CN112142838A - Caripropa artificial antigen and polyclonal antibody and application thereof - Google Patents
Caripropa artificial antigen and polyclonal antibody and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K16/065—Purification, fragmentation
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9473—Anticonvulsants, e.g. phenobarbitol, phenytoin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
Abstract
The invention discloses a carisoprodol artificial antigen and multiple antibodies and application thereof. A Caripropol artificial antigen KAL-BSA is characterized in that the Caripropol is chemically modified and coupled with the BSA to obtain an artificial antigen which can induce the proliferation and differentiation of B cells and then generate specific antibodies. The anti-Carlipp polyclonal antibody is obtained by collecting animal serum of immune Carlipp polyclonal artificial antigen KAL-BSA, and performing two-step affinity chromatography. The modified and coupled KAL-BSA immune antigen has good specificity, can stimulate animals to generate a large amount of anti-Carlipp polyclonal antibodies, but not other unnecessary antibodies, and provides convenience for subsequent purification. The anti-Carlipp polyclonal antibody is purified by a BSA affinity column and a BGG affinity column, the sensitivity is further improved (the detection limit can reach 100ng/ml) under the condition of not greatly reducing the antibody titer sensitivity, and the purity is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a carisoprodol artificial antigen and multiple antibodies and application thereof.
Background
Cariprodal (Carisoprodol) is a muscle relaxant drug, and is mainly used for treating chronic back pain. Caripropat is a clinical prescription drug. In humans, carisoprodol is converted by CYP2C19 enzyme by N-dealkylation (N-dealkylation) into meprobamate, its active metabolite. The rate of metabolism of carisoprodol to its active metabolite, meprobamate, is related to CYP2C19 enzyme polymorphism. The polymorphic properties of the CYP2C19 gene affect the pharmacokinetics of carpiprole.
The molecular formula of carisoprodol: c12H24N2O4Molecular weight: 260.33, CAS RN 78-44-4, having the molecular structure of formula (I):
formula (I)
There are currently few reports of abuse of carphedon, but attention is still paid, especially to those patients prone to substance dependence. Although carphedon has proven to be effective in treating back pain and activating patients, its potential for abuse and its toxic side effects of overdosing have also incurred many criticalities. Common side effects of carisoprodol include dizziness, somnolence, nausea, and psychological damage.
At present, no rapid diagnosis test paper for detecting the carpipropan, no carpipropan immune antigen and anti-carpipropan polyclonal antibody with high titer and strong specificity are available on the market.
Disclosure of Invention
The first purpose of the invention is to provide a carphedon artificial antigen aiming at the defects of the prior art.
The kalipropan has small molecular weight and does not have immunogen, namely lacks T cell epitope and can not induce animal organisms to generate specific antibodies, so the kalipropan is modified and coupled with BSA (bovine serum albumin) to obtain the artificial antigen which can induce the proliferation and differentiation of B cells and further generate the specific antibodies.
The synthesis method of the KAL-BSA (Artificial antigen of Carlipduo) comprises the following steps:
step (1), balancing carbodiimide EDC cross-linking agent to room temperature;
adding BSA (bovine serum albumin) into a coupling buffer solution to obtain the coupling buffer solution loaded with the BSA; wherein the coupling buffer solution is 0.1M MES, and the pH value is 4.8-5.0;
dissolving the carpolippon hapten into a coupling buffer solution loaded with BSA (bovine serum albumin) so as to introduce a carrier protein BSA;
adding EDC into the coupling buffer solution mixed by the Carlipp hapten and the BSA;
reacting at room temperature for 2 hours, and purifying the coupled protein by using a desalting column to obtain KAL-BSA;
and (6) sterile filtering, and storing at-20 ℃.
It is a second object of the present invention to provide a high titer, highly specific anti-carisoprodonic antibody. In the process of immunizing New Zealand big ear rabbits with KAL-BSA, not only the anti-Carlipp polyclonal antibody is generated, but also the anti-BSA antibody and other hybrid proteins are generated, and the specificity and purity of the final antibody are influenced.
The invention adopts an innovative two-step affinity chromatography, can further improve the specificity and purity of the anti-Carlpox polyclonal antibody without greatly influencing the titer of the anti-Carlpox polyclonal antibody, and can be better applied to the detection of the Carlpox colloidal gold immunodiagnosis test strip.
