CN101126760A - All-human source follicle stimulating hormone beta single-chain antibody screening method and its uses - Google Patents
All-human source follicle stimulating hormone beta single-chain antibody screening method and its uses Download PDFInfo
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Abstract
The utility model relates to a whole human anti follicule-stimulating hormone (FSH) Beta single chain antibody. Phage antibody that contains anti-A epitope antibody genes is screened out from the whole human single chain phage antibody library and conducted soluble expression to get the whole human single chain antibody of anti FSH-Beta A epitope; the A epitope is the 33-53 amino acid residue on FSH-Beta subunit and comprises the amino acid sequence of Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg. The utility model also relates to a method that utilizes phage display technology to screen the whole human anti FSH-Beta single chain antibody. The whole human anti FSH-Beta single chain antibody of the utility model can be applied to the purposes of anti-fertility, detection kit preparation and relevant diseases treatment.
Description
Technical field
The present invention relates to monoclonal antibody technique, total man source antibody technique relates in particular to the application all-human source follicle stimulating hormone beta single-chain antibody choosing method that display technique of bacteriophage carried out and the purposes of this antibody thereof.
Background technology
Pituitary gonadotropic hormone (GTH) cell synthesizes and release follicular stimulating hormone (FSH) and lutropin (LH) under hypothalamus GnRH stimulates.In the women, FSH acts on the ovarian follicle of ovary, comprise promote that folliculus generates, before the ovulation folliculus select, by capsule cell originate that androgenic aromatization effect stimulates that estrogen generates, granular cell leuteinization and induce the LH acceptor.The male sex, the androgenic testosterone (T) of the follicular stimulating hormone (FSH) of hypophysis secretion and testis secretion itself is spermatogenetic sharp attemperator.T and FSH acting in conjunction be in sustentacular cell of testis (Sertoli) and peritubular cell, thereby start and keep the spermatogenesis process.FSH is cell-mediated by Sertoli to spermatogenetic regulating action, the Sertoli cell is unique body cell with fsh receptor in various mammals and the human testicle, the main performance of effect: the 1. spermatogenetic startup of induced animal and people or start, 2. cause that hypophysis rat and hibernator spermatogenesis restart, 3. participate in keeping spermatogenesis with T, necessary to spermatogenesis especially in quantity and the normal fully institute of quality.In theory, anti-FSH specific antibody is eliminated the biological action of endogenous FSH, so has the effect of anti-male fertility; And anti-FSH specific antibody do not influence the effect of LH, and T is synthetic unaffected in man's testis, does not need supplemented with exogenous T.The male-contraception of other modes (except that androgen) as merging progestational hormone, GnRH analog, GnRH antibody etc., all needs the androgen of supplemented with exogenous.The research report that anti-FSH antibody of a small amount of application or FSH vaccine antifertility are abroad arranged, present problem has two aspects, the one, anti-FSH antibody is incomplete for the spermatogenetic blocking effect of man, and non-human antibody is applied to human allergy, autoimmunity etc.; Next is that the source of human antibody is extremely difficult.
For the treatment of true precocious puberty, at present mainly round the correlative study and the treatment of maincenter gonadotropin-releasing hormone (GRH) (GnRH) and acceptor thereof.The effect of blocking-up FSH, also can reach the purpose of treatment true precocious puberty, but yet there are no the report that utilizes FSH-β Antybody therapy men and women sex premature, be because non-human antibody is applied to human allergy, autoimmunity etc. equally, and the source of human antibody is extremely difficult.
Often need to detect blood FSH level clinically in reproductive medicine, gynemetrics, andrology, division of endocrinology, paediatrics etc., main at present serum radioimmunoassay and the chemiluminescence method of adopting.These two kinds of methods come with some shortcomings, and because of FSH is pulsatile secretion, 20%～40% fluctuation are arranged from the peak valley to the summit, and the result of single blood sampling often can not react basis or average level, and interior on the same day repeated multiple times is taken a blood sample thick and fast and be infeasible.And, the women of normal menstrual cycle, blood FSH is cyclic fluctuation in each cycle, reaches peak value in the onset of ovulation, but only keeps 1～2 day, usually is difficult to capture, adopt continuous every day or the next day to get the method for blood also infeasible clinically.After the nineties, the someone adopts urine to extract FSH (or LH) back and is measuring with radioimmunoassay, but the drainage of quantitative test urine FSH (or LH), and the hormone in the reflection circulation changes indirectly.But because the FSH instability in the urine is dissociated into α and β subunit easily, so the simple urine FSH that measures may not reflect blood FSH level truly.
Monoclonal antibody is with a wide range of applications.The Monoclonal Antibody technology is to merge bone-marrow-derived lymphocyte (but can not infinitely break up) that can produce antibody and myeloma cell's (but can not produce antibody) that can infinitely break up to get up to form hybridoma, and the latter not only enough produces antibody but also can infinitely break up.But also have significant limitation, particularly the background in its property source, mouse source has greatly limited monoclonal antibody as the application of treatment preparation in human body.Self limitation of mouse endogenous antibody mainly comprise following some: (1) mouse monoclonal antibody has immunogenicity to human body, produces human antimouse antibody and brings out allergic reaction; (2) the effective biological effect function of human activin of mouse monoclonal antibody; (3) half life period of mouse monoclonal antibody in human body also shorter, removed by body easily; (4) the mouse monoclonal antibody molecular weight is bigger, is difficult for passing cell membrane and enters in the endochylema; (5) mouse source property monoclonal antibody complicated process of preparation, immune animal, Fusion of Cells difficulty are bigger, are unfavorable for large-scale production; (6) the mouse monoclonal antibody preparation cost is also higher.
Summary of the invention
Fundamental purpose of the present invention is to provide the anti-follicular stimulating hormone beta single-chain antibody of the total man source with potential medical science and pharmacy value---total man's resource monoclonal antibody of anti-FSH-β " A " epi-position.
The present invention also aims to provide the screening technique of a total man source anti-FSH-beta single-chain antibody.
The present invention also aims to provide the purposes of total man source anti-FSH-beta single-chain antibody in male-contraception.
The present invention also aims to provide the purposes of total man source anti-FSH-beta single-chain antibody in the preparation detection kit.
The present invention also aims to provide the purposes of total man source anti-FSH-beta single-chain antibody in the precocious disease of therapeutic.
The foregoing invention purpose is achieved by the following technical programs.
Total man provided by the invention source anti-follicular stimulating hormone beta single-chain antibody, be by filtering out the phage antibody that contains anti-" A " epitope antibodies gene in the single-chain phage antibody library of total man source and carrying out solubility expression, obtain the total man source single-chain antibody of anti-FSH-β " A " epi-position, described " A " epi-position is the 33-53 amino acid residue in FSH-β subunit, have: Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg (be paddy-paddy-half Guang-essence-phenylpropyl alcohol-half Guang-different bright-Si-different is bright-asparagus fern-Su-Su-Se-half Guang-third-Gan-junket-half Guang-junket-Su-essence) amino acid sequence, molecular weight 2.5Kda.
Anti-FSH-beta single-chain antibody provided by the invention, the variable region of heavy chain of this antibody of encoding and chain variable region gene be preferred SEQ ID No.1 and SEQ ID No.2 respectively, secondly also preferably SEQ ID No.3 and SEQ ID No.4.
The screening technique of total man provided by the invention source anti-FSH-beta single-chain antibody is:
1. the preparation of antigen, antibody library and Related Bacteria strain
Synthetic FSH-β fragment (antigenic peptides), this fragment is one section specific fragment in the FSH-β subunit, promptly " A " epi-position is one of antigenic determinant.This fragment is the FSH-β 33-53 of a subunit amino acid residue, has: the amino acid sequence of Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg.Molecular weight 2.5Kda, proterties is white crystalline powder, and is soluble in water, is used for screening and identifies described antibody.
Obtain: the total man source phage antibody library that a) is used to screen described phage antibody;
B) be used for the helper phage of superinfecting phage antibody library;
C) be used for the Escherichia coli that phasmid increases;
D) be used for the Escherichia coli of antibody solubility expression.
2. the amplification of total man source single-chain phage antibody library
Activate amplification by mode to described phage antibody library inoculation and the superinfection of adding helper phage.
3. screen FSH-phagus beta antibody
With described synthetic FSH-β fragment is antigen, and the single-chain phage antibody library of above-mentioned amplification has been carried out the affine screening of several wheels " absorption-wash-out-amplification " enrichment.
4. identify FSH-phagus beta antibody
Identify anti-FSH-phagus beta antibody with ELISA, identify anti-FSH-phagus beta antibody gene, carry out anti-FSH-phagus beta antibody gene order-checking with PCR.
5. anti-FSH-phagus beta solubility expression and purifying
Adopt anti-FSH-phagus beta antibody to infect the method that E.coli HB2151, IPTG induce E.coli HB2151, will resist FSH-phagus beta antibody solubility expression is anti-FSH-beta single-chain antibody;
The anti-FSH-beta single-chain antibody that adopts His-trap affinity column purification of soluble to express.
