Sandbox Reserved 1474: Difference between revisions
From Proteopedia
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 92: | Line 92: | ||
===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9=== | ===Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9=== | ||
IgG mAbs are typically chimeric, humanized, or fully human proteins. The longest t1/2lamdaz values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity. | |||
IgG2, Kappa;METH KD= 7nM | IgG2, Kappa;METH KD= 7nM | ||
| Line 117: | Line 119: | ||
=Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine= | =Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine= | ||
when a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab or scFv is used.() | |||
==Materials and Methods== | ==Materials and Methods== | ||
| Line 123: | Line 127: | ||
The heavy and light chains cDNA of the mAb were cloned by reverse transcription-polymerase chain reaction, and then they were amplified and ligated into the cloning vector. The resulting plasmids of all mAb cloning was transformed into E. coli strain. All sequence submitted to GEN BANK. | The heavy and light chains cDNA of the mAb were cloned by reverse transcription-polymerase chain reaction, and then they were amplified and ligated into the cloning vector. The resulting plasmids of all mAb cloning was transformed into E. coli strain. All sequence submitted to GEN BANK. | ||
===Sub-cloning and Large-Scale Expression=== | ===Sub-cloning and Large-Scale Expression=== | ||
scFv6H4 was cloned and expressed in yeast. | the coding sequence of scFv6H4 was cloned and expressed in yeast. | ||
===Production and purification=== | ===Production and purification=== | ||
large scale fermentation was done in batches in a 10 liter working volume biostatB bioreactor. ScFv was purified from the supernantant by metal affinity chromatography and metal affinity column. | |||
purified and cormulated as a naturally occurring mixture of monomer (~75%) amd dimer (~25%) | purified and cormulated as a naturally occurring mixture of monomer (~75%) amd dimer (~25%) analyzed with SEC. | ||
===Determination of Kd Values using bead-based radioimmunoassay=== | ===Determination of Kd Values using bead-based radioimmunoassay=== | ||
