CN116903751A

CN116903751A - Anti-phenytoin sodium antibody or antigen binding fragment thereof and application thereof

Info

Publication number: CN116903751A
Application number: CN202311092337.5A
Authority: CN
Inventors: 魏文涛; 上官科科; 成含锋; 汪燕燕; 魏昕洋
Original assignee: Sure Biotech (hangzhou) Ltd
Current assignee: Sure Biotech (hangzhou) Ltd
Priority date: 2023-08-28
Filing date: 2023-08-28
Publication date: 2023-10-20

Abstract

The invention provides an anti-phenytoin sodium antibody or an antigen binding fragment thereof and application thereof, and relates to the technical field of antibodies. An anti-phenytoin sodium antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of 25 th to 32 th amino acid residues, VHCDR2 of 50 th to 57 th amino acid residues and VHCDR3 of 96 th to 106 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region comprises VLCDR1 of 27 th to 37 th amino acid residues, VLCDR2 of 55 th to 57 th amino acid residues and VLCDR3 of 94 th to 103 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO. 2. The antibody or the antigen binding fragment thereof can be detected by immunology, and has good specific binding capacity through verification.

Description

Anti-phenytoin sodium antibody or antigen binding fragment thereof and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-phenytoin sodium antibody or an antigen binding fragment thereof and application thereof.

Background

Phenytoin sodium (Phenytoin sodium) has a highly selective inhibitory effect on the brain cortex motor area, and is believed to have an antiepileptic effect by stabilizing the function of brain cell membranes and increasing the effects of the brain inhibitory neurotransmitters 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) to prevent the spread of abnormal discharge. The mechanism of action against neuralgia may be related to the action of this product with the central nervous system, reduced synaptic transmission or reduced transient stimulation causing neuronal firing. It also has effects in inhibiting ectopic rhythm of atrium and ventricle, accelerating atrioventricular conduction, reducing myocardial autonomy, and resisting arrhythmia. The more common adverse reactions are altered behavior, clumsiness or gait, confusion, impaired pronunciation, tremble hands, nerves or irritability, which tend to be reversible and disappear soon upon cessation. In addition, it is more common to have a thick gum, bleeding, rough complexion and hair hyperplasia. There are occasional cervical or axillary lymphadenectasis (IgA reduction), fever or rash (intolerance or allergy), leukopenia, purpura. Is rare to cause toxic cataract of eyes, amenorrhea, cerebellum injury and atrophy. Phenytoin sodium may cause potentially serious skin lesions such as S-J syndrome and Toxic Epidermonecrosis (TEN). Phenytoin sodium tablet is a clinically common antiepileptic drug. Is suitable for treating general tonic-battle twinning, complicated partitional attack, simple partitional attack and depressive psychosis persistent state. Can also be used for treating trigeminal neuralgia, epidermolysis bullosa due to recessive dystrophy, paroxysmal chorea and athetosis, paroxysmal control disorder, myotonia, and heart conduction disorder due to excessive tricyclic antidepressant.

The liver metabolism (hydroxylation) capacity of phenytoin sodium reaches saturation after a certain dosage of medicine is applied, and even if the dosage is increased by a small amount, the blood concentration is increased in a nonlinear and rapid way, and the poisoning risk exists, so that the blood concentration is monitored. The effective blood concentration is 10-20 mg/L, 300mg is orally taken every day, and the steady-state concentration can be reached in 7-10 days.

The phenytoin sodium can be detected by adopting a detection method based on an immunological principle, such as an immunofluorescence method, an enzyme-linked immunosorbent assay, a colloidal gold immunochromatography technology and the like. The detection method based on the immunological principle can be realized by relying on an antibody capable of being combined with phenytoin sodium, and the phenytoin sodium antibody in the market is relatively lack in supply at present and mainly depends on imported raw materials. The invention is provided for providing stable and reliable raw material sources, reducing the product cost, improving the detection rate and sensitivity of phenytoin sodium rapid diagnosis commodity such as gold-labeled test paper strips, ELISA kits and the like, reducing the false positive rate, improving the competitiveness of self-produced commodity and the like.