6. identify the anti-FSH-beta single-chain antibody of solubility expression
Adopt PCR to identify anti-FSH-beta single-chain antibody gene, adopt non-competing ELISA method to measure the antibody affinity costant, adopt ELISA and Western blotting method to identify antibody specificity.
Total man provided by the present invention source FSH-beta single-chain antibody is applied to the male contraceptive pill of male-contraception or the male contraceptive pill that merge to use, because of its amplification and convenient for production, cost is low, high specificity; Can be directly used in the human body,, reduce the immunogenicity of mouse endogenous antibody greatly, have good development prospect because be total man source antibody.
The invention provides the purposes of another kind of treatment true precocious puberty disease.Because anti-FSH-β antibody combines the effect of antagonism FSH with the FSH of hypophysis secretion in vivo.Ageing, effect specificity that this effect has is especially more effective for the patient of high FSH.Stop using about 3 months, antibody titer reduces final the disappearance, and antagonism FSH effect disappears, and will make the patient recover the process of growing naturally.In clinical practice, easy to use, cost is low; Total man source antibody reduces immunogenicity exogenous or the mouse endogenous antibody greatly.
The invention provides the antibody reagent of the most critical in the detection kit of a kind of formation determination blood and urine FSH-β subunit concentration.Can partly substitute needed clinically blood FSH horizontal detection such as present reproductive medicine, gynemetrics, andrology, division of endocrinology, paediatrics, the method for measuring urine FSH-β subunit concentration is provided.Because the FSH in the urine fully is dissociated into α and β subunit, utilizes anti-FSH-β TPPA of the present invention β subunit wherein, and proofread and correct through UCr.Use TPPA urine FSH-β level of the present invention, can be applied to judge women's onset of ovulation and pregnant woman fetus situation, and the recovery situation of ovulating in postpartum, the low clinical classification diagnosis of clinical classification diagnosis, men and women's sexual dyspenesis and gonad function of men and women's property sex premature can be applied to, Menopause and the infull monitoring of male sex's part androgen can also be applied to.
The present invention adopts phage antibody library technique to make himself to have following advantage: 1) phage antibody library technique, with Escherichia coli as the host, filobactivirus is as the carrier of expressing and showing, makes the DNA of antibody and encoding antibody to be present in the host bacterium jointly; 2) phage antibody library replaces B cell clone expressing antibodies with bacterial clone, without Fusion of Cells, not even through immunity, can prepare the total man source antibody at any antigen; 3) antibody of phage antibody library preparation is the people source fully, and human body is not had immunogenicity, can not cause allergic reaction, and can be directly used in the human body; 4) antibody of phage antibody library preparation is the people source fully, the biological effect function of more effective human activin; 5) phage antibody library can prepare Fab or scFv antibody fragment, and molecular weight is little, and has kept the position (heavy chain and variable region of light chain) of full molecular antibody conjugated antigen, and the easier cell membrane that passes enters in the endochylema; 6) phage antibody library prepares antibody fragment, can utilize the in-vivo diagnostic and the treatment of the guidance quality expansion disease of antibody on this basis further with antibody fragment and medicine, toxin or radioactive nuclide coupling; 7) phage antibody library technique is easy and simple to handle, does not need the process of complicated Fusion of Cells, does not even need immunity, can directly take the peripheral blood of patients mononuclearcell; 8) phage antibody library technique prepares total man source antibody, and is lower with respect to the mouse monoclonal antibody cost.
The advantage of total man of the present invention source anti-FSH-beta single-chain antibody is: 1) utilize synthetic FSH-β fragment as screening antigen, the purity height is one section specific fragment in the FSH-β subunit, is one of antigenic determinant; 2) the anti-FSH-beta single-chain in total man source antibody specificity is better, does not have cross reaction with people FSH-α, GH, HCG; 3) can partly substitute the anti-FSH-β of mouse source property antibody and be applied to FSH in clinical detection blood and the urine; 4) the anti-FSH-beta single-chain in total man source antibody keeps the specificity of complete antibody molecule conjugated antigen, but a little less than the immunogenicity of self, for clinical practice is laid a good foundation, antifertility and treatment relevant disease is had potential application prospect.
Description of drawings
Fig. 1 is bacteriophage tactic pattern figure.
Fig. 2 has shown the phasmid tactic pattern, and heavy chain of antibody gene link position is between Sfi I/Nco I and Xho I restriction enzyme site; Light chain of antibody gene link position is between Sal I and Not I restriction enzyme site.
Fig. 3 is that anti-FSH-phagus beta antibody is taken turns the affine The selection result of " absorption-wash-out-amplification " enrichment through 4, and column diagram is represented relative output capacity, and the 4th relative output capacity of taking turns screening reaches 2.0 * 10
-3
Fig. 4 is the column type figure that anti-FSH-phagus beta antibody ELISA is identified, shows that 10 reactions are the light absorption value in the hole (B3, H5, G6, C11, G5, E11, G11, F2, E7, C1) of strong positive, compares with negative control (N) light absorption value.
Fig. 5 is that anti-FSH-phagus beta antibody 96 hole ELISA Plate ELISA detect actual effect, and the left side is 10 minutes photos of TMB colour developing; The right side is H
2SO
4Photo after the cessation reaction.B3, the H5, G6, C11, G5, E11, G11, F2, the hole positive reactions such as E7, C1 that are positioned on the 96 hole ELISA Plate are strong.
Fig. 6 is anti-FSH-phagus beta antibody V
κGene PCR product gel electrophoresis.Figure left side swimming lane is followed successively by B3, H5, G6, DL2000 molecular weight standard, C11, G5, E11 from top to bottom, and figure right side swimming lane is followed successively by G11, F2, E7, DL2000 molecular weight standard, negative control, C1 from top to bottom.Show above-mentioned 10 strain phage antibody V
κGene exists, and the PCR molecular weight of product is 368bp.
Fig. 7 is anti-FSH-phagus beta antibody V
H+ V
κGene PCR product gel electrophoresis.Figure left side swimming lane is followed successively by B3, H5, G6, C11, G5, E11, G11, DL2000 molecular weight standard from top to bottom, and figure right side swimming lane is followed successively by B3, the G6 of solubility expression, the DL2000 molecular weight standard of F2, E7, C1, negative control, solubility expression from top to bottom.The V that shows above-mentioned 10 strain phage antibodies and 2 strain single-chain antibodies
H+ V
κGene exists, and the PCR molecular weight of product is 935bp.
Fig. 8 is that IPTG induces anti-FSH-beta single-chain antibody SDS-PAGE result.Left side figure is the B3 single-chain antibody, and be followed successively by from left to right: E.coli HB2151, B3-HB2151 do not induce bacterium liquid, and B3 induces supernatant, and B3 induces the ultrasonic supernatant of bacterium, and B3 induces bacterium ultrasound precipitation, low molecular weight protein (LMWP) Marker; Right figure is the G6 single-chain antibody, and be followed successively by from left to right: E.coli HB2151, G6-HB2151 do not induce bacterium liquid, and G6 induces supernatant, and G6 induces the ultrasonic supernatant of bacterium, and G6 induces bacterium ultrasound precipitation, low molecular weight protein (LMWP) Marker.Do not detect albumen at the 30Kda place in the supernatant; E.coli HB2151 and do not induce in the bacterium and all see more weak band at the 30Kda place, the expressed proteins product of expression bacterium own; The bacterium process is induced, ultrasonic degradation, centrifugal, and the cleer and peaceful 30Kda of being deposited in all sees at the place strong positive expression in the cracking, the abduction delivering of expression soluble antibody.
The SDS-PAGE of 0.1M～0.5M imidazoles eluted product when Fig. 9 is the anti-FSH-beta single-chain of His-trap affinity column purifying antibody.Swimming lane is followed successively by from left to right: 0.1M, 0.2M, 0.3M, 0.4M, 0.5M imidazoles eluted product, low molecular weight protein (LMWP) Marker.Showing in 0.2M and the 0.3M imidazoles eluted product has 30Kda albumen, and 0.3M imidazoles eluted product is the unicity protein product.
Figure 10 is that anti-FSH-beta single-chain antibody specificity is identified ELISA column diagram as a result.The 1st group of antigen is Human Fallicle-Stimulating Hormone FSH-α, concentration 500IU/ml (37 μ g/ml); The 2nd group of antigen is human growth hormone (HGH) GH, concentration 100 μ g/ml; The 3rd group of antigen is human chorionic gonadotrophin HCG, concentration 5000IU/ml; The 4th group of antigen is Human Fallicle-Stimulating Hormone FSH-β, concentration 200IU/ml (15 μ g/ml); The 5th group of antigen is synthetic FSH-β fragment, concentration 1000 μ g/ml; The 6th group of antigen is synthetic FSH-β fragment, concentration 500 μ g/ml.Each group column diagram is followed successively by from left to right and uses anti-FSH-beta single-chain antibody titer is 1: 1,1: 2,1: 4, negative control.Represent that anti-FSH-beta single-chain antibody of the present invention only combines with FSH-β specificity, and do not have the Ag-Ab association reaction with FSH-α, HCG, GH.
