Anti-testosterone antibody or functional fragment thereof and application thereofTechnical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-testosterone antibody or a functional fragment thereof and application thereof.
Background
Testosterone (Teststerone, T), also known as testosterone, testosterone or testosterone, is a steroid hormone secreted by the testes of men or the ovaries of women, and testosterone is also secreted in small amounts by the adrenal glands. It is the main male sex hormone and assimilation hormone. Whether male or female, it has important health and wellness effects, including enhancing sexual desire, strength, immune function, combating osteoporosis, etc. The amount of testosterone secreted by adult males is statistically 20 times that of adult females.
Testosterone effects can be divided into anabolic and masculinizing effects, wherein anabolic effects include increases in muscle mass and strength, increases in bone mass density and strength, stimulates linear growth and skeletal maturation, etc., whereas masculinizing effects refer to secondary characteristics of men such as maturation of sexual organs, turnover of acoustic lines, and growth of beard and axilla. Testosterone affects both men and women differently, and testosterone affects the period of natural testosterone production, mainly due to the amount and time of free testosterone delivery. Testosterone is a hormone produced by the human body, if the testosterone is not a pathological cause or special condition, no additional supplement is needed, the human body is affected by excessive testosterone, male sexual precocity and testicular diseases can be caused, female infertility can be caused, and the like.
Furthermore, testosterone is one of the important sex hormones for men, and the prostate has androgen dependence, and androgen levels are closely related to prostate lesions, so diagnosis of testosterone levels plays an important role in early prostate cancer.
Currently, most of the diagnosis of T is murine monoclonal antibody. Sheep have a larger size than mice and rabbits, and are suitable for preparing a large amount of antisera, so that in the biotechnology field, sheep are widely used for producing polyclonal antibodies at present. The prior studies showed that sheep had a higher immune sensitivity and were able to recognize a wider range of epitopes than mice and rabbits, and in addition, the comparison showed that sheep antibodies generally had a higher affinity and specificity than standard mouse, rat, and rabbit antibodies (reference hybrid. 1999Apr;18 (2): 183-91.). The ultrahigh affinity and wide epitope recognition characteristics of the goat monoclonal antibody enable the goat monoclonal antibody to have great advantages in detection of small molecules with very low concentration, such as detection of hormone, drug residue and the like. Phage display technology provides a powerful tool for screening for new proteins (e.g., antibodies) that bind to a particular ligand. In phage display technology, rapid screening to isolate antibody sequences that bind with high affinity to a target antigen of interest is made possible by constructing a large library containing a large number of different antibody sequences. In monovalent phage display systems that have been developed, nucleic acids encoding different antibodies are fused to nucleic acid sequences encoding viral coat proteins (e.g., PIII proteins). In such display systems, the antibody gene fusion will be expressed at low levels in bacterial cells and wild-type PIII protein will also be expressed in the same bacterial cells, thus ensuring infectivity of the newly generated phage particles. Numerous data have disclosed methods for constructing such libraries and performing screening for antibodies of interest, such as (Biochemistry.1991Nov 12;30(45):10832-8.;;J Biol Chem.1997Apr18;272(16):10678-84.;;Methods Enzymol.2016;580:45-87.;;US5723286A;;US5427908A, etc.). This way of expressing polypeptides or antibody fragments on the surface of filamentous phage is the most important feature of phage display technology. A library is constructed by a plurality of phage particles displaying a plurality of polypeptides or antibody fragments of different sequences, respectively. The library can be constructed by cloning a family of related antibody genes, such as the BCR genes extracted after immunization of an animal with a specific antigen, or by isolating all of the BCR components from B cells of an immunized or non-immunized human. After the construction of the display library is successful, antibodies with ideal properties are obtained through different screening modes. The preparation of antibodies using phage display technology has its unique advantage, with which it is possible to develop and obtain the antibody sequence of interest in a short time without using animals, for example, using natural libraries and the like that have been commercially provided. In addition, hybridoma technology commonly used in antibody development is required to develop appropriate myeloma cells when developing antibodies of different species, and phage display technology is not limited by species. In addition, some antigens having high sequence homology with animal-derived proteins may not be able to elicit an immune response in animals, and thus it is difficult to generate target B cells, in which case the target antibodies cannot be developed using hybridoma technology, and phage display technology may bypass the problem of immune tolerance, and screen for suitable antibody sequences.