Disclosure of Invention

The present invention provides an anti-phenytoin sodium antibody or antigen-binding fragment thereof, which is obtained by screening with phenytoin sodium as an antigen, and the CDR region sequences thereof are determined by cloning, identification and analysis of the gene structure, and another object of the present invention is to provide a biological material for the antibody or antigen-binding fragment thereof and uses thereof.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention there is provided an anti-phenytoin sodium antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of 25 th to 32 th amino acid residues, VHCDR2 of 50 th to 57 th amino acid residues and VHCDR3 of 96 th to 106 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO. 1;

the light chain variable region comprises VLCDR1 of amino acid residues 27-37, VLCDR2 of amino acid residues 55-57 and VLCDR3 of amino acid residues 94-103 of the N-terminal amino acid sequence shown in SEQ ID NO. 2.

According to an aspect of the present invention, there is also provided a biomaterial selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding the anti-phenytoin sodium antibody or antigen-binding fragment thereof described above;

(ii) a vector carrying the polynucleotide of (i) above.

(iii) a cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the aforementioned anti-phenytoin sodium antibody or antigen-binding fragment thereof.

According to one aspect of the present invention there is also provided the use of an anti-phenytoin sodium antibody or antigen binding fragment thereof as described above, or of a biological material as described above, for the detection of phenytoin sodium for non-diagnostic and therapeutic purposes, or for the preparation of a product for use in the detection of phenytoin sodium.

According to one aspect of the present invention there is also provided a reagent or kit for detecting sodium phenytoin, the reagent or kit comprising an anti-sodium phenytoin antibody or antigen-binding fragment thereof as described above.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the Phenytoin sodium is used for immunizing Balb/c mice, spleen cells of the mice are fused with myeloma cells, hybridoma cells with high specificity are obtained through specific high-throughput screening, a large amount of mouse ascites is obtained through culture and re-immunization, and then a monoclonal antibody anti-Phenytoin-sodium-mab1 with high purity, high sensitivity and high specificity is obtained through multi-step separation and purification, so that a required raw material is provided for developing an immune test strip for detecting Phenytoin sodium. The anti-Phenytoin sodium monoclonal antibody anti-Phenytoin-sodium-mab1 can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has good specific binding capacity through verification.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of the immunochromatographic test strip coated with the antibody No. A1-1 according to example 2 of the present invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

An "antibody or antigen-binding fragment thereof" as used herein refers to a protein that binds a particular antigen, which broadly refers to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). In addition, "antibody or antigen-binding fragment thereof" also includes naturally occurring antibodies as well as non-naturally occurring antibodies. An "antigen binding fragment" in this context is a substance comprising the CDRs of an antibody which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. The antigen binding fragments of the invention have the effect of specifically recognizing and binding to phenytoin sodium.

"variable region" or "variable domain" of an antibody or antigen binding fragment thereof refers to the domain of an antibody that recognizes and binds to an antigen at the amino terminus of the heavy or light chain of the antibody, the composition and arrangement of the amino acids of the segment determining the specificity of the antibody for recognizing the antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-determining region (CDRs) (also known as hypervariable regions) connected by 4 Frameworks (FR). The framework and CDR ranges have been precisely defined, for example in Kabat (see sequence of immunologically important proteins ((Sequences of Proteins of Immunological Interest), E.Kabat et al) and Chothia), any CDR determination method well known in the art, including combinations of methods, can identify the CDRs of the variable domain the CDRs in each chain are held closely together by the FRs to form the variable region, typically the variable regions VL/VH of the heavy and light chains are obtained by ligating the CDRs numbered FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combination arrangement with the FRs.

The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide comprises a portion encoding the antibody or antigen binding fragment thereof described above, optionally encoding the sense or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. The offspring may not necessarily be identical to the primary cells due to natural, accidental or deliberate mutation, e.g. are morphologically and/or differ from the primary cells in genomic DNA. "transformant" and "transformed cell" include primary test cells and cultures derived therefrom.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising" or the like will be understood to include the stated element or component without excluding other elements or components.

According to one aspect of the present invention there is provided an anti-phenytoin sodium antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 (GYTFTSYY) of amino acid residues 25 to 32, VHCDR2 (IYPGNVNT) of amino acid residues 50 to 57 and VHCDR3 (ARIYYGNYVDY) of amino acid residues 96 to 106 of the N-terminal amino acid sequence shown in SEQ ID NO. 1.