Figure 11 is urine protein SDS-PAGE result, and sample is the menopausal women overnight urine that ammonium sulfate salting-out process concentrates.Swimming lane is respectively 25% saturated ammonium sulfate saltout product, 50% saturated ammonium sulfate saltout product, 75% saturated ammonium sulfate saltout product, 100% saturated ammonium sulfate saltout product, low-molecular-weight Marker from left to right.
Figure 12 is urine protein Western blotting result, use anti-FSH-beta single-chain antibody of the present invention, antigen is the menopausal women overnight urine that ammonium sulfate salting-out process concentrates, swimming lane is respectively 25% saturated ammonium sulfate product, 50% saturated ammonium sulfate product, 75% saturated ammonium sulfate product, the 100% saturated ammonium sulfate product of saltouing of saltouing of saltouing of saltouing from left to right, and two bands are followed successively by full molecule FSH, FSH-β from top to bottom.Show and to saltout menopausal women urine 50% and 75% saturated ammonium sulfate urine FSH-β is positive in the product.
Figure 13 is the Western blotting result who identifies anti-FSH-beta single-chain antibody specificity, and antigen is respectively menopausal women from left to right and urinates 50% saturated ammonium sulfate saltout product, HCG, GH, and two bands are followed successively by full molecule FSH, FSH-β from top to bottom.Represent that anti-FSH-beta single-chain antibody of the present invention only combines with FSH-β specificity in the menopausal women urine, and do not have the Ag-Ab association reaction with HCG, GH.
Figure 14 is that anti-FSH-beta single-chain antibody is used for ELISA method mensuration urine FSH horizontal detection actual effect.The left side is 10 minutes photos of TMB colour developing; The right side is H
2SO
4Photo after the cessation reaction.
Figure 15 is that anti-FSH-beta single-chain antibody is used for the typical curve that the ELISA method is measured urine FSH level.Horizontal ordinate is the logarithm of FSH-beta antigen standard items concentration, and ordinate is a light absorption value; The line of broken line for directly being formed by connecting, the typical curve that smooth curve forms for the SPSS match.
Embodiment
The screening of embodiment one total man source anti-FSH-beta single-chain antibody
1. the preparation of antigen, antibody library and Related Bacteria strain
1) synthetic FSH-β fragment is synthesized 21 amino acid residues of total length (FSH-β subunit 33-53 amino acid residue) by Shanghai Chinese Academy of Sciences biological chemistry and RESEARCH ON CELL-BIOLOGY.Amino acid residue sequence:
Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg, (paddy-paddy-half Guang-essence-phenylpropyl alcohol-half Guang-different is bright-and Si-different is bright-asparagus fern-Su-Su-Se-half Guang-third-Gan-junket-half Guang-junket-Su-essence), molecular weight 2.5Kda, synthetic purity 99.8%.Synthetic FSH-β fragment proterties is white crystalline powder, and is water-soluble.
2) total man source single-chain phage antibody library: storage capacity is about 1 * 10
8, pIT2 phasmid carrier comprises gene III, myc tag and His tag, has ampicillin (Amp) resistance.Available from Britain Camb Medical Research Council (MRC).Bacteriophage structure and phasmid structure are seen Fig. 1, Fig. 2.
3) helper phage KM13: have kanamycins (Kana) resistance, be used for the superinfecting phage antibody library.Available from Britain Camb Medical Research Council (MRC).
4) e. coli tg1, genotype is: K12, supE, hsd, Δ 5, thi, Δ (lac-proAB) F ' [traD36proAB+lacIqlacZ Δ M15].Amber mutation inhibition type is used for the amplification of phasmid.Available from Britain Camb Medical Research Council (MRC).
5) Escherichia coli HB2151, genotype is: K12 (lac-pro), ara, nalr, thi/F ', proAB, lacIq, lacZ, Δ M15.Non-amber mutation inhibition type is used to express soluble antibody.Available from Britain Camb Medical Research Council (MRC).
6) the anti-M13 bacteriophage monoclonal antibody of HRP mark is available from Amersham company.The staphylococcal protein A of HRP mark is available from doctor's moral company.
7) the PCR primer can betting office be synthesized by the Shen, Shanghai.Dna sequence dna is measured by Shanghai Ying Jun Bioisystech Co., Ltd.
8) the His-trap affinity column is available from Amersham company.20 * LumiGLO Kit is available from Cell Signaling.6 orifice plates and 96 hole ELISA Plate are available from Costar company.
9) tryptone, yeast extract are available from Oxoid company.DNA Marker, albumen Marker are epoch company limiteds available from sky, Beijing.
2. the amplification of total man source single-chain phage antibody library
Method I
1) Jiang Yuanku is seeded in the warm 2 * TY nutrient culture media of 200ml and (contains final concentration 100 μ g/ml Amp+1% glucose), 250rpm, and 37 ℃ of joltings are cultured to OD
600nmBe about 0.4.
2) therefrom get 50ml bacterium liquid, add 1 * 10
12Pfu helper phage KM13 superinfection, water-bath 30min in 37 ℃ of constant water bath box.Residue 150ml bacterium liquid prepares secondary phage antibody library.
3) with bacterium liquid with the centrifugal 10min of 3000g, precipitate resuspended with 100ml 2 * TY nutrient culture media (containing final concentration 100 μ g/ml Amp+50 μ g/ml Kana+0.1% glucose), 250rpm, 30 ℃ of jolting overnight incubation.
4) get the centrifugal 30min of bacterium liquid 3300g, collect supernatant and be about 80ml, add 20ml PEG/Nacl solution, mixing is placed on 1h on ice.
5) get the centrifugal 30min of bacterium liquid 3300g, precipitation is resuspended with 4ml PBS, with the abundant mixing of gas bath constant temperature oscillator.
6) get the centrifugal 10min of bacterium liquid 10800g, get supernatant, 4 ℃ of preservations are used for the antibody library screening.
7) phage antibody library titer determination:
A. get bacteriophage supernatant 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
B. get a liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
C. get b liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
D. get c liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
E. get d liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
F. respectively get 10 μ l phage-infects, 200 μ l exponential phase E.coli TG1 from c, d, e liquid, 37 ℃ of water-bath 30min are laid on the TYE culture plate and (contain final concentration 100 μ g/ml Amp), 37 ℃ of overnight incubation.
G. count the bacterial clone number next day and calculate phage titre.
The amplification of antibody library activation as a result reaches 10
12Cfu/ml can screen.
Method II
37 ℃ of joltings of step 1) bacterium among the method I are cultivated can be to OD
600nmBe about 0.4～0.6;
Step 4) among the method I adds PEG/Nacl solution and is placed on ice that the time can change from 0.5h～4h, the longlyest can note keeping 0 ℃ of low temperature to spending the night, and sedimentation effect is good;
Step 7) phage antibody library titer determination among the method I:
A. get bacteriophage supernatant 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
B. get a liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
C. get b liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
D. get c liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
E. get d liquid 10 μ l and add 1ml 2 * TY nutrient culture media, fully mixing.
F. from c, d, e liquid, respectively get 37 ℃ of warm Top Agar that 10 μ l bacteriophages add 3ml, be laid on TYE culture plate top layer (containing final concentration 100 μ g/ml Amp), 37 ℃ of overnight incubation.
G. count plaque quantity next day and calculate phage titre.
All the other are with method I, and this method can directly be identified bacteriophage quantity, and reflects indirectly without bacterial infection quantity.
3. screen FSH-β phagocytosis antibody
Method I
1) be that antigen (100 μ g/ml) wraps by 6 orifice plates with the synthetic FSH-β fragment of 1ml, coating buffer is PBS (pH7.4), and 4 ℃ of wrapper sheets spend the night.
2) PBS washs 6 orifice plates 3 times, each 5min.
3) in 6 orifice plates, fill it up with 2%MPBS, 37 ℃ of sealing 2h.
4) remove confining liquid, add the phage antibody of amplification, hatch 2h for 37 ℃.
5) remove unconjugated phage antibody, the PBST damping fluid washs 6 orifice plates 10 times (first round washing 10 times, later every washing 20 times of taking turns).
6) 1mg/ml trypsase 500 μ l add 6 orifice plates, hatch 30min for 37 ℃, piping and druming repeatedly, the phage antibody of wash-out specificity combination.
7) 500 μ l eluents infect 3.5ml exponential phase E.coli TG1,37 ℃ of water-bath 30min.