Disclosure of Invention
According to the invention, through preparing T immunogen, immunizing female sheep of 6-12 months of age, collecting fresh peripheral blood of sheep with sufficient titer after 6-9 times of immunization, and using the fresh peripheral blood for separating PBMC to construct a gene library. Through the screening of phage display technology, the invention provides a sheep monoclonal antibody with very high recognition reactivity to T protein,
The anti-human T antibody or antigen-binding portion thereof comprises:
The anti-human T sheep monoclonal antibody or a functional fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR 1-HCDR 3 are amino acid sequences consistent with HCDR 1-HCDR 3 of a heavy chain variable region shown in SEQ ID NO. 1, and the LCDR 1-LCDR 3 are amino acid sequences consistent with LCDR 1-LCDR 3 of a light chain variable region shown in SEQ ID NO. 2.
Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact systems.
Preferably, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system,
Wherein, the HCDR1 is shown as SEQ ID NO. 3;
The HCDR2 is shown as SEQ ID NO. 4;
the HCDR3 is shown as SEQ ID NO. 5;
The LCDR1 is shown as SEQ ID NO. 6;
The LCDR2 is shown as SEQ ID NO. 7;
and the LCDR3 is shown as SEQ ID NO. 8.
In this context, the terms "variable region", "CDR" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues contributing to the binding affinity of an antibody or antigen binding fragment to an antigen or epitope recognized thereby.
Herein, the heavy chain variable region (heavy chain complementarity determining region) is denoted by "HCDR" which includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is denoted by "LCDR" which includes LCDR1, LCDR2 and LCDR3. CDR labelling methods commonly used in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the novel standardized numbering system introduced by Lefranc et al in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The present disclosure uses Kabat annotation standards to identify CDR regions, but other methods to identify CDR regions are within the scope of the present invention.
Herein, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, meaning regions other than CDRs in an antibody heavy chain variable region (which may be denoted VH) and a light chain variable region (which may be denoted VL), wherein the heavy chain framework region is denoted by "HFR" and may be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework region is denoted by "LFR" and may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3, and LFR4 framework regions.
Preferably, the antibody or functional fragment thereof further comprises framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region,
Wherein the HFR1 is an amino acid sequence as shown in EQ ID NO 9 or an amino acid sequence having at least 80% homology therewith;
The HFR2 is an amino acid sequence as shown in EQ ID NO 10 or an amino acid sequence having at least 80% homology therewith;
The HFR3 is an amino acid sequence as shown in EQ ID NO. 11 or an amino acid sequence having at least 80% homology therewith;
the HFR4 is an amino acid sequence as shown in EQ ID NO.12 or an amino acid sequence having at least 80% homology therewith;
the LFR1 is an amino acid sequence as shown in EQ ID NO. 13 or an amino acid sequence with at least 80% homology thereto;
The LFR2 is an amino acid sequence as shown in EQ ID NO. 14 or an amino acid sequence with at least 80% homology thereto;
the LFR3 is an amino acid sequence as shown in EQ ID NO. 15 or an amino acid sequence with at least 80% homology thereto;
the LFR4 is an amino acid sequence as shown in EQ ID NO. 16 or an amino acid sequence having at least 80% homology thereto.
In this context, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the corresponding framework region (SEQ ID NO:9, 10, 11, 12, 13, 14, 15 or 16) described above. In alternative embodiments, the antibody or functional fragment thereof binds human T with an affinity of KD.ltoreq.10 10-7M、KD≤10-8M、KD≤10-9M、KD≤10-10 M or KD.ltoreq.10 10-11.
