CN109651508A - Heterophile antibody blocking agent HBT-1 and preparation method thereof - Google Patents
Heterophile antibody blocking agent HBT-1 and preparation method thereof Download PDFInfo
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- CN109651508A CN109651508A CN201811323396.8A CN201811323396A CN109651508A CN 109651508 A CN109651508 A CN 109651508A CN 201811323396 A CN201811323396 A CN 201811323396A CN 109651508 A CN109651508 A CN 109651508A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
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- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
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Abstract
The present invention proposes a kind of heterophile antibody blocking agent HBT-1, amino acid sequence and nucleotide sequence including its variable region, wherein, 3 complementary determining region CDR sequences of heavy chain are respectively, CDR1:Val-Gln-Ser-Ser-Ile-Thr, CDR2:Gln-Asp-Asp-Tyr-Ala-Tyr-Thr-Leu-Tyr, CDR3:Val-Tyr-Lys-Leu；3 complementary determining region CDR sequences of light chain are respectively CDR1:Gly-Asp-Val-Leu-Ser-Lys-Ser-Cys, CDR2:Lys-Asp-Arg-Phe, CDR3:His-Ile-Ile-Ser-Thr-Ser, have good compatibility.The invention also provides the preparation methods of heterophile antibody blocking agent HBT-1 simultaneously.
Description
Technical field
The invention belongs to field of biomedicine technology, especially a kind of heterophile antibody blocking agent HBT-1 and its preparation side
Method.
Background technique
Blocking agent is also receptor antagonist, and referring to can be in conjunction with receptor, and can prevent a kind of ligand of agonist generation effect
Substance.Antagonist has compatibility to corresponding receptor, but does not have efficiency, to inhibit effect of the agonist to receptor.Antagonist
Can be with the active site of bind receptor, can also be in conjunction with other structure site, or even individually combine usually not biological regulation effect
Site reaches effect.Antagonist and receptor binding matter are different, so binding time has with short, in conjunction with invertibity also have
It is different.
Antagonist is divided into the types such as competitive antagonist and noncompetitive antaganist.Most of Drug Antagonists all with it is endogenous
Property ligand or substrate competition receptor binding site reach effect.
Receptor is the large-scale protein molecule that can be activated by ligand binding.Receptor can combine on cell membrane, can also
It is present in cell interior, such as in nucleus or mitochondria.Receptor is in conjunction with active site and ligand are with non-covalent bond, and one
There can be the site of multiple combination different ligands on receptor.Ligand and receptor all may be used after active site or other structure site combine
To adjust the activity of receptor.Antagonist can combination be usually not involved in life by combining active site, other structure site, or individually
The site that object is adjusted tells on.
Summary of the invention
Technical problem solved by the invention is to provide a kind of heterophile antibody blocking agent HBT-1, including its variable region
Amino acid sequence and nucleotide sequence, have good compatibility.
The technical solution for realizing the aim of the invention is as follows:
Heterophile antibody blocking agent HBT-1, including heavy chain and light chain, wherein 3 complementary determining region CDR sequences of heavy chain
Respectively, CDR1:Val-Gln-Ser-Ser-Ile-Thr, CDR2:Gln-Asp-Asp-Tyr-Ala-Tyr-Thr-Leu-Tyr,
CDR3:Val-Tyr-Lys-Leu；3 complementary determining region CDR sequences of light chain are respectively CDR1:Gly-Asp-Val-Leu-
Ser-Lys-Ser-Cys, CDR2:Lys-Asp-Arg-Phe, CDR3:His-Ile-Ile-Ser-Thr-Ser.
Further, heterophile antibody blocking agent HBT-1, the heavy chain variable amino acid sequence such as SEQ of the invention
Shown in ID NO.1.
Further, heterophile antibody blocking agent HBT-1, the chain variable region amino acid sequence such as SEQ of the invention
Shown in ID NO.2.
Further, heterophile antibody blocking agent HBT-1, the weight chain variable region nucleotide sequence such as SEQ of the invention
Shown in ID NO.3.
Further, heterophile antibody blocking agent HBT-1, the light chain variable region nucleotide sequence such as SEQ of the invention
Shown in ID NO.4.