8) quantity of titration wash-out bacteriophage:
A. get previous step bacterium liquid 40 μ l and add 360 μ l, 2 * TY nutrient culture media, fully mixing.
B. get a liquid 4 μ l and add 396 μ l, 2 * TY nutrient culture media, fully mixing.
C. get b liquid 4 μ l and add 396 μ l, 2 * TY nutrient culture media, fully mixing.
D. get c liquid 4 μ l and add 396 μ l, 2 * TY nutrient culture media, fully mixing.
E. b, c, d liquid respectively being got 100 μ l is laid on the TYE culture plate and (contains final concentration 100 μ g/ml Amp), 37 ℃ of overnight incubation.9) bacterium liquid removes supernatant with the centrifugal 5min of 10800g, precipitates resuspendedly with 1ml 2 * TY nutrient culture media (containing final concentration 100 μ g/ml Amp+1% glucose), is laid on the TYE culture plate 37 ℃ of overnight incubation behind the mixing.
10) add 2ml 2 * TY nutrient culture media (containing final concentration 15% glycerine) next day on the TYE culture plate, gently scrape all bacterium colonies, mixing with the glass hairclipper.
11) get 50 μ l bacterium liquid and add 50ml 2 * TY nutrient culture media (containing final concentration 100 μ g/ml Amp+1% glucose), 250rpm, 37 ℃ of joltings are cultured to OD
600nmBe about 0.4.All the other bacterium liquid add final concentration 15% glycerine ,-70 ℃ of preservations.
12) from 50ml 2 * TY nutrient culture media, get 10ml, add 5 * 10
10Pfu helper phage KM13,37 ℃ of water-bath 30min.
13) the centrifugal 30min of 3300g removes supernatant, precipitate resuspended with 50ml 2 * TY nutrient culture media (containing final concentration 100 μ g/ml Amp+50 μ g/mlKana+0.1% glucose), 250rpm, 30 ℃ of jolting overnight incubation.
14) the centrifugal 15min of 3300g, honest and upright and thrifty 40ml in the collection adds 10ml PEG/Nael solution, and mixing is placed on 1h on ice.
15) the centrifugal 30min of 3300g, precipitation is resuspended with 2ml PBS, fully mixing.
16) the centrifugal 10min of 10800g gets honest and upright and thrifty 2ml, and titration adds the quantity of bacteriophage, and the 1ml supernatant is used for the next round screening, remains about 1ml supernatant and places 4 ℃ of preservations.
17) repeat above screening step, carry out four-wheel " absorption-wash-out-amplification " enrichment screening altogether.
The result is an antigen with synthetic FSH-β fragment, and single-chain phage antibody library has been carried out the affine screening of four-wheel " absorption-wash-out-amplification " enrichment.After each took turns screening, the phage antibody yield rate all had increase, and the phage antibody yield rate of four-wheel screening has improved 2000 times than the first round, saw Table 1 and Fig. 3.
Table 1 four-wheel phage antibody screening yield rate relatively
| The screening wheel number | Add bacteriophage quantity | Wash-out bacteriophage quantity | Relative output capacity * |
| 1 2 3 4 | 1.0×10 12 1.0×10 12 1.0×10 12 1.0×10 12 | 1.0×10 6 5.0×10 7 3.0×10 8 2.0×10 9 | 1.0×10 -6 5.0×10 -5 3.0×10 -4 2.0×10 -3 |
*Relative output capacity=wash-out bacteriophage quantity/adding bacteriophage quantity
Other method
Method I 1) antigen concentration can be between 10～100 μ g/ml in the step, coating buffer can change the sodium bicarbonate solution of pH9.6 into, the wrapper sheet time can be at 37 ℃ of bags by 2h;
Method I 6) elution process can change additive method in the step, comprises soda acid eluant, eluent etc.;
Method I 16) identify that bacteriophage quantity can adopt direct method (directly identifying bacteriophage quantity) or indirect method (reflection indirectly behind the phage-infect bacterium) in the step;
Whole screening technique not only can use the solid phase screening method, and other wrap screened method can also to use liquid phase, magnetic bead etc.
Embodiment two FSH-phagus beta antibody mediated immunity activity identification
1.ELISA identify anti-FSH-phagus beta antibody
Method I
1) last is taken turns to screen to be laid on the TYE culture plate after the bacterium that obtains is diluted and (contains final concentration 100 μ g/ml Amp), 37 ℃ of overnight incubation.
2) every hole adds 2 * TY nutrient culture media 100 μ l (containing final concentration 100 μ g/ml Amp+1% glucose) in 96 orifice plates, from the TYE culture plate at random 90 colony inoculations of picking in 96 orifice plates (remain 6 holes and do not add bacterium), 250rpm, 37 ℃ of jolting overnight incubation.
3) get one 96 orifice plate again, every hole adds 2 * TY nutrient culture media 200 μ l (containing final concentration 100 μ g/ml Amp+1% glucose), distinguishes transferase 45 μ l bacterium liquid to the second block plate from first 96 each hole of orifice plate, 250rpm, and 2h are cultivated in 37 ℃ of joltings.The first 96 every hole of orifice plate glycerol adding to final concentration is 15% ,-70 ℃ of preservations.
4) the second 96 every hole of orifice plate adds 2 * TY nutrient culture media 25 μ l (containing final concentration 100 μ g/ml Amp+1% glucose), and every hole adds 1 * 10 again
9Pfu helper phage KM13 superinfection, 250rpm, 1h is cultivated in 37 ℃ of joltings.
5) the centrifugal 10min of 1800g, precipitate resuspended with 200 μ l, 2 * TY nutrient culture media (containing final concentration 100 μ g/ml Amp+50 μ g/ml Kana), 250rpm, 30 ℃ of jolting overnight incubation.
6) the centrifugal 10min of 1800g gets supernatant and carries out the ELISA detection.
7) be antigen coated the 3rd 96 hole ELISA Plate with the synthetic FSH-β fragment (100 μ g/ml) of 100 μ l, coating buffer is PBS (pH7.4), and 4 ℃ of wrapper sheets spend the night.
8) PBS washs 96 hole ELISA Plate 3 times, and 2%MPBS is filled it up with in every hole, 37 ℃ of sealing 2h.
9) remove confining liquid, the every hole of 96 hole ELISA Plate adds 2%MPBS 150 μ l, and 95 holes add phage antibody 50 μ l (the A1 hole does not add antibody, adds 50 μ l 3%BSA and compares) more respectively, and mixing is hatched 2h for 37 ℃.
10) washing of PBST damping fluid is 3 times, removes unconjugated phage antibody.
11) every hole adds the anti-M13 bacteriophage monoclonal antibody of the HRP mark of dilution in 1: 3000, hatches 1h for 37 ℃.
12) washing of PBST damping fluid is 3 times, and every hole adds 100 μ l TMB colour developing liquid, and room temperature is placed 10min.
13) every hole, the clear back of colour developing adds the sulfuric acid cessation reaction of 50 μ l 1mol/L.
14) measure every hole 450nm light absorption value, positive criteria is that P/N value (Positive/Negative) is greater than 2.1.
15) negative control is that 3%BSA alternative is anti-.
The result is 95 phage antibodies of picking at random, measure every hole A
450nmLight absorption value (seeing Table 2), positive criteria are that P/N value (Positive/Negative) is greater than 2.1.Have 17 feminine genders (P/N＜2.1), a little less than 11 positive (P/N=2.1-5.0), 17 positives (P/N=5.0-10.0), 31 strong positives (P/N=10.0-20.0), 19 strong positives (P/N＞20.0).10 the strongest positive holes are followed successively by B3, H5, G6, C11, G5, E11, G11, F2, E7, C1 (representing with underscore respectively), and the negative contrast in A1 hole is referring to Fig. 4, Fig. 5.
The anti-FSH-phagus beta of table 2 antibody ELISA detects
1 2 3 4 5 6 7 8 9 10 11 12
A 0.057 0.447 1.047 0.820 1.398 0.243 0.583 0.618 1.150 0.088 0.087 0.502
B 0.995 0.595
2.344 0.776 0.617 0.462 0.654 0.165 0.147 0.753 0.458 0.408
C
1.506 0.921 1.387 0.169 1.484 0.526 0.500 0.076 0.861 1.554
1.972 0.791
D 0.083 0.136 0.874 0.095 0.572 0.756 0.092 0.084 0.154 0.856 0.090 0.090
E 1.288 0.109 1.002 1.046 0.874 0.480
1.560 1.029 1.052 0.111
1.822 0.761
F 0.635
1.589 0.104 0.099 0.423 1.070 0.863 0.195 0.347 1.921 0.403 0.587
G 0.764 0.845 1.312 0.649
1.858 1.986 1.373 0.518 0.105 0.127
1.807 0.145
H 0.434 0.375 0.546 0.100
2.270 0.338 0.120 0.414 0.701 0.233 0.097 0.090
Method II
The antigen concentration of step 7) can be between 10～100 μ g/ml among the method I, and coating buffer can change the sodium bicarbonate solution of pH9.6 into, and the wrapper sheet time can be at 37 ℃ of bags by 2h;
Available 1%～the 3%BSA of sealing in the step 8) among the method I;
Two anti-concentration can be between 1: 1000～1: 10000 in the step 11) among the method I;
Step 14) can be measured every hole 650nm light absorption value in addition among the method I, and every hole light absorption value is pressed A
450nm-A
650nmCalculate;
All the other are with method I.