Preferably, the constant region is also included;
optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;
Alternatively, the heavy chain constant region is selected from the group consisting of IgG1, igG2, igG3, igG4, igA, T, igE, and IgD heavy chain constant regions;
alternatively, the constant region is of a species source of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock or human;
alternatively, the heavy chain constant region is an amino acid sequence as shown in EQ ID NO. 17 or an amino acid sequence having at least 80% homology thereto, and the light chain constant region is an amino acid sequence as shown in EQ ID NO. 18 or an amino acid sequence having at least 80% homology thereto.
Preferably, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above-described functional fragments are readily available to those skilled in the art based on the structure of the intact antibodies of the disclosure.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by applied biosystems, and the like.
In another aspect, the invention also discloses an antibody conjugate comprising the antibody or a functional fragment thereof and a coupling moiety coupled thereto;
Optionally, the coupling moiety is selected from one or more of a purification tag or a detectable label, such as colloidal gold, a radiolabel, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron dense label, such as a radioisotope, a fluorophore, rhodamine and derivatives thereof, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin-labeling;
Alternatively, the coupling moiety is selected from the group consisting of solid supports, such as magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.
In another aspect, the invention features a nucleic acid encoding the antibody or a functional fragment thereof.
Nucleic acids are typically RNA or DNA, and nucleic acid molecules may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are used when they are incorporated into vectors.
Some embodiments of the present disclosure also provide vectors containing the above nucleic acid molecules.
Some embodiments of the present disclosure also provide cells comprising the above vectors.
Some embodiments of the present disclosure also provide a method of preparing an antibody or functional fragment thereof, comprising culturing a cell as described above, and isolating and purifying the antibody or functional fragment thereof from the culture product.
Based on the amino acid sequence of the antibody or functional fragment thereof in the present disclosure, it is easy for a person skilled in the art to prepare the antibody or functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or functional fragment thereof as described in any one of the above, and it is within the scope of the present disclosure to prepare the antibody or functional fragment thereof by any one of the techniques based on this.
In another aspect, the invention discloses a detection reagent or kit comprising said antibody or functional fragment thereof, said antibody conjugate.
In alternative embodiments, the blocking agent contained in the kit may be in the form of a liquid solution, attached to a solid support, or as a dry powder. When the blocking agent is a liquid solution, the liquid solution may be an aqueous solution. When the immune blocking agent is in the form of an attached solid support, the preferred solid support may be a chromatographic medium such as a film, test strip, plastic bead or plate, or a microscope slide. When the blocking agent is a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
Preferably, the kit is a chemiluminescent detection kit.
In another aspect, the invention discloses the use of said antibody or functional fragment thereof, said antibody conjugate, or said reagent or kit for the preparation of a testosterone protein assay product.
In another aspect, the invention features a method of detecting testosterone protein in a sample, the method comprising:
a) Contacting testosterone protein antigen in said test sample with said antibody or functional fragment thereof, said antibody conjugate, or said reagent or kit under conditions sufficient for an antibody/antigen binding reaction to occur, and
B) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the antigen in the test sample;
Optionally, in step a), a second antibody is further included in the immunocomplex, the second antibody being bound to the antibody or antigen binding fragment;
Optionally, in step a), a second antibody is further included in the immunocomplex, the second antibody binding to the testosterone protein antigen.
Advantageous effects
The present disclosure provides anti-T sheep monoclonal antibodies that have high affinity, high reactivity, high sensitivity or specificity to human T. The kit is used for immunodetection, has good specificity and high affinity, and provides a detection means for immunodiagnosis of T. Therefore, the anti-T antibody, the immunoassay reagent and the kit provided by the disclosure have excellent practical performance and wide market application prospect.
Drawings
FIG. 1 is a schematic diagram of an antibody gel disclosed by the invention, wherein the left side is a marker, and the right side is a purified antibody reduction band;
FIG. 2 shows the results of an experiment for detecting the interaction of antibodies with human T antigen using an enzyme-linked immunosorbent assay (ELISA);
FIG. 3 shows the results of detection of clinical samples using the chemiluminescent method for the antibodies of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. The specific conditions are not noted in the examples or embodiments and are carried out according to conventional conditions or conditions suggested by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are fully explained in the literature, such as the molecular cloning laboratory Manual (Molecular Cloning: ALaboratoryManual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (OligonucleotideSynthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I.Freshney, 1987), enzymatic methods (Methodsin Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), laboratory immunology handbook (Handbookof Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), gene transfer vectors for mammalian cells (GENE TRANSFER Vectors forMammalian Cells) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (CurrentProtocols in Molecular Biology) (F.M.Ausubel et al, 1987), PCR chain reactions (The Polymerase Chain Reaction) and PCR (Current Protocols in Immunology) are explicitly incorporated by reference in each of the literature, J.M.Miller and M.P.Calos.