The present invention also proposes the preparation method of heterophile antibody blocking agent HBT-1, comprising the following steps:
Step 1: prepare reagent: equilibration buffer, dissociation buffer, elution buffer, pH neutralizer prepare equipment:
Protein G column, low-temperature and high-speed centrifuge, balance, peristaltic pump；
Step 2: the ascites of freezing is melted at 10-15 DEG C of proxima luce (prox. luc), filters removal grease with the absorbent cotton for soaking tri-distilled water,
4 DEG C of 10000rPm centrifugation 30min of filtered ascites collect supernatant；Column prepares: loading Protein G filler, is washed with purifying
5-10 times of column volume is washed, ethyl alcohol is removed；
Step 3: being balanced, loading, balance, dissociation, balance, dialysis, recycling, save Protein A column, cleaning is wriggled
Pump sample feeding pipe；
Step 4: reagent preparation: 10mM Tris-NaCl, 0.1M glycine buffer, 20mM Tris-NaCl, 2M
Tris, elution buffer.
The invention adopts the above technical scheme compared with prior art, has following technical effect that
The present invention filters out heterophile antibody blocking agent HBT-1, and obtains its heavy chain and light chain variable region, obtains after sequencing
Its unique nucleotide sequence has good compatibility.
Detailed description of the invention
Fig. 1 is affinity data result figure of the invention.
Specific embodiment
Embodiments of the present invention are described below in detail, the example of the embodiment is shown in the accompanying drawings, wherein from beginning
Same or similar element or element with the same or similar functions are indicated to same or similar label eventually.Below by ginseng
The embodiment for examining attached drawing description is exemplary, and for explaining only the invention, and is not construed as limiting the claims.
Embodiment 1
Heterophile antibody blocking agent HBT-1, including heavy chain and light chain, wherein 3 complementary determining region CDR sequences of heavy chain
Respectively, CDR1:Val-Gln-Ser-Ser-Ile-Thr, CDR2:Gln-Asp-Asp-Tyr-Ala-Tyr-Thr-Leu-Tyr,
CDR3:Val-Tyr-Lys-Leu；3 complementary determining region CDR sequences of light chain are respectively CDR1:Gly-Asp-Val-Leu-
Ser-Lys-Ser-Cys, CDR2:Lys-Asp-Arg-Phe, CDR3:His-Ile-Ile-Ser-Thr-Ser.
Heavy chain variable amino acid sequence is as shown in SEQ ID NO.1, chain variable region amino acid sequence such as SEQ ID
Shown in NO.2.Weight chain variable region nucleotide sequence is as shown in SEQ ID NO.3, light chain variable region nucleotide sequence such as SEQ ID
Shown in NO.4.
As shown in Figure 1, heterophile antibody blocking agent HBT-1 of the invention has good affinity, with other blockings
Agent is compared to as shown in the table:
| Loading Sample ID | Response | KD(M) | kon(1/Ms) | kdis(1/s) |
| Blocking agent of the present invention | 0.7359 | 1.08E-10 | 4.51E+05 | 4.87E-05 |
| Other blocking agents | 1.0146 | 1.04E-09 | 4.50E+05 | 4.70E-04 |
The preparation method of heterophile antibody blocking agent HBT-1 specifically includes the following steps:
Step 1: prepare reagent: equilibration buffer, dissociation buffer, elution buffer, pH neutralizer prepare equipment:
Protein G column, low-temperature and high-speed centrifuge, balance, peristaltic pump；
Step 2: the ascites of freezing is melted at 10-15 DEG C of proxima luce (prox. luc), filters removal grease with the absorbent cotton for soaking tri-distilled water,
4 DEG C of 10000rPm centrifugation 30min of filtered ascites collect supernatant；Column prepares: loading Protein G filler, is washed with purifying
5-10 times of column volume is washed, ethyl alcohol is removed；
Step 3: being balanced, loading, balance, dissociation, balance, dialysis, recycling, save Protein A column, cleaning is wriggled
Pump sample feeding pipe；
Step 4: reagent preparation: 10mM Tris-NaCl, 0.1M glycine buffer, 20mM Tris-NaCl, 2M
Tris, elution buffer.
Embodiment 2
Heterophile antibody blocking agent HBT-1's the preparation method is as follows:
One, prepare:
1, reagent prepares:
Equilibration buffer: 10mM Tris-NaCl (pH 8.3)
Dissociate buffer: 0.1M glycine (pH 2.7)
Elution buffer: 20mM Tris-NaCl (pH7.5) or 10mM PBS (pH7.4)
PH neutralizer: 2M Tris (pH8.0)
2, equipment prepares:
Protein G column, low-temperature and high-speed centrifuge, balance, peristaltic pump.
Two, preparation manipulation
1, preparation of samples: the ascites of freezing is melted at 10-15 DEG C of proxima luce (prox. luc), filters removal with the absorbent cotton for soaking tri-distilled water
Grease, 4 DEG C of 10000rPm centrifugation 30min of filtered ascites collect supernatant.