2.PCR identify anti-FSH-phagus beta antibody gene
Method
1) chooses the darkest 10 clone: B3, H5, G6, C11, G5, E11, G11, F2, E7, the C1 of above-mentioned ELISA colour developing.From first 96 orifice plate, get 20 μ l bacterium liquid, add 2 * TY nutrient culture media 2ml (containing final concentration 100 μ g/ml Amp+1% glucose), 37 ℃ of jolting overnight incubation.
2) get bacterium liquid and carry out bacterium liquid PCR, E.coli TG1 compares.
(a) primer:
V
κUpstream: DPK5FR15 '-CAT CTG TAG GAG ACA GAG TC-3 '
Downstream: pHEN 5 '-CTA TGC GGC CCC ATT CA-3 '
V
H+ V
κUpstream: LMB35 '-CAG GAA ACA GCT ATG AC-3 '
Downstream: pHEN 5 '-CTA TGC GGC CCC ATT CA-3 '
(b) reaction system:
10×Ex Taq Buffer 2.5μl
2.5mmol/L dNTPs 2.5μl
25mmol/L MgCl
2 2.5μl
Upstream primer 0.5 μ l
Downstream primer 0.5 μ l
Bacterium liquid (template) 1 μ l
Ex Taq 0.5μl
H
2O 15μl
Cumulative volume 25 μ l
(c) reaction conditions: * 30 circulations
95℃ 5min
95℃ 1min
56℃ 1min
72℃ 1min(V
κ)/2min(V
H+V
κ)
72℃ 10min
4℃ 10min
3) 1% agarose gel electrophoresis is identified antibody gene.
The result
368bp band (V all appears in 10 clones
κ), see Fig. 6: figure left side swimming lane is followed successively by B3, H5, G6, DL2000 molecular weight standard, C11, G5, E11 from top to bottom, and figure right side swimming lane is followed successively by G11, F2, E7, DL2000 molecular weight standard, negative control, C1 from top to bottom.Show above-mentioned 10 strain phage antibody V
κGene exists, and the PCR molecular weight of product is 368bp.
935bp band (V all appears in 10 clones
H+ V
κ), see Fig. 7: figure left side swimming lane is followed successively by B3, H5, G6, C11, G5, E11, G11, DL2000 molecular weight standard from top to bottom, and figure right side swimming lane is followed successively by B3, the G6 of solubility expression, the DL2000 molecular weight standard of F2, E7, C1, negative control, solubility expression from top to bottom.The V that shows above-mentioned 10 strain phage antibodies and 2 strain single-chain antibodies
H+ V
κGene exists, and the PCR molecular weight of product is 935bp.
3. anti-FSH-phagus beta antibody gene order-checking
Method
Each 500 μ l of B3-HB2151, G6-HB2151 bacterium liquid serve extra large Ying Jun Bioisystech Co., Ltd and measure sequence, and primer is synthetic again by the said firm.
The result
B3 inserts fragment: variable region of heavy chain V
H: 339bp (113aa); Variable region of light chain V
κ: 324bp (108aa).
G6 inserts fragment: variable region of heavy chain V
H: 339bp (113aa); Variable region of light chain V
κ: 324bp (108aa).
Sequential analysis shows that B3 and G6 sequence have 12 amino acid differences, all to be positioned at V
HAnd V
κThe hypervariable region (hypervariable region, HV): V
H52,54,56,57 amino acid positions (HV2), V
H99,101 amino acid positions (HV3).V
κ50-51 amino acid position (HV2), V
H91-94 amino acid position (HV3).
The B3 sequence
V
H(SEQ ID No.1)：
GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA
GCC TCT GGA TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG
TGG GTC TCA TCG ATT TAG AAG CTT GGT CGG CAT ACA AGG TAC GCA GAC TCC GTG AAG GGC CGG TTC ACC
ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC
GTA TAT TAC TGT GCG AAA CCT GGG AGG AGG TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC
V
κ(SEQ ID No.2)：
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC CGG
GCA AGT CAG AGC ATT AGC AGC TAT TTA AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC
TAT GCT GCA TCC AGT TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT GGG ACA GAT TTC ACT
CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TAC TGT CAA CAG AGT TAC AGT ACC CCT AAT
ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
The G6 sequence
V
H(SEQ ID No.3)：
GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA
GCC TCT GGA TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG
TGG GTC TCA TCG ATT GAG AAG TAG GGT AAT AAG ACA AGG TAC GCA GAC TCC GTG AAG GGC CGG TTC ACC
ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC
GTA TAT TAC TGT GCG AAA AAG GGG GCT AGG TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC
V
κ(SEQ ID No.4)：
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC CGG
GCA AGT CAG AGC ATT AGC AGC TAT TTA AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC
TAT CGT GCA TCC CGT TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT GGG ACA GAT T7C ACT
CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TAC TGT CAA CAG GGG GAT AGG ACT CCT AAT
ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
4. the solubility expression of anti-FSH-phagus beta antibody
4.1 anti-FSH-phagus beta antibody infects E.coli HB2151
Method I
1) identifies 2 clone: B3 of selection and G6 positive the strongest preceding several clones from above-mentioned ELISA, respectively get 10 μ l phage antibodies (non-TG1 bacterium), infect 200 μ l exponential phase E.coli HB2151 respectively, 37 ℃ of water-bath 30min.
2) quantity of bacteriophage is infected in titration: doubling dilution is measured infection rate.
A. get phage-infect E.coli HB2151 10 μ l and add 100 μ l, 2 * TY nutrient culture media, fully mixing.
B. get a liquid 10 μ l and add 100 μ l, 2 * TY nutrient culture media, fully mixing.
C. get b liquid 10 μ l and add 100 μ l, 2 * TY nutrient culture media, fully mixing.
D. get c liquid 10 μ l and add 100 μ l, 2 * TY nutrient culture media, fully mixing.
E. get d liquid 10 μ l and add 100 μ l, 2 * TY nutrient culture media, fully mixing.
F. get e liquid 10 μ l and add 100 μ l, 2 * TY nutrient culture media, fully mixing.
G. get c, d, e, f liquid is laid on the TYE culture plate and (contains final concentration 100 μ g/ml Amp), 37 ℃ of overnight incubation.The E.coli HB2151 that gets 100 μ l is as negative control.
H. count the bacterial clone number next day and calculate infection rate.
3) add 2 * TY nutrient culture media 2ml (containing final concentration 100 μ g/ml Amp+1% glucose), 250rpm, 37 ℃ of jolting overnight incubation respectively.
4) getting B3-HB2151, G6-HB2151 bacterium liquid respectively makes PCR and identifies.
The result
B3:5 * 10
9Cfu, G6:6 * 10
9Cfu, both all infect success, and bacterium liquid PCR the results are shown in shown in Figure 7.
Method II
Can identify that infecting back E.coli HB2151 discharges bacteriophage and judge and infect successfully;
The evaluation of efficiency of infection can be adopted direct method (directly identifying bacteriophage quantity) or indirect method (reflection indirectly behind the phage-infect bacterium);
Its solubility expression adopts: anti-FSH-phagus beta antibody infection E.coli HB2151, the order-checking of anti-FSH-phagus beta antibody gene, IPTG induce E.coli HB2151 to produce the method for anti-FSH-beta single-chain antibody, adopt the anti-FSH-beta single-chain of His-trap affinity column purifying antibody.
4.2IPTG induce E.coli HB2151 to produce anti-FSH-beta single-chain antibody
Method
1) learn from else's experience B3-HB2151, the G6-HB2151 bacterium liquid 200 μ l of sequencing add 2 * TY nutrient culture media 20ml, inoculation in 1: 100,37 ℃ of jolting overnight incubation.
2) 20ml is spent the night bacterium liquid adds to 1000ml 2 * TY nutrient culture media, 1: 50 switching E.coli HB2151, and 37 ℃ of joltings are cultured to OD
600nmBe about 0.9.
3) adding final concentration is the IPTG of 1mmol/L, 250rpm, and 16h is cultivated in 30 ℃ of joltings.
4) 12000rpm, 4 ℃ of centrifugal 10min get bacterial precipitation.
5) bacterial precipitation is resuspended with 10ml PBS, places ultrasonic-wave crushing on ice, 50 watts of power, ultrasonic time 30s, quiescent interval 30s, work number of times 50 times.
6) 12000rpm, 4 ℃ of centrifugal 10min get supernatant.Adding PMSF is 100 μ g/ml to final concentration, and Sodium azide to final concentration is 0.02%.
7) get 50 μ l+2 * sample-loading buffers, 50 μ l+1mol/L DTT, 10 μ l, mixing, boiling water bath 5min.
8) go up sample 15 μ l in the 12%SDS-PAGE gel, 100V constant voltage electrophoresis 2h.Coomassie brilliant blue R250 dyeing 30min, destainer decolours transparent to background color.
