate solution.
3) Preparation of alkaline phosphatase-labeled IgG antibody
1ml of alkaline phosphatase (1 mg/ml) was added to 1mg/ml of carbodiimide (EDC), 1mg of N-hydroxysuccinimide (NHS) and stirred at room temperature for 3 hours at 30r/min, then added to 1mg/ml of the antibody intermediate solution and stirred at room temperature for 1 hour at 30r/min, followed by blocking with 10mg of BSA and stirring continued for 1 hour.
Finally dialyzing in 0.01M PBS (pH 7.2) for 24 hours (molecular weight cut-off is 10 ten thousand D), placing in a penicillin bottle, adding 5 microliter of Proclin300, adding 1 wt% BSA and 1ml glycerol, mixing, and storing at-20 deg.C.
4) Preparation of Biotin-labeled IgG antibodies
Taking 1mg of human IgG coated antibody, and dialyzing in PBS (0.01M, pH7.2) for 30 min; dissolving esterified biotin 1mg in 0.1mL of purified water; the two were mixed and reacted at room temperature for 1 hour.
The solution after 1 hour of reaction was transferred to a dialysis bag (cut-off molecular weight of 7000D), and dialyzed with 0.01M PBS buffer solution (pH7.2) for 24 hours with 1-time intermediate exchange.
Transferring the dialyzed solution into a bottle, adding 10g of 1 wt% BSA and 1ml glycerol, mixing uniformly, and storing at-20 ℃.
5) Preparation of streptavidin-labeled magnetic microparticles
Taking 300mg of streptavidin, adding 30mL of purified water, and preparing into 10 mg/mL; 300mg of 10mg/mL magnetic microparticles purchased from semer fly were washed repeatedly 5 times with 0.02M PBS buffer, and then diluted to 100mg/mL with 3mL of 0.02M PBS buffer; finally, the two are mixed, 30mg of carbodiimide is added, and the mixture is uniformly mixed again and reacts for 1 hour at room temperature.
After the reaction, the reaction mixture was washed 5 times with 0.02M PBS buffer, and finally diluted again to 100mg/mL with 1 wt% bovine serum albumin, pH7.0, 0.02M Tris buffer.
2. Detection of
Adding the biotin-labeled coating antibody obtained in example 1 and the alkaline phosphatase-labeled antibody into a marker diluent according to the volume ratio of 1:1000 and 1:3000 respectively; 0.1mL of streptavidin-labeled magnetic microparticle solution with a concentration of 100mg/mL was added to 19.9mL of magnetic microparticle diluent to prepare a working concentration of 0.5 mg/mL.
Marker dilutions were prepared as shown in table 1 below.
TABLE 1 formulation of marker dilutions
(2) According to the sample adding mode: after 10 μ L of calibrator is pre-diluted 500 times, 10 μ L +50 μ L of incubation solution +50 μ L of marker → incubation for 15min, +50 μ L of magnetic particle reagent → incubation for 5min, and washing 5 times; → 200. mu.L of luminescent liquid, and detection.
Placing a sample to be detected (centrifuging at 3000 r/min for 3min after taking the sample from a blood collection tube, separating to obtain serum, namely the sample to be detected) into a test tube rack, placing into a full-automatic chemiluminescence determinator, simultaneously placing a magnetic bead reagent, an incubation solution and a marker reagent of the kit into the full-automatic chemiluminescence determinator, arranging program selection items, detecting for 30 minutes to obtain a detection result, and obtaining the detection result on a detection record page.
3. Establishment of a Standard Curve
Preparing human IgG standard substances (0, 10, 100, 500, 2000, 10000ng/mL) with different concentrations, detecting by using the human IgG kit prepared in the embodiment 2 of the invention, wherein the detection result is shown in Table 2, the detection range is 10-5000 ng/mL, four parameters are adopted for curve fitting, and the standard curve is as follows: y ═ 8011604.0000-1532.0400)/(1+ (X/16443.3800) ^ -1.0467) +1532.0400, and the correlation coefficient r ═ 1.0000, as shown in fig. 1. As can be seen from Table 2 and FIG. 1, as the concentration increases, the signal value (light emission value) also increases, and S2 is higher than S0 by an order of magnitude, which proves that the signal-to-noise ratio is better, the fitting curve is better, and the data is reliable.
TABLE 2 test data
4. Performance evaluation
The kit obtained in example 2 was used to test 40 different samples (20 normal persons and 20 positive patients), and then the specificity of the kit was 100%, the sensitivity was 100%, the CUTOFF value CUTOFF was 65.67ng/ml, and the AUC was 1.0000 by Medcalc ROC curve analysis, as shown in FIG. 2. The specific data are shown in Table 3. The kit is proved to have high sensitivity and small accurate error in measurement.