Example 1T antibody preparation
1. Preparation of human T immunogens
Human T was digested with pepsin in sodium citrate buffer solution at 25-37 ℃ and ph=3.5 for 2 hours to obtain Fc end. Purifying by His tag to obtain a T-Fc end with purity of more than 75%, constructing the T-Fc end into pcDNA3.1 (+) vector, transfecting into HEK293 cells (the plasmid dosage is 1 ug/mL), culturing for 5 days, and purifying by Ni excel column to obtain the human T-Fc immunogen.
2. Immunization of animals
3 Healthy female sheep with the age of 6-12 months are selected for carrying out immunization operation. The primary immunization was carried out by using 250ug of human T-Fc immunogen, mixing Freund's complete adjuvant in a ratio of 1:1 and emulsifying, and multipoint immunization was carried out on the left/right side underarm, around the cervical lymph node, shoulder and back, etc. Thereafter, booster immunizations were performed at 3 week intervals using Freund's incomplete adjuvant. Serum detection titers were collected after each immunization until the serum titers were >100 ten thousand qualified (6-9 immunizations). One week after the last immunization, sheep peripheral blood was collected and PBMCs were isolated.
3 Healthy female sheep with the age of 6-12 months are selected for carrying out immunization operation. The primary immunization was carried out by using 250ug of human T-Fc immunogen, mixing Freund's complete adjuvant in a ratio of 1:1 and emulsifying, and multipoint immunization was carried out on the left/right side underarm, around the cervical lymph node, shoulder and back, etc. Thereafter, booster immunizations were performed at 3 week intervals using Freund's incomplete adjuvant. Serum detection titers were collected after each immunization until the serum titers were >100 ten thousand qualified (6-9 immunizations). One week after the last immunization, sheep peripheral blood was collected and PBMCs were isolated.
3. Peripheral blood PBMC preparation
The immunized sheep with higher titer was selected, 50mL of fresh blood was collected, and the blood sample was diluted with PBS 1:1. 5 50mL centrifuge tubes were taken, 20mL of Histopaque (Sigma) separation solution was added, and 20mL of diluted blood sample was carefully added to each tube above the liquid surface of the Histopaque separation solution, and the mixture was centrifuged at 400g for 35 minutes at room temperature to separate PBMC. After centrifugation, granulocytes, platelets, red blood cells pass through the separation liquid to the bottom of the centrifuge tube, while PBMCs are above the Histopaque separation liquid. The collected PBMCs were washed 2 times with PBS and cells were collected by centrifugation at 300 g.
4. Phage display library construction
Using freshly collected sheep PBMC cells, 1mL TRIzolTM Reagent was added per 1E7 cells. The mixing was repeated upside down until all cells were lysed without apparent precipitation and left at room temperature for 10 minutes. Adding chloroform with the volume of 1/5 of the solution, shaking vigorously and mixing for 1min, and fully mixing. The mixture was centrifuged at 12000g for 10min at 4℃and the supernatant collected. The supernatant was carefully added to an equal volume of isopropanol, mixed well upside down, and left to stand at room temperature for 30 minutes to precipitate RNA. RNA pellet was collected by centrifugation at 12000g for 10min at 4℃and washed with 800ul of 75% ethanol, then ethanol was evaporated, and RNA was dissolved and quantified with 50ul of RNase-free water. cDNA was prepared by reverse transcription as described in the specification using PRIMESCRIPTTM II REVERSE TRANSCRIPTASE (2690A). Sheep scFv immune repertoire against human T-Fc immunogen was constructed according to the published experimental methods. (reference) J Immunol Methods.2000Mar 6;236(1-2):133-46.;;Phage Display—A Practical Approach.Edited by T.Clacksonand H.B.Lowman Oxford University Press,Oxford,UK;2004;)
The PCR primer of the cloned sheep V region gene adopted in the invention is shown as SEQ ID NO. 19-39, wherein VHF1、VHF2、VHF3、VHF4、VHR1、VHR2、VHR3、VLF1、VLF2、VLR1、VLR2、VLR3、VLR4、VLR5、VLR6、VLR7、VKF1、VKF2、VKR1、VKR2、VKR3 corresponds to SEQ ID NO. 19-39 in sequence.