The result
IPTG induces anti-FSH-beta single-chain antibody SDS-PAGE result referring to Fig. 8: the left side is a B3 antibody, is followed successively by from left to right: E.coli HB2151, B3-HB2151 do not induce bacterium liquid, B3 induces supernatant, B3 induces the ultrasonic supernatant of bacterium, and B3 induces bacterium ultrasound precipitation, low molecular weight protein (LMWP) Marker; The right side is a G6 antibody, is followed successively by from left to right: E.coli HB2151, G6-HB2151 do not induce bacterium liquid, and G6 induces supernatant, and G6 induces the ultrasonic supernatant of bacterium, and G6 induces bacterium ultrasound precipitation, low molecular weight protein (LMWP) Marker.Do not detect albumen at the 30Kda place in the supernatant; E.coli HB2151 and do not induce in the bacterium and all see more weak band at the 30Kda place, the expressed proteins product of expression bacterium own; The bacterium process is induced, ultrasonic degradation, centrifugal, and the cleer and peaceful 30Kda of being deposited in all sees at the place strong positive expression in the cracking, the abduction delivering of expression soluble antibody.
5.His-trap the anti-FSH-beta single-chain of affinity column purifying antibody
Method
Get B3 as the purifying object.
1) the ultrasonic broken bacterium supernatant of B3 prevents to stop up affinity column with 0.22 μ m filter membrane degerming.
2) Binding Buffer 4ml+0.5mol/L EDTA 400 μ l add affinity column.
3) distilled water 4ml adds affinity column.
4) 0.1mol/L nickelous sulfate 2ml adds affinity column.
5) distilled water 4ml adds affinity column.
6) Binding Buffer 2ml adds affinity column.
7) bacteria-removing liquid of 2ml method 5 (1) adds affinity column.
8) Binding Buffer 4ml adds affinity column, flush away non-specific binding albumen.
9) get 1ml Eluting Buffer (0.1M-0.5M imidazoles) and add affinity column, wash-out antibody respectively.
10) get 50 μ l+2 * sample-loading buffers, 50 μ l+1mol/L DTT, 10 μ l, mixing, boiling water bath 5min.
11) go up sample 15 μ l in the 12%SDS-PAGE gel, 100V constant voltage electrophoresis 2h.Coomassie brilliant blue R250 dyeing 30min, destainer decolours transparent to background color.
The result
0.1M～0.5M imidazoles eluted product is carried out SDS-PAGE, the results are shown in Figure 9.Swimming lane is followed successively by from left to right among the figure: 0.1M, 0.2M, 0.3M, 0.4M, 0.5M imidazoles eluted product, low molecular weight protein (LMWP) Marker.Showing in 0.2M and the 0.3M imidazoles eluted product has 30Kda albumen, and 0.3M imidazoles eluted product is the unicity protein product.
6. identify anti-FSH-beta single-chain antibody
6.1 the antibody affinity costant is measured
Method
(1) 1.45A by formula
280nm-0.74A
260nmCalculate 4 batches purifying B3 antibody concentration, use 3%BSA doubling dilution to 1: 128.
(2) be that antigen (concentration is respectively 100 μ g/ml, 50 μ g/ml) wraps respectively by 96 hole ELISA Plate with synthetic FSH-β fragment, coating buffer is PBS (pH7.4), and 4 ℃ of wrapper sheets spend the night.(seeing table 3 for details)
(3) PBS washs 96 hole ELISA Plate 3 times, and 3%BSA is filled it up with in every hole, 37 ℃ of sealing 2h.
(4) remove confining liquid, add the anti-FSH-beta single-chain antibody of doubling dilution, hatch 2h for 37 ℃.3%BSA substitutes antibody and makes negative control.
(5) washing of PBST damping fluid is 3 times, removes unconjugated antibody.
(6) every hole adds the staphylococcal protein A of the HRP mark of dilution in 1: 800, hatches 1h for 37 ℃.
Table 3 antibody affinity costant is measured the wrapper sheet synoptic diagram
| 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| Antigen | 100 | 50 | 100 | 50 | 100 | 50 | 100 | 50 | 100 | |
| |
1∶2 | 1∶2 | 1∶2 | 1∶2 | ||||||
| |
1∶4 | 1∶4 | 1∶4 | 1∶4 | ||||||
| |
1∶8 | 1∶8 | 1∶8 | 1∶8 | ||||||
| |
1∶16 | 1∶16 | 1∶16 | 1∶16 | ||||||
| |
1∶32 | 1∶32 | 1∶32 | 1∶32 | | |||||
| G | ||||||||||
| 1∶64 | 1∶64 | 1∶64 | 1∶64 | |||||||
| |
1∶128 | 1∶128 | 1∶128 | 1∶128 | N | |||||
| 0.3M 8-29 | 0.5M1 8-31-1 | 0.5M2 8-31-1 | 0.2M 8-31-2 | |||||||
(7) washing of PBST damping fluid is 3 times, and every hole adds 100 μ l TMB colour developing liquid, and room temperature is placed 10min.
(8) every hole, the clear back of colour developing adds the sulfuric acid cessation reaction of 50 μ l 1mol/L.
(9) measure every hole 450nm and 630nm light absorption value, press A
450nm-A
630nmCalculate every hole light absorption value.Positive criteria is that P/N value (Positive/Negative) is greater than 2.1.
When (10) SPSS13.0 software is analyzed two kinds of antigen concentrations respectively, the logarithm of antibody concentration and relation between the light absorption value and matched curve.
(11) according to two curves of match, the actual concentrations [Ab] of antibody when calculating 1/2 maximum OD value respectively
t[Ab ']
t
(12) K by formula
Aft=1/{2[Ab ']
t-[Ab]
tThe calculating affinity costant.
The result
Every hole light absorption value sees table 4 for details.
Table 4 antibody affinity costant is measured light absorption value
| 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| B | 0.812 | 0.769 | 0.891 | 0.973 | 0.958 | 1.076 | 1.222 | 1.336 | ||
| C | 0.688 | 0.605 | 0.865 | 0.884 | 0.951 | 0.907 | 1.139 | 1.162 | ||
| D | 0.642 | 0.462 | 0.851 | 0.791 | 0.769 | 0.700 | 1.036 | 1.061 | ||
| E | 0.333 | 0.288 | 0.737 | 0.787 | 0.568 | 0.485 | 0.827 | 0.832 | ||
| F | 0.288 | 0.230 | 0.628 | 0.712 | 0.416 | 0.449 | 0.589 | 0.515 | 0.144 | |
| G | 0.240 | 0.227 | 0.516 | 0.524 | 0.268 | 0.274 | 0.696 | 0.340 | ||
| H | 0.284 | 0.216 | 0.474 | 0.349 | 0.225 | 0.215 | 0.305 | 0.286 | 0.144 | |
| Numbering | 0.3M 8-29 | 0.5M1 8-31-1 | 0.5M2 8-31-1 | 0.2M 8-31-2 | N | |||||
The Bradford method is measured the protein concentration of antibody:
0.3M 8-29:0.24mg/ml is about 8.0 μ mol/L with molecular weight 30kD conversion.
0.5M1 8-31-1:1.26mg/ml is about 42 μ mol/L with molecular weight 30kD conversion.
0.5M2 8-31-1:0.23mg/ml is about 7.7 μ mol/L with molecular weight 30kD conversion.
0.2M 8-31-2:1.02mg/ml is about 33.9 μ mol/L with molecular weight 30kD conversion.
Non-competing ELISA method is measured the antibody affinity costant, sees table 5 for details.
The non-competing ELISA method of table 5 is measured the antibody affinity costant
| The antibody numbering | Ka(L/mol) | Kd(mol/L) |
| 0.3M 8-29 0.5M1 8-31-1 0.5M2 8-31-1 0.2M 8-31-2 | 3.79×10 6 4.00×10 6 4.05×10 6 4.32×10 6 | 2.64×10 -7 2.50×10 -7 2.47×10 -7 2.31×10 -7 |
6.2 the specificity of anti-FSH-beta single-chain antibody is identified
Method
1) taking off the row hormone is respectively antigen coated 96 hole ELISA Plate and makes antibody specificity and identify that positive control is the FSH-beta antigen fragment of 100 μ g/ml.Various antigens are all got 100 μ l wrapper sheets, and coating buffer is PBS (pH7.4), and 4 ℃ of wrapper sheets spend the night.(seeing table 6 for details)
The anti-FSH-beta single-chain of table 6 antibody specificity is identified the wrapper sheet synoptic diagram
| 5 | 6 | 7 | 11 | 12 | ||
| Fruit receives sweet smell | Pu Likang | FSH- | ||||
| A | ||||||
| 1∶1 | 1∶1 | 1/1 | 1/2 | |||
| |
1∶2 | 1∶2 | ||||
| |
1∶4 | 1∶4 | ||||
| D | | BSA | ||||
| E | ||||||
| 1∶1 | 1∶1 | |||||
| |
1∶2 | 1∶2 | ||||
| |
1∶4 | 1∶4 | ||||
| H | BSA | BSA | ||||
| Think true | Pregnyl | |||||
A. human chorionic gonadotrophin HCG (Pregnyl Pregnyl, Ou Jianong), concentration 5000IU/ml.
B. human growth hormone (HGH) GH (think true Saizen, Xue Lannuo), concentration 100 μ g/ml.
C. Human Fallicle-Stimulating Hormone FSH-α (fruit that fragrant Gonal-f, Xue Lannuo), concentration 500IU/ml (37 μ g/ml).