The preparation method of the anti-carisop polyclonal antibody comprises the following steps:
diluting an artificial antigen KAL-BSA, mixing the diluted artificial antigen with an adjuvant, and then immunizing an animal for multiple times;
step (2), collecting serum of immune animals, measuring the titer of the serum by ELISA, continuing to immunize if the titer does not reach the standard, and purifying the serum in the next step if the titer reaches the standard;
preparing an affinity column coupled with BSA macromolecules and an affinity column coupled with BGG macromolecules;
specifically, 100ml of GE-Sepharose-4B filling glue is activated by hydrogen bromide and then is equally divided into two parts; respectively dissolving BSA and BGG protein in 0.01M carbonate buffer (pH 9.6), immediately adding into activated filling gel, stirring well, and standing overnight; blocking was then performed with ethanolamine and the equilibrium was washed with 0.01M PBS.
Purifying the serum by ammonium sulfate to obtain an antibody semi-finished product;
step (5), enabling the obtained antibody semi-finished product to pass through a BSA (bovine serum albumin) affinity column and a BGG (bovine serum albumin) affinity column, collecting flow-through liquid, and measuring titer and inhibition rate by ELISA (enzyme-linked immuno sorbent assay) in the middle;
and (6) dialyzing the concentrated flow-through liquid for multiple times, collecting the volume of the measured concentration, and storing at-20 ℃.
The third purpose of the invention is to provide the application of the anti-Cariprep polyclonal antibody in preparing a Cariprep polyclonal detection kit; or in the preparation of test strips for the detection of carpriptan.
Further, the colloidal gold immunodiagnosis test strip for detecting the carphedron comprises a sample pad, a marker pad, a reaction membrane and a sample sucking pad; the marker pad is coated with the colloidal gold-labeled polyclonal antibody against the carisoprodol, the detection line (T line) of the reaction membrane is coated with a carisoprodol-bovine serum albumin complex, and the quality control line (C line) of the reaction membrane is coated with a secondary antibody.
The test strip is prepared by a competitive inhibition principle, and the coating amount of the Cariprep-bovine serum albumin compound is also suitable for the detection limit of the anti-Cariprep polyclonal antibody on Cariprep. During detection, if only C line is developed, the sample to be detected contains the carphedon and the content of the carphedon detected is higher than the detection limit; if the C line and the T line are both colored, the content of the carisoprodol in the sample to be detected is lower than the detection limit, or the sample to be detected does not contain the carisoprodol; if the C line and the T line do not develop color, the test strip is failed.
Compared with the prior art, the invention has the beneficial effects that:
1. the modified and coupled KAL-BSA immune antigen has good specificity, can stimulate animals to generate a large amount of anti-Carlipp polyclonal antibodies, but not other unnecessary antibodies, and provides convenience for subsequent purification.
2. The anti-carriepridopril antibody obtained by immunization is purified by the BSA affinity column and the BGG affinity column, so that the sensitivity (the detection limit can reach 100ng/ml) is further improved and the purity is improved under the condition that the titer sensitivity of the antibody is not greatly reduced.
Drawings
FIG. 1 shows a test strip for detecting Cariprodione colloidal gold.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments.
Example 1 preparation of Caripropat Immunity antigen
1. Solution preparation
(1) A carrier protein: 10mgBSA (bovine serum Albumin), purchased from SIGMA
(2) Coupling buffer: 0.1M MES pH4.8-5.0
(3).EDC 50mg
(4) A hapten: caripropa 10mg
(5) Desalting column
2. Procedure of experiment
(1) EDC was equilibrated to room temperature, 10mg BSA was added to 1000ul coupling buffer and stirred well until completely dissolved.
(2) Dissolve 10mg of the carpropods half-antigen to 2500ul of the coupling buffer, stir well, add to 1000ul of carrier protein, and continue stirring.
(3) 50mg of EDC was dissolved in 5ml of ultrapure water, 500. mu.l of the solution was added to the carrier-hapten solution immediately after the dissolution, and the state of the solution was observed, and if precipitation occurred, the amount of EDC used was reduced.
(4) The reaction was carried out at room temperature for 2 hours, followed by centrifugation at 12000 for 15 minutes to take the supernatant, and the conjugated protein was purified by using a desalting column.