The light chain variable region comprises VLCDR1 (QSLFDSDGKTY) of amino acid residues 27-37, VLCDR2 (LVS) of amino acid residues 55-57 and VLCDR3 (WQGTHFPHGR) of amino acid residues 94-103 of the N-terminal amino acid sequence shown in SEQ ID NO. 2.

In alternative embodiments, the heavy chain variable region is of the structure VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4; wherein the amino acid sequence of VHFR1 is shown as SEQ ID NO.3 (VKLQESGPELVKPGASVRISCKAS), and/or the amino acid sequence of VHFR2 is shown as SEQ ID NO.4 (IHWVKQRPGQGLEWIGW), and/or the amino acid sequence of VHFR3 is shown as SEQ ID NO.5 (KYNEKFKGKATLTADKSSSTAYIQLSSLTSEDSAVYFC), and/or the amino acid sequence of VHFR4 is shown as SEQ ID NO.6 (WGQGTTLTVSS).

In an alternative embodiment, the light chain variable region has the structure VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4; wherein the amino acid sequence of VLFR1 is shown as SEQ ID NO.7 (DIVITQTPLTLSVTIGQPASISCKSS), and/or the amino acid sequence of VLFR2 is shown as SEQ ID NO.8 (LNWLLQRPGQSPKRLIY), and/or the amino acid sequence of VLFR3 is shown as SEQ ID NO.9 (KLDSGVPDRFTGSGSGTDFTLKISGVEAEDLGVYYC), and/or the amino acid sequence of VLFR4 is shown as SEQ ID NO.10 (SVEAPSWKSN).

IN an alternative embodiment, the amino acid sequence of the heavy chain variable region is shown IN SEQ ID NO. 1; and/or the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

In alternative embodiments, the antibody or antigen binding fragment thereof further comprises the sequence of a portion or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD.

In alternative embodiments, the antigen binding fragment comprises F (ab') ₂ One or more of Fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units.

In alternative embodiments, the antibody or antigen binding fragment thereof removes CDR regions, and the remaining sequence-derived species include, but are not limited to, one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

In alternative embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region. In alternative embodiments, the heavy chain constant region and/or the light chain constant region are derived from mice.

In alternative embodiments, the light chain of the antibody is a kappa chain;

in an alternative embodiment, the antibody is an IgG antibody.

According to another aspect of the present invention, there is also provided a biomaterial selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding the aforementioned antibody or antigen binding fragment thereof; in an alternative embodiment, the polynucleotide encodes the heavy chain variable region and the nucleotide sequence is shown in SEQ ID NO. 11; in an alternative embodiment, the polynucleotide encodes the light chain variable region and the nucleotide sequence is set forth in SEQ ID NO. 12.

(ii) a vector carrying the polynucleotide of (i) above.

(iii) a cell carrying said polynucleotide (i), or comprising said vector (ii), or expressing the aforementioned anti-phenytoin sodium antibody or antigen-binding fragment thereof.

According to another aspect of the present invention there is also provided the use of an anti-phenytoin sodium antibody or antigen binding fragment thereof as described above, or of a biological material as described above, for the detection of phenytoin sodium for non-diagnostic and therapeutic purposes, or for the preparation of a product for use in the detection of phenytoin sodium.

According to another aspect of the present invention, there is also provided a reagent or kit for detecting phenytoin sodium, comprising the above-mentioned anti-phenytoin sodium antibody or antigen-binding fragment thereof.

In alternative embodiments, a label comprising one or more of an enzyme, a fluorescent molecular label, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin is also included.

In alternative embodiments, the reagent or kit is used for immunodetection. In alternative embodiments, the reagent or kit comprises an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic microparticle detection reagent or kit, an immunofluorescent detection reagent or kit, or an immunoblotting detection reagent or kit.

In an alternative embodiment, the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip. The competitive immunochromatography test paper is coated with the antibody or the antigen binding fragment thereof in any one of the above embodiments, and is used for capturing phenytoin sodium in a sample to be detected.

