Sandbox Reserved 1474: Difference between revisions

The heavy and light chains cDNA of the mAb were cloned by reverse transcription-polymerase chain reaction<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, and then they were amplified and ligated into the cloning vector<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. The resulting plasmids of all mAb cloning was transformed into E. coli strain and then sequenced and submitted to Genbank<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.

After the sequence confirmation, the plasmid was linearized with SacI <ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> and the coding sequence of scFv6H4 was transformed to P.pastoris strain(yeast) by electroporation<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. It is expressed in yeast colony which exhibited high zeocin resistance<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.  

Large scale fermentation was done in batches in a 10 liter working volume biostatB bioreactor to express scFv6H4 in large scale for in vivo pharmacokinetic studies<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. ScFv was purified from the supernantant by metal affinity chromatography and metal affinity column<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> in a naturally occurring mixture of monomer (~75%) amd dimer (~25%)<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> analyzed with SEC.                             

Changes have been made to RIA using TALON beads which binds to the His6 tag at the carboxyl terminus of the VL region of scFv6H4<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, which will orient the binding site of METH distal to the beads, allowing unhindered access to METH<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. Standard curve was constructed with [H3]METH competing with unlabeled METH. The KD was reported to be 10nM nearly identical to parent IgG mAb6H4 11nM<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. It was reported just like parent mAb6H4, there is no cross reactivity with related compounds<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> including pseudo-ephedrine, norepinephrine, dopamine, and serotonin, even at 100 microMolar<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.

After METH infusion 3.2mg/kg/day<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/> by osmotic minipumps which resulted average steady state serum concentration of 25ng/ml after 24h<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, mice were administered scFv6H4 which has led to drastic increase of serum concentration of METH<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, determined by liquid chromatography-tandem mass spectrometry as described previously<ref name ="Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum"/>. The compare of the first 480 min METH concentration with control group was reported<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. scFv6H4 was reported stable in serum in vitro yet unstable in Urine in vitro<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. The concentration of scFv in serum in vivo was determined by SEC<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, the method has been previously described <ref name ="Pharmacokinetic mechanisms for obtaining high renal coelimination of phencyclidine and a monoclonal antiphencyclidine antigen-binding fragment of immunoglobulin G in the rat"/>. Monomer of scFv was reportd to have been completely eliminated in the serum within the first 30 minutes<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>, yet the multivalent larger proteins persisted for >240 mins corresponding to the reported t1/2lamdaz to be 228 +/- 38 min<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>. It is interesting that it was reported the divalent form did not decrease for the first 10 minutes, as if while it is been eliminated, it is also been formed from the mono scFvs. Pharmacokinetic parameters were reported for mono and multi scFvs respectively<ref name="Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine"/>.