(5) Dialyzing and collecting samples, centrifuging the solution 12000, taking supernatant, measuring the concentration, performing sterile filtration, and storing at-20 ℃.
EXAMPLE 2 preparation of anti-Carlipp polyclonal antibody
1. Animal immunization and serum collection
(1-1) the invention needs healthy New Zealand big ear rabbits, purchased from professional cooperative society of Hangzhou Kefeng rabbit industry, more than 8 weeks.
(1-2) the adjuvants required by the invention are Freund's complete adjuvant and Freund's incomplete adjuvant (SIGMA, cat # F5506-10ml), and the Freund's complete adjuvant and the immunogen are mixed according to the volume ratio of 1:1 for the primary immunization, and the immunization amount is 500 ug. Freund's incomplete adjuvant was subsequently used, and the immunization dose was 500 ug.
The adjuvant is a non-specific immunopotentiator, and the mechanism of the adjuvant for enhancing the immune response is to prolong the retention time of the antigen in the body by changing the physical shape of the antigen; stimulating the antigen presenting ability of mononuclear phagocytes; stimulate lymphocyte differentiation and increase the ability of amplifying immune response. When injected with an antigen or pre-injected into a body, the immune response of the body to the antigen can be enhanced or the type of immune response can be altered. There are a wide variety of adjuvants; such as aluminum hydroxide adjuvant, corynebacterium parvum, lipopolysaccharide, cytokine, alum, etc. Freund's complete adjuvant and Freund's incomplete adjuvant are currently the most commonly used adjuvants in animal testing.
(1-3) the invention adopts subcutaneous 5-point immunization of the back, the four needles are separated by one week before the immune cycle, the two weeks after the immune cycle, and a large amount of anti-carisoprodol antibody can be continuously obtained in the later period.
The interval time between two injections is appropriate, too short to achieve the effect of re-reaction, and too long to lose the sensitivity of the previous excitation. Preferably, the four needles before each immunization are shortened to be separated by one week, so that the immune response reaction can be rapidly triggered, the titer is rapidly improved, and the time for preparing the antibody is shortened.
(1-4) after one week of immunizing antigen, rabbit ear arterial blood was collected using a 3ml syringe and 6 ml blood was collected at a time. Standing in refrigerator at 4 deg.C for more than 4 hr, centrifuging at 6000 rpm for 5 min, and collecting serum.
2. ELISA assay for serum
(2-1) diluting KAL-BSA to 1ug/ml with 0.01M PBS buffer solution (pH 7.4), adding 100ul of diluted KAL-BSA antigen to each well of 96-well enzyme-labeled plate, and coating overnight at 4 ℃. The following day, 300. mu.L of 1% BSA (in TBST) was added to each well for blocking, and the wells were incubated at 37 ℃ for 6 hours, after which the blocking solution was discarded.
(2-2) adding 50ul of Carlipp polyclonal antibody standard substance with the concentration of 100ng/ml into a longitudinal row of enzyme-labeled holes, diluting the collected anti-Carlipp polyclonal antibody serum sample by 1000 times, and then diluting by 1:3 times, wherein each hole is 100 ul. Finally, a control well is left, and 50. mu.L of the control well is added to a longitudinal enzyme-labeled well containing a standard.
(2-3.) Add 50ul of 0.01PBS to the original double diluted wells as a control.
(2-4), incubating for 30min at 36-38 ℃, washing for 3 times, adding an enzyme-labeled secondary antibody, incubating for 30min at 36-38 ℃, washing, adding a color development solution for color development, and finally stopping the reaction. The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled secondary antibody, and the color development liquid is TMB color development liquid.
(2-5) measuring the OD value at 450nm, and calculating the competitive inhibition rate according to the formula:
TABLE 1 competitive inhibition ELISA results at various dilution times
| Dilution factor | Control OD value | Competitive inhibition OD value | Rate of competitive inhibition |
| 1：1000 | 2.077 | 0.824 | 60.32％ |
| 1：3000 | 1.877 | 0.732 | 61.00％ |
| 1：9000 | 1.522 | 0.683 | 55.12％ |
| 1：27000 | 1.143 | 0.521 | 54.41％ |
| 1：81000 | 0.872 | 0.398 | 54.35％ |
| 1：243000 | 0.678 | 0.301 | 55.60％ |
| 1：729000 | 0.587 | 0.273 | 53.49％ |
| Control PBS | 0.050 | 0.047 | Is free of |
(2-6) the measured titer of serum collected after immunization, as seen in Table 1, reached 1: 729000, the comprehensive inhibition rate is about 55%.
