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All-human source follicle stimulating hormone beta single-chain antibody screening method and its uses

A follicle-stimulating hormone and single-chain antibody technology, applied in biochemical equipment and methods, material inspection products, measuring devices, etc., can solve the problems of unfavorable large-scale production, short half-life, and high cost of mouse-derived monoclonal antibody preparation, Achieve convenient amplification and production, reduce immunogenicity, and strong specificity

Inactive Publication Date: 2008-02-20
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also great limitations, especially the background of its murine origin, which greatly limits the application of monoclonal antibodies as therapeutic agents in humans
The limitations of mouse-derived antibodies mainly include the following points: (1) mouse-derived monoclonal antibodies are immunogenic to humans, and the production of human anti-mouse antibodies can induce allergic reactions; (2) mouse-derived monoclonal antibodies cannot be effective Activate the biological effect function of the human body; (3) The half-life of the mouse-derived monoclonal antibody in the human body is also short, and it is easy to be cleared by the body; (4) The molecular weight of the mouse-derived monoclonal antibody is large, and it is difficult to pass through the cell membrane and enter the cytoplasm (5) The preparation process of mouse-derived monoclonal antibody is complicated, and it is difficult to immunize animals and cell fusion, which is not conducive to large-scale production; (6) The preparation cost of mouse-derived monoclonal antibody is also relatively high

Method used

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  • All-human source follicle stimulating hormone beta single-chain antibody screening method and its uses
  • All-human source follicle stimulating hormone beta single-chain antibody screening method and its uses
  • All-human source follicle stimulating hormone beta single-chain antibody screening method and its uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Screening of fully human anti-FSH-β single-chain antibody

[0053] 1. Preparation of Antigen, Antibody Library and Related Bacterial Strains

[0054] 1) Synthesize the FSH-β fragment, synthesized by the Institute of Biochemistry and Cell Biology, Shanghai Chinese Academy of Sciences, with a full length of 21 amino acid residues (33-53 amino acid residues of the FSH-β subunit). Amino acid residue sequence:

[0055] Glu-Glu-Cys-Arg-Phe-Cys-Ile-Ser-Ile-Asn-Thr-Thr-Trp-Cys-Ala-Gly-Tyr-Cys-Try-Thr-Arg, (valley-valley-cysteine- Ethyl-phenylpropanoid-cysteine-isoluminous-silk-isoluminous-asparagus-threo-threo-color-cysteine-propyl-glycerin-cysteine-tyrosine-threo-arthrin), molecular weight 2.5Kda, synthetic purity 99.8%. The synthesized FSH-β fragment is in the form of white crystalline powder, soluble in water.

[0056] 2) Fully human single-chain phage antibody library: the library capacity is about 1×10 8 , pIT2 phagemid vector, including gene III, myc tag an...

Embodiment 2

[0125] Example 2 Identification of immune activity of FSH-β phage antibody

[0126] 1. ELISA identification of anti-FSH-β phage antibody

[0127] Method I

[0128] 1) The bacteria obtained in the last round of screening were diluted and spread on TYE culture plates (with a final concentration of 100 μg / ml Amp), and cultured overnight at 37°C.

[0129] 2) Add 100 μl of 2×TY medium to each well of the 96-well plate (containing a final concentration of 100 μg / ml Amp+1% glucose), and randomly pick 90 colonies from the TYE culture plate to inoculate the 96-well plate (the remaining 6 wells No bacteria), 250rpm, 37°C shaking culture overnight.

[0130] 3) Take another 96-well plate, add 200 μl of 2×TY medium (containing a final concentration of 100 μg / ml Amp+1% glucose) to each well, and transfer 5 μl of bacterial solution from each well of the first 96-well plate to the second Plates were shaken at 250 rpm at 37°C for 2 hours. Add glycerol to each well of the first 96-well plate ...

Embodiment 3

[0322] Embodiment 3 Anti-FSH-β single chain antibody identification

[0323] method

[0324] 1) Menopausal women urine 4000ml overnight, refer to Table 8 to extract total protein in urine by salting out with saturated ammonium sulfate.

[0325] Table 8 Saturated ammonium sulfate salting-out protein table

[0326] Ammonium sulfate final concentration at 25°C, % saturation

sulfuric acid

ammonium initial

strong

Spend,

% full

Harmony

10

20

25

30

33

35

40

45

50

55

60

65

70

75

80

90

100

The number of grams of solid ammonium sulfate added per 1000ml solution

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

...

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PUM

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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 [0001] The present invention relates to monoclonal antibody technology and fully human antibody technology, in particular to a screening method for fully human anti-follicle-stimulating hormone beta single-chain antibody using phage display technology and the application of the antibody. Background technique [0002] Under the stimulation of GnRH in the hypothalamus, pituitary gonadotropin (GTH) cells synthesize and release follicle stimulating hormone (FSH) and luteinizing hormone (LH). In women, FSH acts on the follicles of the ovary, including promotion of follicle formation, preovulatory follicle selection, stimulation of estrogen production by aromatization of cystic cell-derived androgens, luteinization of granulosa cells, and induction of LH receptors. In males, follicle-stimulating hormone (FSH) secreted by the pituitary gland and the androgen testosterone (T) secreted by the testes themselves are hormonal regulators of spermatogenesis. T and FSH act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/53G01N33/577G01N33/569C12Q1/02
Inventor 崔毓桂丁贵鹏钱思轩刘嘉茵
Owner JIANGSU PROVINCE HOSPITAL
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