Column prepares: loading Protein G filler, with purifying 5-10 times of column volume of water washing, removes ethyl alcohol；
2, balance: Protein G column balances 5 times of column volumes, column with equilibration buffer 10mM Tris-NaCl (pH 8.3)
Albumen Ultraviolet Detector show value is adjusted to 0 after balance is good, as baseline；
3, loading: ascites uses the equilibration buffer 10mM Tris-NaCl (pH 8.3) of two volumes to dilute as on sample
Sample, using peristaltic pump under the conditions of 20-25 DEG C slow loading；
4, it balances: balancing 5-10 times of cylinder with equilibration buffer 10mM Tris-NaCl (pH 8.3) again after completion of the sample
Product, balance to no albumen flow out, i.e., albumen Ultraviolet Detector show value is 0；
5, it dissociates: being dissociated after the completion of balance using 0.1M glycine (pH 2.7), when albumen Ultraviolet Detector is shown
Value starts to collect albumen when being greater than 0.3, stops collecting albumen when albumen Ultraviolet Detector show value is less than 0.3；
6, it balances: continuing to be flowed out with 0.1M glycine (pH 2.7) column scrubber to no albumen after the completion of dissociation, it is slow with balance
Fliud flushing 10mM Tris-NaCl (pH 8.3) about 5-10 times column volume of balance columns；
7, it dialyses: merging and collect protein component, use elution buffer 20mM Tris-NaCl (pH7.5) or 10mM PBS
(pH7.4) it dialyses, dialysis ratio is 1:50, is changed liquid 3 times, and each dialysis time is not less than 4 hours；
8, antibody is handled: being recycled antibody after the completion of dialysis and is saved egg with 0.09%NaN3 is added after 0.22 μm of membrane filtration
It is white.
Three, Protein G column saves
The Protein G column length phase, 4-8 DEG C saved without using 20% ethyl alcohol is filled in then column.
Four, the cleaning of peristaltic pump sample feeding pipe
Same peristaltic pump should use the sample sample-loading buffer to rinse 5-10 and carry out again all over silicone tube when handling other samples
Other operations.
Five, preparation of reagents
1、10mM Tris-NaCl(pH 8.3)
| The name of an article | Raw material sources | 1L standard volume |
| Tris | Beijing benefit benefit | 1.21g |
| NaCl | Beijing benefit benefit | 11.688g |
| Purified water | Self-control, quality inspection are qualified | 1L |
Mentioned reagent is weighed with electronic balance, is poured into 1L beaker, appropriate purified water, stirring and dissolving, with dense salt are added
Acid adjusts pH to 8.3, is settled to 1L.
2,0.1M glycine buffer (pH2.7)
| The name of an article | Raw material sources | 1L standard volume |
| Glycine | Beijing benefit benefit | 7.51g |
| Purified water | Self-control, quality inspection are qualified | 1L |
Mentioned reagent is weighed with electronic balance, is poured into 1L beaker, appropriate purified water, stirring and dissolving, with dense salt are added
Acid adjusts pH to 2.7, is settled to 1L.
3、20mM Tris-NaCl(pH7.5)
| The name of an article | Raw material sources | 1L standard volume |
| Tris | Beijing benefit benefit | 2.4218g |
| NaCl | Beijing benefit benefit | 11.688g |
| Purified water | Self-control, quality inspection are qualified | 1L |
Mentioned reagent is weighed with electronic balance, is poured into 1L beaker, appropriate purified water, stirring and dissolving, with dense salt are added
Acid adjusts pH to 7.5, is settled to 1L.
4、2M Tris(pH8.0)
| The name of an article | Raw material sources | 1L standard volume |
| Tris | Beijing benefit benefit | 242.18g |
| Purified water | Self-control, quality inspection are qualified | 1L |
Mentioned reagent is weighed with electronic balance, is poured into 1L beaker, appropriate purified water, stirring and dissolving, with dense salt are added
Acid adjusts pH to 8.0, is settled to 1L.
5, elution buffer: 10mM PBS (pH7.4)
| The name of an article | Ingredient requirement | 1L standard volume |
| Na2HPO4·12H2O | Traditional Chinese medicines | 2.87g |
| NaH2PO4·2H2O | Traditional Chinese medicines | 0.31g |
| NaCl | Traditional Chinese medicines | 9g |
| Purified water | Self-control, quality inspection are qualified | 1L |
Mentioned reagent is weighed with electronic balance, is poured into 1L beaker, appropriate purified water, stirring and dissolving, with dense salt are added
Acid adjusts pH to 7.4, is settled to 1L.