In an alternative embodiment, the immunochromatographic test paper comprises a sample pad, a combination pad and a detection pad along the flowing direction of the sample, wherein the detection pad is provided with a detection line and a quality control line; the binding pad is coated with the anti-phenytoin sodium antibody or antigen binding fragment thereof labeled by a label; the detection line is coated with a phenytoin sodium standard substance; coating the quality control line with an antibody which specifically binds to the anti-phenytoin sodium antibody or the antigen binding fragment thereof; when the sample contains sodium phenytoin, the sodium phenytoin in the sample is competitively combined with the sodium phenytoin coated on the detection line and the anti-sodium phenytoin antibody or antigen binding fragment thereof on the binding pad, the higher the sodium phenytoin content in the sample is, the less the anti-sodium phenytoin antibody or antigen binding fragment thereof is combined with the detection line, and the shallower the detection line is. And the anti-phenytoin sodium antibody or the antigen binding fragment thereof marked by the marker flows to the quality control line to be captured and developed by the antibody coated by the quality control line, so that the immunochromatographic test paper can be used.

In an alternative embodiment, the binding pad is coated with colloidal gold-labeled said anti-phenytoin sodium antibody or antigen-binding fragment thereof. In an alternative embodiment, the immunochromatography test paper is coated with a marker-labeled antibody combined with phenytoin sodium, the amino acid sequence of the heavy chain variable region of the antibody combined with phenytoin sodium is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

Example 1

Monoclonal antibodies were prepared:

preparation of sodium phenytoin-PHT antigen as shown in table 1:

TABLE 1

Antigen sources	Antigen name	Lot number	Concentration of	Total amount of	Use of the same
						Self-producing antigens	PHT	#D22012701	6.2mg/ml	5.0mg	For immunization and screening

The chemically synthesized phenytoin sodium antigen was used for stepwise immunization of experimental mice.

(II) preparation of monoclonal antibody anti-phenyl-sodium-mab 1:

healthy female mice of 6-8 weeks old Balb/c were selected and immunized according to the immunization protocol described below. The phenytoin sodium is used as an immunogen to immunize a BALB/c mouse, spleen lymphocytes of the mouse which are successfully immunized are extracted, the lymphocytes are fused with myeloma cells SP2/0 of the mouse through a cell fusion technology, and a hybridoma cell strain which stably secretes a monoclonal antibody of the phenytoin sodium is obtained after two rounds of subcloning and screening, so that the monoclonal antibody of the phenytoin sodium is obtained.

2.1, carrying out stepwise immunization on experimental mice by using chemically synthesized phenytoin sodium antigen, wherein the specific steps of animal immunization experiments comprise:

(1) Balb/c mice with consistent weight and age average were randomly divided into 2 groups, with aluminum adjuvant (aluminum hydroxide adjuvant) and without aluminum adjuvant.

(2) Before the start of the experiment, mice were each collected preimmune serum (preimmune serum was collected on the fifth day, blood was taken through eyeballs, and a proper amount of blood was taken to ensure the normal state of the mice), and the collected serum was stored at 80 ℃.

(3) The preparation mode of the aluminum adjuvant (aluminum hydroxide adjuvant) group comprises the following steps: prior to immunization, each antigen was diluted to the corresponding dose (75 μg/mouse) in 75 μl PBS, respectively, and mixed with alum adjuvant (1 mg/mouse) at a volume antigen: adjuvant=3:1 (i.e., 25 μl adjuvant was added to 75 μl immunogen dilution); shaking the adjuvant before use, and slowly dripping the injected adjuvant (25 μl) into the immunogen solution; after the adjuvant and the immunogen dilution were thoroughly mixed, the two were thoroughly mixed for 30 minutes. Allowing the adjuvant to effectively adsorb the antigen; the subsequent experiments were performed according to the experimental procedure of immunized animals.

(4) Group without aluminum adjuvant: the antigens were diluted in 100 μl PBS at the corresponding doses (75 μg/mouse) in the above table, 100 μl of immunogen, followed by the following immunization animal experimental procedure.

(5) Subcutaneous injections at 2 week intervals: the experiment was designed in 3 immunization modes, but after 7 days of each immunization, the eyeball was taken out of blood, part of the mouse supernatant was obtained by centrifugation, serum titer was detected first, and after 7 days of the last immunization, the heart was taken out of blood to obtain the maximum blood volume, and the supernatant was obtained by centrifugation and stored at 80 ℃.

2.2 detection of serum titers.

(1) 3 mice were immunized and the mice were numbered A0, A1, A2 in this order. Serum titers were measured after 3 immunizations were completed. The test data are shown in Table 2.

And respectively using phenytoin sodium as antigen competition detection, and using phenytoin sodium as coating antigen to perform indirect ELISA and competition ELISA to detect serum titers of immunized mice.