D. Human Fallicle-Stimulating Hormone FSH-β (Pu Likang Puregon, Ou Jianong), concentration 200IU/ml (15 μ g/ml).
2) PBS washs 96 hole ELISA Plate 3 times, and 3%BSA is filled it up with in every hole, 37 ℃ of sealing 2h.
3) remove confining liquid, add the anti-FSH-beta single-chain antibody (1/1,1/2,1/4) of doubling dilution, hatch 2h for 37 ℃.
4) washing of PBST damping fluid is 3 times, removes unconjugated antibody.
5) every hole adds the staphylococcal protein A of the HRP mark of dilution in 1: 800, hatches 1h for 37 ℃.
6) washing of PBST damping fluid is 3 times, and every hole adds 100 μ l TMB colour developing liquid, and room temperature is placed 10min.
7) every hole, the clear back of colour developing adds the sulfuric acid cessation reaction of 50 μ l 1mol/L.
8) measure every hole 450nm and 630nm light absorption value, press A
450nm-A
630nmCalculate every hole light absorption value.Positive criteria is that P/N value (Positive/Negative) is greater than 2.1.
The result
Anti-FSH-beta single-chain antibody specificity is identified, is shown that this antibody only combines with FSH-β specificity, and all do not combine with FSH-α, HCG, GH.The result sees table 7, Figure 10 for details.
The anti-FSH-beta single-chain of table 7 antibody specificity is identified ELISA result
| 5 | 6 | 11 | 12 | |||
| Fruit receives sweet smell | Pu Likang | FSH-β fragment | ||||
| A | 0.197 | 0.683 | 1.767 | 1.701 | ||
| B | 0.173 | 0.400 | ||||
| C | 0.147 | 0.306 | ||||
| D | 0.096 | 0.107 | ||||
| E | 0.203 | 0.148 | ||||
| F | 0.159 | 0.064 | ||||
| G | 0.112 | 0.031 | ||||
| H | 0.110 | 0.046 | ||||
| Think true | Pregnyl | |||||
Embodiment three anti-FSH-beta single-chain antibody are identified
Method
1) menopausal women overnight urine 4000ml extracts urine total protein with reference to table 8 saturated ammonium sulfate salting out method.
The table 8 saturated ammonium sulfate albumen table of saltouing
| In 25 ℃ of ammonium sulfate final concentrations, % saturation degree | ||||||||||||||||||
| The ammonium sulfate initial concentration, the % saturation degree | 10 | 20 | 25 | 30 | 33 | 35 | 40 | 45 | 50 | 55 | 60 | 65 | 70 | 75 | 80 | 90 | 100 | |
| Every 1000ml solution adds the gram number of |
||||||||||||||||||
| 0 | 56 | 114 | 144 | 176 | 196 | 209 | 243 | 277 | 313 | 351 | 390 | 430 | 472 | 516 | 561 | 662 | 767 | |
| 10 | 57 | 86 | 118 | 137 | 150 | 183 | 216 | 251 | 288 | 326 | 365 | 406 | 449 | 494 | 592 | 694 | ||
| 20 | 29 | 59 | 78 | 91 | 123 | 155 | 189 | 225 | 262 | 300 | 340 | 382 | 424 | 520 | 619 | |||
| 25 | 30 | 49 | 61 | 93 | 125 | 158 | 193 | 230 | 267 | 307 | 348 | 390 | 485 | 583 | ||||
| 30 | 19 | 30 | 62 | 94 | 127 | 162 | 198 | 235 | 273 | 314 | 356 | 449 | 546 | |||||
| 33 | 12 | 43 | 74 | 107 | 142 | 177 | 214 | 252 | 292 | 333 | 426 | 522 | ||||||
| 35 | 31 | 63 | 94 | 129 | 164 | 200 | 238 | 278 | 319 | 411 | 506 | |||||||
| 40 | 31 | 63 | 97 | 132 | 168 | 205 | 245 | 285 | 375 | 469 | ||||||||
| 45 | 32 | 65 | 99 | 134 | 171 | 210 | 250 | 339 | 431 | |||||||||
| 50 | 33 | 66 | 101 | 137 | 176 | 214 | 302 | 392 | ||||||||||
| 55 | 33 | 67 | 103 | 141 | 179 | 264 | 353 | |||||||||||
| 60 | 34 | 69 | 105 | 143 | 227 | 314 | ||||||||||||
| 65 | 34 | 70 | 107 | 190 | 275 | |||||||||||||
| 70 | 35 | 72 | 153 | 237 | ||||||||||||||
| 75 | 36 | 115 | 198 | |||||||||||||||
| 80 | 77 | 157 | ||||||||||||||||
| 90 | 79 | |||||||||||||||||
2) saltout and adopt 0-25%, 25%-50%, 50%-75%, 75%-100% saturated ammonium sulfate.8000rpm, 4 ℃ of centrifugal 10min after saltouing, supernatant gives over to next round and saltouts, and precipitation is resuspended with 2ml PBS.Be labeled as 25%, 50%, 75%, the 100% saturated ammonium sulfate urine protein of saltouing respectively.
3) 25%, 50%, 75%, the 100% saturated ammonium sulfate urine protein 12%SDS-PAGE constant voltage 100V electrophoresis 2h that saltouts, the 100V constant voltage goes to pvdf membrane.Use two kinds of negative contrasts of hormone: human chorionic gonadotrophin HCG (Pregnyl Pregnyl, Ou Jianong), concentration 50IU/ μ l; Human growth hormone (HGH) GH (think true Saizen, Xue Lannuo), concentration 0.1 μ g/ μ l.
4) the 3%BSA sealing is spent the night.One is anti-with 1: 100 anti-FSH-beta single-chain antibody, and two is anti-with 1: 1000HRP-albumin A, chemiluminescence.
The result
Urinary concentration protein 12 %SDS-PAGE, as shown in figure 11: swimming lane is respectively 25% saturated ammonium sulfate saltout product, 50% saturated ammonium sulfate saltout product, 75% saturated ammonium sulfate saltout product, 100% saturated ammonium sulfate saltout product, low-molecular-weight Marker from left to right.
Urinary concentration albumen Western blotting, as shown in figure 12: swimming lane is respectively 25% saturated ammonium sulfate product, 50% saturated ammonium sulfate product, 75% saturated ammonium sulfate product, the 100% saturated ammonium sulfate product of saltouing of saltouing of saltouing of saltouing from left to right, and two bands are followed successively by full molecule FSH, FSH-β from top to bottom.Show and to saltout menopausal women urine 50% and 75% saturated ammonium sulfate urine FSH-β is positive in the product.
Identify the Western blotting result of anti-FSH-beta single-chain antibody specificity, as shown in figure 13: antigen is respectively menopausal women from left to right and urinates 50% saturated ammonium sulfate saltout product, HCG, GH, and two bands are followed successively by full molecule FSH, FSH-β from top to bottom.Represent that anti-FSH-beta single-chain antibody of the present invention only combines with FSH-β specificity in the menopausal women urine, and do not have the Ag-Ab association reaction with HCG, GH.
Embodiment four anti-FSH-beta single-chain TPPA urine FSH levels
Method
1) synthetic FSH-β fragment is antigen standard items (concentration is respectively 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, 31.2 μ g/ml, 15.6 μ g/ml, 7.8 μ g/ml).
2) 5 menopausal womens are left and taken overnight urine.Adopt above-mentioned saturated ammonium sulfate salting out method to extract the urine samples total protein, recombinated by 1000: 1 with PBS.
3) bag is by 96 hole ELISA Plate, and coating buffer is PBS (pH7.4), and 4 ℃ of wrapper sheets spend the night.
4) PBS washs 96 hole ELISA Plate 3 times, and 3%BSA is filled it up with in every hole, 37 ℃ of sealing 2h.
5) remove confining liquid, add the anti-FSH-beta single-chain antibody of dilution in 1: 10, hatch 2h for 37 ℃.3%BSA substitutes antibody and makes negative control.
6) washing of PBST damping fluid is 3 times, removes unconjugated antibody.
7) every hole adds the staphylococcal protein A of the HRP mark of dilution in 1: 2000, hatches 1h for 37 ℃.
8) washing of PBST damping fluid is 3 times, and every hole adds 100 μ l TMB colour developing liquid, and room temperature is placed 10min.
9) every hole, the clear back of colour developing adds the sulfuric acid cessation reaction of 50 μ l 1mol/L.
10) measure every hole 450nm and 630nm light absorption value, press A
450nm-A
630nmCalculate every hole light absorption value.