Claims (6)
1. heterophile antibody blocking agent HBT-1, which is characterized in that including heavy chain and light chain, wherein 3 of heavy chain are complementary to be determined
Area's CDR sequence is respectively CDR1:Val-Gln-Ser-Ser-Ile-Thr, CDR2:Gln-Asp-Asp-Tyr-Ala-Tyr-
Thr-Leu-Tyr, CDR3:Val-Tyr-Lys-Leu；3 complementary determining region CDR sequences of light chain are respectively CDR1:Gly-
Asp-Val-Leu-Ser-Lys-Ser-Cys, CDR2:Lys-Asp-Arg-Phe, CDR3:His-Ile-Ile-Ser-Thr-
Ser。
2. heterophile antibody blocking agent HBT-1 according to claim 1, which is characterized in that the heavy chain variable region amino
Acid sequence is as shown in SEQ ID NO.1.
3. heterophile antibody blocking agent HBT-1 according to claim 1, which is characterized in that the light chain variable region amino
Acid sequence is as shown in SEQ ID NO.2.
4. heterophile antibody blocking agent HBT-1 according to claim 1, which is characterized in that the heavy chain variable region nucleosides
Acid sequence is as shown in SEQ ID NO.3.
5. heterophile antibody blocking agent HBT-1 according to claim 1, which is characterized in that the light chain variable region nucleosides
Acid sequence is as shown in SEQ ID NO.4.
6. the preparation method of heterophile antibody blocking agent HBT-1, which comprises the following steps:
Step 1: prepare reagent: equilibration buffer, dissociation buffer, elution buffer, pH neutralizer prepare equipment: Protein
G column, low-temperature and high-speed centrifuge, balance, peristaltic pump；
Step 2: the ascites of freezing is melted at 10-15 DEG C of proxima luce (prox. luc), filters removal grease, filtering with the absorbent cotton for soaking tri-distilled water
4 DEG C of 10000rPm centrifugation 30min of ascites afterwards collect supernatant；Column prepares: Protein G filler is loaded, with purifying water washing 5-
10 times of column volumes remove ethyl alcohol；
Step 3: be balanced, loading, balance, dissociation, balance, dialysis, recycling, save Protein A column, cleaning peristaltic pump into
Sample pipe；
Step 4: reagent preparation: 10mM Tris-NaCl, 0.1M glycine buffer, 20mM Tris-NaCl, 2M Tris, thoroughly
Analyse buffer.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811323396.8A CN109651508A (en) | 2018-11-08 | 2018-11-08 | Heterophile antibody blocking agent HBT-1 and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811323396.8A CN109651508A (en) | 2018-11-08 | 2018-11-08 | Heterophile antibody blocking agent HBT-1 and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111393523A (en) * | 2020-01-27 | 2020-07-10 | 江苏帆博生物制品有限公司 | Preparation method of HA blocking agent and antibody detection system |
| CN114213544A (en) * | 2021-12-21 | 2022-03-22 | 江苏帆博生物制品有限公司 | Heterophilic antibody blocking agent HBR-7 and preparation method thereof |
| CN114835808A (en) * | 2022-06-10 | 2022-08-02 | 郑州伊美诺生物技术有限公司 | Blocking agent capable of directionally eliminating false positive and preparation method thereof |
| CN116948034A (en) * | 2023-08-10 | 2023-10-27 | 南京珀尔泰生物技术有限公司 | Active type heterophagy antibody blocker Block-5S and preparation method thereof |
-
2018
- 2018-11-08 CN CN201811323396.8A patent/CN109651508A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111393523A (en) * | 2020-01-27 | 2020-07-10 | 江苏帆博生物制品有限公司 | Preparation method of HA blocking agent and antibody detection system |
| CN114213544A (en) * | 2021-12-21 | 2022-03-22 | 江苏帆博生物制品有限公司 | Heterophilic antibody blocking agent HBR-7 and preparation method thereof |
| CN114213544B (en) * | 2021-12-21 | 2023-05-23 | 江苏帆博生物制品有限公司 | Anisotropic antibody blocker HBR-7 and preparation method thereof |
| CN114835808A (en) * | 2022-06-10 | 2022-08-02 | 郑州伊美诺生物技术有限公司 | Blocking agent capable of directionally eliminating false positive and preparation method thereof |
| CN114835808B (en) * | 2022-06-10 | 2023-07-21 | 郑州伊美诺生物技术有限公司 | Blocker capable of directionally eliminating false positive and preparation method thereof |
| CN116948034A (en) * | 2023-08-10 | 2023-10-27 | 南京珀尔泰生物技术有限公司 | Active type heterophagy antibody blocker Block-5S and preparation method thereof |
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Application publication date: 20190419 |
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