The amplification of each gene fragment and the splice assembly of scFv were performed using Phanta Max Super-FIDELITY DNA Polymerase (P505-d 3) manufactured by Nanjinouzan Biotechnology Co., ltd.
The PCR amplification conditions were 95℃pre-denaturation for 3min (1 cycle), 95℃denaturation 13s,56℃annealing 15s,72℃extension 30s (30 cycles), 72℃final extension for 5min (1 cycle). Each amplified fragment was purified and recovered using a DNA agarose gel purification kit (OMEGA, D2500-02). The resulting scFv fragments after splicing need to be ligated into the pComb3X phagemid vector (addgene, # 63890) after cleavage with SfiI restriction enzyme (NEB, R0123L). Ligation products were purified using a DNA purification kit (OMEGA, D6492-02) and used to shock transform TG1 competent cells (Agilent, 200123). After recovery culture, all TG1 cells obtained by transformation were plated on plates containing 100ug/mL Amp antibiotics and cultured overnight, and the resulting culture was a gene library. The TG1 gene library is subjected to infection by M13K07 helper phage (NEB, N0315S) and then is subjected to overnight culture, and a culture supernatant is subjected to polyethylene glycol precipitation to prepare the phage display library.
5. Phage-antibody panning and clone identification
To panning to obtain the appropriate antibodies set forth in this invention, the antigen human T-Fc immunogens were diluted to 100ug/ml,10ug/ml,1ug/ml and 0.1ug/ml, respectively, using PBS, and the immune tubes (NUNC MaxiSorpTM) were coated, respectively. 5E12 phage-scFv particles were incubated with coated antigen and washed with PBS containing 0.1% Tween 20. Each round of panning gradually decreased the concentration of coated antigen and increased the number of washes. The bound phage of interest was eluted using 10mM HCl and the eluted solution was neutralized using 1M Tris-HCl, pH 8.3. The eluted phage of interest was infected with TG1 cells that proliferated in the fresh exponential phase and plated on YTAG plates (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 100ug/mL ampicillin, 2% glucose) to obtain a monoclonal. The monoclonal cultures were picked to OD600 of 0.5, infected M13K07 helper phage (NEB, N0315S), cultured overnight in YTAK medium (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 100ug/mL ampicillin, 50ug/mL kanamycin) with a large number of phage-scFv monoclonal particles present in the culture supernatant. The culture supernatant was used for ELISA to identify clones that specifically bind to human T-Fc immunogenic proteins. The gene coding sequence of the phage-scFv is obtained by gene sequencing.
6. Antibody expression purification
1. The gene sequence is synthesized by Shanghai, and is constructed on a pCDNA3.4 vector;
2. resuscitating and passaging the Expi293 cells, and inoculating 24 hours before transfection with a density of 1E 6;
3. On the day of transfection, the cell density reaches 2-2.5E6, and the cell activity is more than 98 percent to start transfection;
4. the plasmid addition was 1ug/ml, the heavy to light chain ratio was 1:1, and the plasmid to PEI ratio was 1:3. Mixing uniformly and then at room temperature;
5. standing for 15min, and adding into cell sap prepared in advance. Suspension culture at 37℃and 120 rpm;
6. After 24 hours, adding the feed 1 with the addition amount of 1%;
7. after 48 hours, adding the feed supplement 2 with the addition amount of 2 percent;
8. after 5 days of fermentation, the cell viability is more than 70%, and the sample is collected by centrifugation at 5000rpm and 4 ℃;
9. prepare G column, PBS equilibrate 5 column volumes;
10. 2ml/min flow rate through the column, and after the sample is passed through the column, balancing the volume of 5 columns by PBS;
11. Eluting with 0.5M glycine pH2.7, and dialyzing against PBS buffer;
12. After concentration in the ultrafiltration tube, the BCA method was used for quantification to give a final concentration of 5mg/mL of T-mAb antibody, and the result of SDS-PAGE after reduction was shown in FIG. 1.