TABLE 3
| Type of sample | Normal human (ng/ml) | Positive patient (ng/ml) |
| Serum | 40.81 | 334.72 |
| Serum | 62.34 | 739.43 |
| Serum | 52.06 | 107.45 |
| Serum | 59.22 | 796.11 |
| Serum | 49.69 | 873.98 |
| Serum | 56.25 | 814.38 |
| Serum | 37.4 | 278.34 |
| Serum | 40.33 | 729.61 |
| Serum | 51.91 | 961.18 |
| Serum | 54.62 | 211.07 |
| Serum | 32.17 | 101.64 |
| Serum | 65.67 | 710.97 |
| Serum | 60.79 | 621.71 |
| Serum | 54.06 | 430.6 |
| Serum | 29.14 | 171.41 |
| Serum | 29.64 | 941.18 |
| Serum | 47.58 | 905.15 |
| Serum | 34.47 | 796.85 |
| Serum | 42.28 | 693.36 |
| Serum | 44.06 | 539.92 |
The kit obtained in example 2 was used to test 40 different samples (20 normal persons and 20 positive patients) prepared by the non-directional coupling technique of comparative example 1, wherein the detection was performed by Medcalc analysis, and no significant difference was observed between the clinical sample and the normal persons before the non-directional coupling labeling, and the sensitivity of the kit of comparative example 1 was 55%, the specificity was 100%, the cut-off value CUTOFF was 25.26, and the AUC was 0.783, whereas the detection was performed by the non-directional coupling technique of comparative example 1, and the sensitivity of the kit of example 2 was 100%, the specificity was 100%, the cut-off value CUTOFF was 65.97, and the AUC was 1.000, as shown in FIGS. 2 and 3, and the specific data are shown in Table 4. Comparing the two groups of analysis results, the result of the non-directional coupling in the comparative example 1 is found to be inaccurate due to steric hindrance, and the basic performance of the kit cannot be met.
TABLE 4
The above-mentioned embodiments are merely examples provided to fully illustrate the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (8)
1. A human IgG magnetic particle chemical detection kit is characterized by comprising an IgG coated antibody marked by biotin, a magnetic bead marked by streptavidin and an IgG antibody marked by a substance containing amino or carboxyl groups, wherein the weight ratio of the IgG coated antibody marked by biotin to the magnetic bead marked by streptavidin to the IgG antibody marked by a substance containing amino or carboxyl groups is (0.5-1) to (0.5-1);
the IgG antibody marked by the substance containing amino or carboxyl groups is prepared by the following steps:
1) dissolving the IgG monoclonal antibody in an acidic buffer solution, and then adding pepsin to carry out enzyme digestion reaction; the mass ratio of the IgG antibody, the pepsin, the acidic buffer solution is (0.5-1): (0.5-1): 10000; the temperature of the enzyme digestion reaction is 37-55 ℃; the time of the enzyme digestion reaction is 0.5 to 2 hours;
2) dialyzing the mixed solution subjected to enzyme digestion in the step 1) by using a neutral buffer solution overnight;
3) subjecting the liquid obtained in step 2) to Protein A affinity purification and DEAE cellulose anion exchange chromatography to obtain IgG-F (ab')2；
4) Covalently coupling the IgG-F (ab') 2 obtained in step 3) with HOOC- (CHCHO) n-NH2 to form an antibody intermediate; n in the HOOC- (CHCHO) n-NH2 is 12-16; the mass ratio of the IgG-F (ab') 2 to the HOOC- (CHCHO) n-NH2 is (0.5-1): 1;
5) covalently combining the antibody intermediate obtained in the step 4) with a substance containing amino or carboxyl groups to obtain an IgG antibody marked by the substance containing amino or carboxyl groups; the mass ratio of the antibody intermediate to the substance containing amino or carboxyl groups is 1: (5-10).
2. The human IgG magnetic particle chemical detection kit of claim 1, wherein the human IgG magnetic particle chemical detection kit comprises an incubation solution, a marker reagent, a magnetic bead reagent, a substrate, a washing solution, a calibrator and a quality control product, and the volume ratio of the incubation solution to the marker reagent is (0.5-1): (0.5-1): (0.5-1): (2-4): (2-4): (0.1-0.2): (0.1-0.2).
3. The kit for chemical detection of human IgG magnetic particles according to claim 2, wherein the incubation solution is Tris buffer solution of 0.05mol/L, pH7.0, containing biotin-labeled human IgG-coated antibody and 0.5 wt% bovine serum albumin; the weight ratio of the biotin-labeled IgG coating antibody to the Tris buffer solution is 1: (1000-3000); the marker reagent is Tris buffer solution with 0.05mol/L, pH7.0, containing IgG antibody marked by amino-group-containing or carboxyl-group-containing substances and 0.5 wt% of bovine serum albumin; the weight ratio of the IgG antibody marked by the substance containing amino or carboxyl groups to the Tris buffer solution is 1: (1000-3000); the magnetic bead reagent is a Tris buffer solution with 0.05mol/L and pH7.0, and contains streptavidin marked magnetic beads and 0.5 wt% of bovine serum albumin, and the ratio of the streptavidin marked magnetic beads to the Tris buffer solution is 0.5-1 mg/ml.
