Antibodies that immunospecifically bind to trail receptors
A technology of antibodies and immunoglobulins, applied in the direction of antibodies, immunoglobulins, anti-animal/human immunoglobulins, etc., can solve problems such as increased susceptibility to microbial infections
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Embodiment 1
[1397] Example 1: Isolation and characterization of the scFv shown in Table 1
[1398] Rescue large scFv libraries
[1399] Use a close to 1×10 11 Antibodies specific to TR4 were selected from a cloned scFv library that was the 1.38 x 10 10 An expanded version of the library (Vaughan et al. (1996), Nature Biotechnology 14:309-314). Rescue the phage by taking 3 × 10 10 Cells were grown in 2YTAG (2YT medium supplemented with 100 μg / ml ampicillin and 2% (w / v) glucose) at 37° C. for 2 hours with shaking. M13K07 helper phage (Stratagene) was added to the cultures at a multiplicity of infection (moi) of approximately 10. The culture was incubated stationary at 37° C. for 15 minutes, followed by gentle aeration (200 rpm) for 45 minutes at the same temperature. The culture was centrifuged and the cells were resuspended in 500 ml 2YTAK (2YT medium supplemented with 100 μg / ml kanamycin) and incubated overnight at 30° C. with good aeration (300 rpm). Phage particles were purified...
Embodiment 2
[1418] Embodiment 2: Biacore analysis of the affinity of TR4 binding polypeptide
[1419] Material
[1420] BIAcore2000 equipment
[1421] BIAcore2000 control software, version 3.1.1
[1422] BIAevaluation, version 3.1
[1423] BIAcore CM5 sensor chip, Cat#BR-1000-14Lot#0364 (BIAcore)
[1424] HBS-EP buffer
[1425] Amine Coupling Kit Cat#BR-1000-50 (BIAcore)
[1426] EDC, #1048-950345 (BIAcore)
[1427] NHS, #1048-950345 (BIAcore)
[1428] Ethanolamine, #1048-950345 (BIAcore)
[1429] 10mM Acetic Acid, pH4.0, Cat#BR1003-50Lot#1821-9503844 (BIAcore)
[1430] TRAIL-FLAG (Alexis Biochemical Cat#522-003-C010#L04793 / a)
[1431] All experiments were performed at 25°C.
[1432] general method
[1433] TR4, TR5, TR7 and TR10 (in the form of Fc fusion protein) were immobilized on the respective flow cells of the BIAcore sensor chip. The TR4-Fcc fusion protein comprises M1-I240 residues of TR4 (SEQ ID NO: 1). Post-translational processing of the entire fusion protein resul...
Embodiment 3
[1448] Example 3: Inhibition of biotinylated TRAIL binding to TR4
[1449] I. Materials:
[1450] 10×PBS (Quality Biological Cat 130-069-161, Lot 708712) Immulon 4 microplate (Dynex Cat 3855, Lot ND540319)
[1451] Bovine serum albumin fraction V (Sigma, #58H0456)
[1452] Trishydroxymethylaminomethane (TRIS BASE)
[1453] Tween20 (Sigma)
[1454] Goat anti-human Fc (Sigma, I-2136, #89H4871)
[1455] TR-4: Fc (as above)
[1456] Biotinylated TRAIL (AM100200-Peprotech)
[1457] HRP-streptavidin (Vector, #L0328)
[1458] TMB Peroxidase Microwell Substrate System (KPL, Kirkegaard & Perry Laboratories Inc.)
[1459] h 2 SO 4 (Fisher)
[1460] 96-well dilution plate (Costar)
[1461] II. Buffer
[1462] Coating buffer (1×PBS)
[1463] Blocking buffer (3% BSA in PBS)
[1464] Universal Diluent (1% BSA in PBST)
[1465] Wash buffer (0.1% Tween20 and 1×PBS)
[1466] III. Method
[1467] Goat anti-human Fc was diluted to 0.1 μg / ml in coating buffer. Immulon 4 microplate...
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