The indirect ELISA method comprises the steps of adding 1 mu g/ml of coating antigen diluted by coating liquid into each hole of an ELISA plate, coating the plate for 3 times at 4 ℃ after coating, adding 200 mu l of sealing liquid (5% skimmed milk powder) into each hole, standing for 2 hours in a 37 ℃ incubator, taking out the plate, adding 50 mu l of diluted serum into each hole, reacting for 30 minutes in the 37 ℃ incubator, adding 50 mu l of goat anti-mouse IgG-HRP solution after washing, and reacting for 30 minutes in the 37 ℃ incubator. Washing, adding 100 μl of substrate solution, developing in a 37 ℃ incubator for 10min in dark, and finally adding 2mol/L H ₂ SO ₄ The reaction was stopped at 50. Mu.L and the A450 values were read on a microplate reader. Three post-exemption orbital blood titers of 3 mice>62500。

The competition ELISA procedure was largely identical to the indirect ELISA procedure except that 50. Mu.l of diluted 50ng/ml and 300ng/ml small molecule phenytoin sodium standard solution was added after blocking with blocking solution and washing the ELISA plate, and 50. Mu.l of diluted serum antibody was added, and the remaining steps were identical. The competition detection of 50ng/ml and 300ng/ml small molecule phenytoin sodium can reach more than 50% only when 3 mice are 1:12500, and fusion can be arranged.

TABLE 2 serum potency assay data

2.3 cell fusion: the immune spleen cells are fused with myeloma cell line SP2/0 cells, the fused cells are screened by a HAT selection medium (the HAT selection medium contains hypoxanthine, aminopterin and thymine), and ELISA positive screening and subcloning are carried out on the fused cells; the screened positive monoclonal is taken as ascites, protein A/G antibody purification column is used for purifying antibody, and ELISA titer of the purified antibody is more than 1:128,000, and purity is more than 90%.

(III) ELISA detection of binding Activity to recognize phenytoin sodium

The IgG antibody titer detection method comprises the following steps:

(1) And (3) coating a bottom plate: the antigen used was diluted to 3. Mu.g/ml with coating dilution, 100. Mu.l of the prepared coating was added to each well, and the mixture was placed in a refrigerator at 4℃for 24 hours.

(2) After 24h, taking out from the refrigerator, balancing at 37 ℃ for 30min, and then discarding the liquid in the holes; washing the wells with washing liquid for 3 times and 3min each time.

(3) Closing the enzyme-labeled reaction hole: 200 μl of 5% calf serum is added into each well, and the wells are sealed for 90min at 37 ℃, and after sealing, the wells are washed 3 times with washing liquid for 3min each time.

(4) Adding a sample to be detected: diluting the sample according to the required proportion, adding the diluted sample into enzyme-labeled reaction holes, placing 100 mu l of each hole at 37 ℃ for 90min; washing the wells with washing liquid for 3 times and 3min each time.

(5) Adding enzyme-labeled antibody: adding secondary antibody with proper concentration according to the specification; 100 μl of wash was applied to each well at 37deg.C for 90 min.

(6) Adding a substrate solution: the substrate is added in an amount of 100 mu l per hole, and the mixture is placed at 37 ℃ and protected from light for 15-30 min.

(7) Terminating the reaction: the reaction was stopped by adding 50. Mu.l of stop solution to each well, and the experimental results were measured within 20 min.

(IV) detecting binding Activity of monoclonal antibody for recognizing phenytoin sodium

4.1 cell fusion and clone screening data:

the serial numbers of the mice are A0, A1 and A2 in sequence, and four rounds of fusion are completed.

A0 mice are subjected to fusion screening to pick 18 positive holes, subcloning is carried out, finally 2 cell strains are completed, and 48 OD (origin-destination) are selected through fusion screening ₄₅₀ Value of>Positive clones above 2.2 were subjected to multiple dilution detection titers and then to secondary subclone screening. 2 cell lines were obtained, designated A0-1 to A0-2, respectively.

And (3) carrying out fusion screening on the A1 mice to pick 22 positive holes, subcloning, carrying out fusion screening on the 22 positive holes, carrying out multiple ratio dilution detection titer on the selected positive holes with the OD450 value of more than 2.1, carrying out secondary and third subcloning screening, and finally, completely obtaining 4 cell strains which are named as A1-1 and A1-4 respectively.