3. Polyclonal antibody purification
(3-1) affinity column preparation: 100ml of the GE-Sepharose-4B filled gel was activated with hydrogen bromide and then divided equally into two portions. BSA and BGG proteins were dissolved in 0.01M carbonate buffer PH 9.6, added immediately to the activated filled gel, stirred well and left overnight. Blocking with ethanolamine the following day, and rinsing with 0.01M PBS to equilibrate for use.
(3-2) pretreating the BSA affinity column with PB at a concentration of 0.01M, pH7.4, at a flow rate of 60 mL/h; adding 50% saturated ammonium sulfate treated anti-Cariprdopoly-antibody solution into a pretreated chromatographic column, then using 100mL of PB with concentration of 0.01M and PH7.4 to flow through at a flow rate of 60mL/h, connecting an ultraviolet detector, taking flow-through liquid after the degree is more than 10, and stopping taking after the degree falls back to less than 10.
(3-3) the protein solution after flowing through was concentrated with 50% saturated ammonium sulfate solution, and then dialyzed against 0.01M PB solution having a pH of 7.4 for 3 times each for 6 hours or more. The affinity column was eluted with 0.1M glycine-HCl, pH2.5, and then equilibrated with 0.01M PB, pH 7.4.
(3-4) the collected protein was diluted to 1ug/ml for ELISA as described in example 2.2, and the results are shown in Table 2.
TABLE 2 competitive inhibition ELISA results at various dilution times after BSA affinity column purification
| Dilution factor | Control OD value | Competitive inhibition OD value | Rate of competitive inhibition |
| 1：1 | 1.988 | 0.585 | 70.57％ |
| 1：3 | 1.781 | 0.525 | 70.52％ |
| 1：9 | 1.502 | 0.460 | 69.37％ |
| 1：27 | 1.103 | 0.377 | 65.82％ |
| 1：81 | 0.799 | 0.241 | 69.83％ |
| 1：243 | 0.654 | 0.201 | 69.26％ |
| 1：729 | 0.498 | 0.175 | 64.85％ |
| Control PBS | 0.059 | 0.044 | Is free of |
The antibody titer measured after the BSA affinity column purification still reached 1: 729000, the comprehensive inhibition rate is about 70%, the antibody titer is not obviously reduced, and the inhibition rate is obviously improved.
(3-5) pretreating the BGG affinity chromatography column by using PB with the concentration of 0.01M, the pH value of 7.4 and the flow rate of 60 mL/h; adding the anti-Carlipp polyclonal antibody protein solution treated by the BSA affinity column into a pretreated chromatographic column, then using 100mL of PB with the concentration of 0.01M and the pH of 7.4 to flow through the column at the flow rate of 60mL/h, connecting an ultraviolet detector, taking a flow-through liquid after the degree is more than 10, and stopping taking after the degree falls below 10.
(3-6) the protein solution after flowing through was concentrated with 50% saturated ammonium sulfate solution, and then dialyzed against 0.01M PB solution having a pH of 7.4 for 3 times each for 6 hours or more. The affinity column was eluted with 0.1M glycine-HCl, pH2.5, and then equilibrated with 0.01M PB, pH 7.4.
(3-7) the collected protein was diluted to 1ug/ml for ELISA as described in example 2.2, and the results are shown in Table 3.
TABLE 3 competitive inhibition ELISA results at various dilution times after purification of BGG affinity column
As analyzed in table 3, the detectable titer of the antibody after BSA affinity column purification still reached 1: 729000, the comprehensive inhibition rate is about 87%, the antibody titer is slightly reduced, but the inhibition rate is obviously improved.
(3-8) storing the antibody after final purification at-20 ℃.