4. The kit for the chemical detection of magnetic particles of human IgG according to claim 3, wherein said substrate is Tris buffer containing 0.2 wt% lucigenin at 0.05mol/L, pH 7.0; the washing solution is Tris buffer solution which contains 0.1 wt% of Tween-20 and 8 wt% of NaCl and is 0.05mol/L and pH7.0; the calibrator is a Tris buffer solution with the pH value of 7.0 and the concentration of 0.05mol/L containing human IgG antigen and 0.5 wt% of bovine serum albumin, and the calibrator comprises five liquid calibrators with the concentrations of the human IgG antigen of 10ng/mL, 100ng/mL, 500ng/mL, 2000ng/mL and 10000ng/mL respectively; the quality control product is Tris buffer solution with 0.05mol/L and pH7.0 containing human IgG antigen and 0.5 wt% bovine serum albumin, and comprises two liquid quality control products with human IgG antigen concentrations of 100ng/ml and 2000ng/ml respectively.
5. The kit for chemical detection of human IgG magnetic particles according to claim 3, wherein the substance containing amino or carboxyl groups is a protein, an antibody, an antigen, an enzyme, an amino acid, a polystyrene microsphere, a quantum dot, a magnetic particle, a metal particle, or colloidal gold.
6. The kit for chemical detection of human IgG magnetic particles according to claim 1, wherein in step 1), the pH value of the acidic buffer is between 1 and 7, and the acidic buffer is selected from one of a citric acid buffer, an acetic acid buffer, a phosphate buffer, a Tris-phosphate buffer, an organic acid buffer, an amino acid buffer and a MES buffer.
7. The kit for chemical detection of human IgG magnetic particles according to claim 1, wherein in step 2), the pH value of the neutral dialysis buffer is between 6 and 8, and the neutral dialysis buffer is phosphate buffer, Tris-hydrochloric acid buffer, barbital sodium-hydrochloric acid buffer, boric acid-borax buffer or MES buffer.
8. The kit for chemical detection of human IgG magnetic particles according to claim 1, wherein in step 5), the substance containing amino or carboxyl groups is a protein, an antibody, an antigen, an enzyme, an amino acid, polystyrene microspheres, quantum dots, magnetic particles, metal particles, or colloidal gold.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101529251A (en) * | 2006-11-01 | 2009-09-09 | 贝克曼考尔特公司 | Binding surfaces for affinity assays |
| CN104198723A (en) * | 2014-08-11 | 2014-12-10 | 南京普朗医疗设备有限公司 | Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm |
| CN104498576A (en) * | 2014-12-05 | 2015-04-08 | 重庆乾德生物技术有限公司 | Immobilized enzyme digestion based preparation method of Fab antibody and application thereof |
| CN107764992A (en) * | 2017-10-16 | 2018-03-06 | 南京诺唯赞医疗科技有限公司 | The orientation coupling method of a kind of microballoon and antibody and application |
| CN109970850A (en) * | 2019-04-03 | 2019-07-05 | 北京纳晶生物科技有限公司 | The method of purification tag antibody |
| CN110922476A (en) * | 2019-12-16 | 2020-03-27 | 蓝怡科技集团股份有限公司 | Biotin coupled antibody and preparation method and application thereof |
| CN111089965A (en) * | 2019-11-18 | 2020-05-01 | 宁波瑞源生物科技有限公司 | Antigen detection kit, preparation method thereof and antigen detection method |
| CN111707835A (en) * | 2020-07-01 | 2020-09-25 | 珠海丽珠试剂股份有限公司 | Composition for detecting IgG4, kit, application and detection method |
-
2021
- 2021-03-12 CN CN202110271943.8A patent/CN113030461B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101529251A (en) * | 2006-11-01 | 2009-09-09 | 贝克曼考尔特公司 | Binding surfaces for affinity assays |
| CN104198723A (en) * | 2014-08-11 | 2014-12-10 | 南京普朗医疗设备有限公司 | Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm |
| CN104498576A (en) * | 2014-12-05 | 2015-04-08 | 重庆乾德生物技术有限公司 | Immobilized enzyme digestion based preparation method of Fab antibody and application thereof |
| CN107764992A (en) * | 2017-10-16 | 2018-03-06 | 南京诺唯赞医疗科技有限公司 | The orientation coupling method of a kind of microballoon and antibody and application |
| CN109970850A (en) * | 2019-04-03 | 2019-07-05 | 北京纳晶生物科技有限公司 | The method of purification tag antibody |
| CN111089965A (en) * | 2019-11-18 | 2020-05-01 | 宁波瑞源生物科技有限公司 | Antigen detection kit, preparation method thereof and antigen detection method |
| CN110922476A (en) * | 2019-12-16 | 2020-03-27 | 蓝怡科技集团股份有限公司 | Biotin coupled antibody and preparation method and application thereof |
| CN111707835A (en) * | 2020-07-01 | 2020-09-25 | 珠海丽珠试剂股份有限公司 | Composition for detecting IgG4, kit, application and detection method |
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