A2 mice were screened for fusion to pick 56 positive wells, subcloned, and screened for second and third subcloning, and finally 4 cell lines were completed, designated A2-1 to A2-4, respectively.

A total of 10 cell lines were obtained after four cell fusions.

4.2 ascites preparation and detection data

3F 1 mice were challenged with each complete cell line, 10 ascites were prepared in total, and all ascites test titers were as follows:

TABLE 3 Table 3

4.3 antibody purification conditions fumbling and detection data:

the above ascites was purified by 3.3% n-octanoic acid-thiamine precipitation to obtain 10 antibodies in total, and the potency detection data of all antibodies are shown in Table 4.

TABLE 4 Table 4

The data show that the monoclonal antibodies of 10 cell lines have good specific binding capacity to phenytoin sodium antigen, and the phenytoin sodium colloidal gold product is detected as a urine sample, so that competition experiments are carried out by using the standard phenytoin sodium, and the result shows that the antibodies A1-1 and A2-2 have competition effects on small molecule phenytoin sodium, so that the antibodies A1-1 and A2-2 are used for testing the phenytoin sodium colloidal gold product.

Example 2

The phenytoin sodium antibodies A1-1 and A2-2 were validated by an immune colloidal gold platform.

Specifically, the immunochromatography test paper comprises a sample pad, a combination pad and a detection pad, wherein the detection pad is provided with a detection line and a quality control line. The binding pad is coated with the screened monoclonal antibody marked by colloidal gold, the coating concentration is 1mg/ml, the detection line is coated with phenytoin sodium standard substance, the coating concentration is 1000ng/ml, and the quality control line is coated with goat anti-mouse antibody. The experiment group is to use the Phenytoin sodium antibodies A1-1 and A2-2 obtained by the experiment to detect Phenytoin sodium standard product Phenytoin-sodium of Hangzhou An Xu biotechnology limited company. The negative control is a urine sample. The above reagents were applied, and then the results were detected using POCT detection instrument ACG1000 (ID-A003) from Hangzhou An Xu Biotechnology Co., ltd., and the results of the experimental data are shown in Table 5 and FIG. 1.

TABLE 5

In the above table, G4-G8 represent the level of the strip color shade of the test strip, with higher values representing darker colors and +/-representing a slightly darker or lighter color than that level. The low value of the added phenytoin sodium standard substance indicates that the small molecule has a competitive action, and the antibody can be combined with the phenytoin sodium standard substance. The results show that the A1-1 and A2-2 antibodies have better gradient, can be used in phenytoin sodium products, have cut-off value of 1000ng/ml, and then perform stability evaluation of the A1-1 antibody, prepare 3 batches of antibody samples, and the evaluation results are shown in the following table.

TABLE 6

The A1-1 antibody is named as anti-phenyl-sodium-mab 1 according to the evaluation result of the product, and can be used for detecting the Phenytoin sodium antigen detection kit.

Example 3

Monoclonal antibody anti-phenyl-sodium-mab 1 heavy chain V region (VH) and light chain V region (VL) sequence analysis:

the analysis method comprises the following steps:

(1) Primers for amplifying heavy chain V region (VH) and light chain V region (VL) genes were designed as follows:

heavy chain variable region forward primer (VH-FOR, SEQ ID No. 13):

CGATGGGCCCTTGGTGCTAGCGGATCCGACAGTGGATARACMGATGG；

heavy chain variable region reverse primer (VH-BACK, SEQ ID No. 14):

CTGTTTCGGGGAGTGCAGTGCCTCGAGGAGGTSMARCTGCAGSAGTCWGG；

light chain variable region forward primer (VL-FOR, SEQ ID No. 15):

CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC；

light chain variable region reverse primer (VL-BACK, SEQ ID No. 16):

GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG。

(2) Hybridoma cell lines (about 10 ⁷ And (3) extracting total RNA of the cells according to the instruction of the Trizol RNA extraction kit, performing reverse transcription to synthesize a first strand of cDNA by taking the total RNA as a template, and amplifying the VH/VL genes of the antibody by taking the amplified product as the template through PCR.

(3) The heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-phenyl-sodium-mab 1 were recovered and sequenced by the company.

(4) The VH/VL gene sequences were then analyzed.