Example 3 application of anti-Carlipp polyclonal antibody in Rapid diagnostic test paper strip
1. The anti-carriedout polyclonal antibody obtained by purification can be applied to a carriedout colloidal gold test strip, which comprises a sample pad, a marker pad, a reaction membrane and a sample sucking pad; the marker pad is coated with the colloidal gold-labeled polyclonal antibody against the carisoprodol, the detection line (T line) of the reaction membrane is coated with a carisoprodol-bovine serum albumin complex, and the quality control line (C line) of the reaction membrane is coated with a secondary antibody. See in particular fig. 1.
2. During detection, one end of the test strip sample pad is inserted into a sample to be detected at room temperature, the liquid level of the sample to be detected does not exceed the MAX line on the test strip, the liquid level is observed by naked eyes after waiting for 10 minutes, and the color development conditions of the C line and the T line are recorded.
3. If only C line is developed, indicating that the sample to be detected contains the carphedon and the content of the detected carphedon is higher than 100 ng/ml; if the C line and the T line are developed, the content of the detected carisoprodol in the sample to be detected is lower than 100 ng/ml; if the C line and the T line do not develop color, the test strip is failed.
4. The preparation method of the test strip is a conventional method in the field, and this example is only an application description.
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (10)
1. A Carlipduous artificial antigen KAL-BSA is characterized in that the Carlipduous is chemically modified, and coupled with the BSA (bovine serum albumin), an artificial antigen capable of inducing the proliferation and differentiation of B cells and then generating specific antibodies is obtained.
2. A method of synthesising a carphedon artificial antigen as claimed in claim 1 comprising the steps of:
step (1), balancing carbodiimide EDC cross-linking agent to room temperature;
adding BSA (bovine serum albumin) into a coupling buffer solution to obtain the coupling buffer solution loaded with the BSA; wherein the coupling buffer solution is 0.1M MES, and the pH value is 4.8-5.0;
dissolving the carpolippon hapten into a coupling buffer solution loaded with BSA (bovine serum albumin) so as to introduce a carrier protein BSA;
adding EDC into the coupling buffer solution mixed by the Carlipp hapten and the BSA;
and (5) reacting at room temperature for a period of time, and purifying the coupled protein by using a desalting column to obtain KAL-BSA.
3. An anti-carisoproduced antibody obtained by collecting serum from an animal immunized with the carisoproduced artificial antigen KAL-BSA according to claim 1 and performing two-step affinity chromatography.
4. An anti-kallidoprol polyclonal antibody according to claim 3, wherein the affinity layer used in the two-step affinity chromatography is an affinity column coupled to a BSA macromolecule and an affinity column coupled to a BGG macromolecule.
5. A method for synthesizing an anti-Callipp polyclonal antibody, which is characterized by comprising the following steps:
a step (1) of diluting the artificial antigen KAL-BSA of claim 1, mixing with an adjuvant, and then immunizing an animal for a plurality of times;
collecting serum of an immune animal, and purifying the serum by using ammonium sulfate to obtain an antibody semi-finished product;
sequentially passing the obtained antibody semi-finished product through an affinity column coupled with a BSA (bovine serum albumin) macromolecule and an affinity column coupled with a BGG macromolecule; collecting the flow-through liquid, concentrating and dialyzing for many times.
6. The method of claim 5, wherein the affinity column for BSA and the affinity column for BGG macromolecules are prepared by activating 100ml of GE-Sepharose-4B packing gel with hydrogen bromide, and dividing into two parts; respectively dissolving BSA and BGG protein in 0.01M carbonate buffer (pH 9.6), immediately adding into activated filling gel, stirring well, and standing overnight; blocking was then performed with ethanolamine and the equilibrium was washed with 0.01M PBS.
7. Use of an anti-carisoprodol polyclonal antibody of claim 3 in preparing a carisoprodol detection kit.
8. The use of an anti-carisoprodol polyclonal antibody of claim 3 in the preparation of a test strip for detecting carisoprodol.
9. Use according to claim 7 or 8, wherein the detection threshold reaches 100 ng/ml.
10. The use of claim 8, wherein the test strip for detecting carphedron comprises a sample pad, a marker pad, a reaction membrane and a sample suction pad; the marker pad is coated with a colloidal gold-labeled anti-Carlpox polyclonal antibody, the detection line of the reaction membrane is coated with a Carlpox-bovine serum albumin complex, and the quality control line of the reaction membrane is coated with a secondary antibody.
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