When 11) SPSS13.0 software is analyzed 7 kinds of antigen concentrations respectively, the logarithm of antigen concentration and relation between the light absorption value and matched curve.
12), calculate the FSH concentration of each testing sample according to the curve of match.
The result
The light absorption value of each FSH-β fragment antigen standard items sees Table 9 and Figure 14.
The light absorption value of table 9 antigen standard items
| Standard items concentration (μ g/ml) | Standard items logarithm concentration | The standard items light absorption value |
| 500 250 125 62.5 31.25 15.625 7.8125 | 2.699 2.398 2.097 1.796 1.495 1.194 0.893 | 1.182 1.211 0.998 0.992 0.908 0.805 0.761 |
Figure 14 is that anti-FSH-beta single-chain antibody is used for ELISA method mensuration urine FSH horizontal detection actual effect.The left side is 10 minutes photos of TMB colour developing; The right side is H
2SO
4Photo after the cessation reaction.
Typical curve according to table 9 match is seen Figure 15: horizontal ordinate is the logarithm of FSH-beta antigen standard items concentration, and ordinate is a light absorption value; The line of broken line for directly being formed by connecting, the typical curve that smooth curve forms for the SPSS match.
5 experimenter's urine samples calculate the concentration of urine FSH-β respectively, and are as shown in table 10.
Table 10 testing sample light absorption value and concentration conversion
| Numbering | The sample light absorption value | Sample logarithm concentration | Urine FSH-β concentration (μ g/ml) | Urine FSH-β concentration (IU/L) |
| 1 2 3 4 5 | 1.002 0.917 0.812 0.858 0.926 | 1.88 1.59 1.19 1.38 1.62 | 75.9 38.9 15.5 24.0 41.7 | 285 146 58 90 156 |
Embodiment five measures FSH-β reagent box for detecting content in blood or the urine sample
With anti-FSH-beta single-chain antibody of the present invention packing, freeze drying, preparation becomes drying, white powder, and is soluble in water.This antibody is the key reagents of FSH-β reagent box for detecting content in efficiency test blood or the urine sample.Relevant matched reagent also comprises FSH-β standard items, two anti-, damping fluid etc.4 ℃ of preservations of whole kit, the term of validity＞6 month.If antibody-20 ℃ preservation can keep the active longer time.One anchorage FSH-beta single-chain antibody is arranged in each kit, add the damping fluid dilution during use, the titre scope is set 1: 50～1: 500, adds the amount of 50～100 microlitres in each reacting hole or the reaction tube.
Embodiment six preparation male-contraception medicines
With anti-FSH-beta single-chain antibody of the present invention packing, freeze drying, preparation becomes drying, white powder, and is soluble in water.4 ℃ of preservations, the term of validity 6 months; If-20 ℃ of preservations can keep active more than 1 year.Add 1～2 milliliter of dissolving of physiological saline when anti-FSH-beta single-chain antibody uses, the titre scope is set 1: 25～1: 100, adds in 250～500 ml physiological salines drip-feed.
Embodiment seven preparation treatment true precocious puberty medicines
With anti-FSH-beta single-chain antibody of the present invention packing, freeze drying, preparation becomes drying, white powder, and is soluble in water, 4 ℃ of preservations, the term of validity 6 months is if-20 ℃ of preservations can keep active more than 1 year.Add 1～2 milliliter of dissolving of physiological saline during use, the titre scope is set 1: 25～1: 100, adds in 250～500 ml physiological salines drip-feed.
Sequence table
＜110〉Jiangsu Prov. People's Hospital
＜120〉all-human source follicle stimulating hormone beta single-chain antibody choosing method and uses thereof
<160>4
<210>1
<211>339bp
<212>DNA
＜213〉people
<400>1
GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT
GCA GCC TCT GGA TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG
CTG GAG TGG GTC TCA TCG ATT TAG AAG CTT GGT CGG CAT ACA AGG TAC GCA GAC TCC GTG AAG GGC
CGG TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC
GAG GAC ACG GCC GTA TAT TAC TGT GCG AAA CCT GGG AGG AGG TTT GAC TAC TGG GGC CAG GGA ACC
CTG GTC ACC
<210>2
<211>324bp
<212>DNA
＜213〉people
<400>2
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT
TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT TTA AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT
AAG CTC CTG ATC TAT GCT GCA TCC AGT TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA
TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TAC TGT
CAA CAG AGT TAC AGT ACC CCT AAT ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
<210>3
<211>339bp
<212>DNA
＜213〉people
<400>3
GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT
GCA GCC TCT GGA TTC ACC TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG
CTG GAG TGG GTC TCA TCG ATT GAG AAG TAG GGT AAT AAG ACA AGG TAC GCA GAC TCC GTG AAG GGC
CGG TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC
GAG GAC ACG GCC GTA TAT TAC TGT GCG AAA AAG GGG GCT AGG TTT GAC TAC TGG GGC CAG GGA ACC
CTG GTC ACC
<210>4
<211>324bp
<212>DNA
＜213〉people
<400>4
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT
TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT TTA AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT
AAG CTC CTG ATC TAT CGT GCA TCC CGT TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA
TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TAC TGT
CAA CAG GGG GAT AGG ACT CCT AAT ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
Claims (4)
1. the anti-follicular stimulating hormone beta single-chain in total man source antibody, it is characterized in that described antibody is by filtering out the phage antibody that contains anti-" A " epitope antibodies gene in the single-chain phage antibody library of total man source and carrying out solubility expression, obtain the total man source single-chain antibody of anti-FSH-β " A " epi-position, described " A " epi-position is the 33-53 amino acid residue in FSH-β subunit, has: the amino acid sequence of Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg.
2. total man according to claim 1 source anti-follicular stimulating hormone beta single-chain antibody, the variable region of heavy chain and the chain variable region gene of this antibody of encoding is respectively SEQ ID No.1 and SEQ ID No.2, or variable region of heavy chain and chain variable region gene are respectively SEQ ID No.3 and SEQ ID No.4.
3. the screening and the authentication method of the anti-follicular stimulating hormone beta single-chain in total man source antibody is characterized in that its method comprises:
3.1 the preparation of antigen, antibody library and Related Bacteria strain
Synthetic FSH-β fragment is used for screening and identifies that described antibody, this fragment are FSH-β albumen 33-53 amino acid residue, have:
The amino acid sequence of Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg,
Obtain: be used to screen the total man source single-chain phage antibody library of described phage antibody, be used for the helper phage of superinfecting phage antibody library, be used for the Escherichia coli and the Escherichia coli that are used for the antibody solubility expression of phasmid amplification;
3.2 the amplification of total man source single-chain phage antibody library
Activate amplification by the mode of described antibody library being inoculated and add the helper phage superinfection;
3.3 screening FSH-phagus beta antibody
With described synthetic FSH-β fragment is antigen, and the phage antibody library of above-mentioned amplification has been carried out the affine screening of several wheels " absorption-wash-out-amplification " enrichment;
3.4 identify FSH-phagus beta antibody
Adopt ELISA to identify anti-FSH-phagus beta antibody, adopt PCR to identify anti-FSH-phagus beta antibody gene, carry out anti-FSH-phagus beta antibody gene order-checking;
3.5 anti-FSH-phagus beta antibody solubility expression and purifying
Adopt anti-FSH-phagus beta antibody to infect the method that E.coli, IPTG induce E.coli, will resist FSH-phagus beta antibody solubility expression is anti-FSH-beta single-chain antibody;
The anti-FSH-beta single-chain antibody that adopts His-trap affinity column purification of soluble to express;
3.6 identify the anti-FSH-beta single-chain antibody of solubility expression
Adopt non-competing ELISA method to measure the antibody affinity costant, adopt ELISA and Western blotting method to identify antibody specificity.
4. the described antibody of claim 1 is in antifertility and the purposes for preparing in detection kit and the treatment relevant disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107167596A (en) * | 2017-07-13 | 2017-09-15 | 深圳市亚辉龙生物科技股份有限公司 | A kind of immunochromatography reagent bar of fluorogenic quantitative detection FSH and preparation method thereof |
| CN107255725A (en) * | 2017-07-07 | 2017-10-17 | 江西科技师范大学 | A kind of homologous competitive enzyme-linked immune analytic approach detected with quantifying Human Fallicle-Stimulating Hormone's In vitro biological activity |
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| EP2067788B1 (en) * | 1999-05-18 | 2015-07-22 | Dyax Corp. | Fab fragment libraries and methods for their use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107255725A (en) * | 2017-07-07 | 2017-10-17 | 江西科技师范大学 | A kind of homologous competitive enzyme-linked immune analytic approach detected with quantifying Human Fallicle-Stimulating Hormone's In vitro biological activity |
| CN107167596A (en) * | 2017-07-13 | 2017-09-15 | 深圳市亚辉龙生物科技股份有限公司 | A kind of immunochromatography reagent bar of fluorogenic quantitative detection FSH and preparation method thereof |
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