(II) analysis results:

(1) Heavy chain variable region:

the heavy chain variable region amino acid sequence is:

VKLQESGPELVKPGASVRISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGNVNTKYNEKFKGKATLTADKSSSTAYIQLSSLTSEDSAVYFCARIYYGNYVDYWGQGTTLTVSS (SEQ ID NO.1, wherein the underlined parts represent VHCDRs 1 to 3, respectively, in sequence).

The heavy chain variable region nucleotide sequence is:

GTGAAGCTGCAGGAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATACAACTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAATCTA CTATGGTAACTACGTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO.11, wherein the underlined parts represent VHCDRs 1 to 3, respectively, in sequence).

(2) Light chain variable region:

the amino acid sequence of the light chain variable region is as follows:

DIVITQTPLTLSVTIGQPASISCKSSQSLFDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISGVEAEDLGVYYCWQGTHFPHGRSVEAPSWKSN (SEQ ID NO.2, wherein the underlined parts represent VLCDRs 1 to 3, respectively, in sequence).

The light chain variable region nucleotide sequence is:

GATATTGTGATAACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTTGATAGTGATGGAAAGACATATTTGAATTGGTTGTTGCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCGGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTT TCCTCATGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID NO. 12), wherein the underlined parts represent VLCDRs 1 to 3, respectively, in sequence.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An anti-phenytoin sodium antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of 25 th to 32 th amino acid residues, VHCDR2 of 50 th to 57 th amino acid residues and VHCDR3 of 96 th to 106 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO. 1;

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of VHFR1 in the heavy chain variable region is shown as SEQ ID No.3 and/or the amino acid sequence of VHFR2 is shown as SEQ ID No.4 and/or the amino acid sequence of VHFR3 is shown as SEQ ID No.5 and/or the amino acid sequence of VHFR4 is shown as SEQ ID No. 6;

and/or the amino acid sequence of VLFR1 in the light chain variable region is shown as SEQ ID NO.7, and/or the amino acid sequence of VLFR2 is shown as SEQ ID NO.8, and/or the amino acid sequence of VLFR3 is shown as SEQ ID NO.9, and/or the amino acid sequence of VLFR4 is shown as SEQ ID NO. 10.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of the heavy chain variable region is set forth IN SEQ IN No. 1; and/or the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, further comprising the sequence of a portion or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD;

preferably, the antigen binding fragment comprises F (ab') ₂ One or more of Fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units;

preferably, the antibody or antigen binding fragment thereof removes CDR regions, and the remaining sequence-derived species include one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human;

preferably, the antibody further comprises a heavy chain constant region and a light chain constant region;

preferably, the light chain of the antibody is a kappa chain;

preferably, the antibody is an IgG antibody.

5. A biomaterial, characterized in that the biomaterial is selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding the anti-phenytoin sodium antibody or antigen-binding fragment thereof of any one of claims 1-4;

(ii) a vector carrying the polynucleotide of (i) above.

(iii) a cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the aforementioned anti-phenytoin sodium antibody or antigen-binding fragment thereof;

preferably, the polynucleotide encodes the heavy chain variable region, and the nucleotide sequence is shown in SEQ ID NO. 11;

preferably, the polynucleotide encodes the light chain variable region and the nucleotide sequence is shown in SEQ ID NO. 12.

6. Use of an anti-phenytoin sodium antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a biomaterial according to claim 5 for the detection of phenytoin sodium for non-diagnostic and therapeutic purposes, or for the preparation of a product for the detection of phenytoin sodium.

7. A reagent or kit for detecting phenytoin sodium, comprising the anti-phenytoin sodium antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. The reagent or kit of claim 7, further comprising a label comprising one or more of an enzyme, a fluorescent molecular label, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin.

9. The reagent or kit of claim 8, wherein the reagent or kit comprises a reagent or kit for immunodetection;

preferably, the reagent or kit comprises an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic particle detection reagent or kit, an immunofluorescent detection reagent or kit, or an immunoblotting detection reagent or kit.

10. The reagent or kit according to claim 9, wherein the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip;

preferably, the immunochromatography test paper is coated with a marker-labeled antibody combined with phenytoin sodium, the amino acid sequence of a heavy chain variable region of the antibody combined with phenytoin sodium is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the antibody combined with phenytoin sodium is shown as SEQ ID NO. 2.