Example 2 antibody Performance validation
This example demonstrates the binding properties of antibodies of the invention by their interaction with human T-Fc.
The specific procedure was as follows, the antibody was diluted to 1ug/mL using a coating solution (0.05M pH 9.5 carbonate and bicarbonate buffer) and coated onto a conventional 96-well ELISA plate. The purified human T-Fc obtained after recombinant expression was subjected to biotin modification using a biotinylation reagent (final commercial reagent). The biotin-modified human T-Fc was diluted to the respective target concentrations (0.3 ng/ml,0.8ng/ml,2.5ng/ml, 200 ng/ml). To ELISA wells coated with the antibody of interest, a specific concentration of human T-Fc was added, and 100. Mu.l per well was incubated at 37℃for 30min. After washing the plates with plate wash (150 mM NaCl solution containing 0.05% Tween-20), streptavidin-horseradish peroxidase conjugate (finished commercial reagent) was added at a dilution of 1:5000, and 100. Mu.l per well was added for 15min incubation at 37 ℃. Finally, ELISA color development is performed, and an enzyme-labeled instrument is used for detecting OD450 absorbance.
The results are shown in Table 1 and FIG. 2, and are obtained by testing the interaction of antibodies with recombinant expression purified human T-Fc using an enzyme-linked immunosorbent assay (ELISA) assay. The abscissa indicates the concentration of human T-Fc used, and the ordinate indicates the detection result of ELISA experiments, i.e. the absorbance detected by the spectrophotometer at 450nm (OD 450), with higher OD450 values indicating stronger detected interactions. The results show that the interaction between the antibody and the human T-Fc is strong, and the antibody is suitable for detecting the human T-Fc.
TABLE 1 Linear experiment results
| Human T-Fc (ng/mL) | T-mab |
| 200 | 2.297 |
| 66.7 | 2.111 |
| 22.2 | 1.706 |
| 7.4 | 0.98 |
| 2.5 | 0.446 |
| 0.8 | 0.185 |
| 0.3 | 0.109 |
| 0 | 0.042 |
EXAMPLE 3 chemiluminescent functional assay of T-mAb antibodies
In order to verify the effect of the purified T antibody applied to an immunoassay, the detection of the antibody at present mainly comprises immunonephelometry, immunochromatography, chemiluminescence and the like, wherein the chemiluminescence has the most strict requirements on the performance of the antibody, so that the chemiluminescent competition method is selected for measurement in the embodiment. Chemiluminescent kits are commercially available.
This example selects the antibodies purified in 1 for the assay.
Assay reagents, including R1, R2 and magnetic bead solutions.
The R1 comprises biotin-labeled T-mAb, and the using concentration is 1ug/ml;
the R2 comprises avidin-labeled magnetic beads, and the concentration is 0.1ug/ml.
The specific operation steps are as follows:
1. The experiment preparation comprises 5ml of R1 and R2 working solutions, 4ml of magnetic bead solution, calibrator and T-mAb.
2. The components are respectively placed into a kit, and then the kit is placed into a full-automatic chemiluminescence immunoassay analyzer EXI1800 (from Zhongyuan Hui Biotechnology Co., ltd.) to select a T item (10 ul, 160ul, 2125ul and 30ul of magnetic bead solution) for experiment.
Clinical samples 40 random clinical samples can be collected in various hospitals.
TABLE 2 clinical sample test results
The results are shown in Table 2 and FIG. 3, and the consistency of the detection results and clinical results of the reagent of the present invention is high, which indicates that the antibody of the present invention has excellent clinical detection performance.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.