Sandbox Reserved 1474: Difference between revisions
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Four(12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study (147 d);however, this response did not appear to be dose related.<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers " /> | Four(12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study (147 d);however, this response did not appear to be dose related.<ref name="First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers " /> | ||
=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= | ||
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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. | |||
===Production and purification=== | ===Production and purification=== | ||
purified and cormulated as a naturally occurring mixture of monomer (~75%) amd dimer (~25%) | |||
===Determination of Kd Values using bead-based radioimmunoassay=== | ===Determination of Kd Values using bead-based radioimmunoassay=== | ||
===Pharmacokinetic Studies of METH and scFv6H4 in Rats=== | ===Pharmacokinetic Studies of METH and scFv6H4 in Rats=== | ||
The very small molecular size(27k Da) of scFv monomers leads to rapid clearance (40) mins. | |||
The very small molecular size(27k Da) of scFv monomers leads to rapid clearance (40) mins.scFv6H4 was engineered from mAb6H4. Monomer was reported to be quickly cleared or converted to multivalent forms with an apparent t1/2lamdaz of 5.8 min while multivalent forms showed a much longer t1/2lamdaz (228min). Multimers instead of monomers were considered the cause for the prolonged redistribution of METH into the serum. | |||
==Structural highlights from crystal structure of scFv6H4== | ==Structural highlights from crystal structure of scFv6H4== | ||