Antibodies for chelated radionuclides and clearing agents
A technology of scavenger and chelating agent, which is applied in the direction of radioactive carriers, radioactive preparations in vivo, antibodies, etc., and can solve problems such as unstable binding of radionuclides, slow complex formation rate, etc.
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Embodiment 1
[0868] Example 1: Description of Immunization
[0869] Immunization of Rabbits
[0870] The 2 enantiomers Pb-DOTAM-Alkyl-PEG 4 - A 1:1 mixture of KLH fractions (MS2-DOTAM KLH fraction 1 and MS2-DOTAMKLH fraction 2) for immunization of New Zealand white rabbits or transgenic rabbits containing human immunoglobulin loci, as in WO 2000 / 46251, WO 2002 / 12437, WO 2005 / 007696, WO 2006 / 047367, US 2007 / 0033661, and WO 2008 / 027986. Each rabbit was immunized with 500ug of the immunogen mixture emulsified in complete Freund's adjuvant by intradermal application on day 0 and 500ug each day by alternating intramuscular and subcutaneous application on days 7, 14, 28, 56 . Thereafter, rabbits received monthly subcutaneous immunizations of 500ug, and aliquots of blood were collected 7 days after immunization for serum titer determination. Larger blood samples (estimated 10% of total blood volume) were collected during month 3 of immunization and during month 9 (5-7 days after immunizati...
Embodiment 2
[0873] Example 2: Cloning B cells from rabbits
[0874] Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC)
[0875] Blood samples were obtained from immunized rabbits. Whole blood containing EDTA was diluted twofold in 1x PBS (PAA, Pasching, Austria) followed by density centrifugation using mammalian lymphocytes (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's instructions. PBMCs were washed twice with 1x PBS.
[0876] EL-4 B5 medium
[0877] Supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM Glutamin, 1% penicillin / streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and RPMI 1640 (Pan Biotech, Aidenbach, Germany) in 0.05 mM b-mercaptoethanol (Gibco, Paisley, Scotland).
[0878] coating of the board
[0879] Sterile cell culture 6-well plates were coated with 2 μg / ml KLH in carbonate buffer (0.1 M sodium bicarbonate, 34 mM sodium bicarbonate, pH 9...
Embodiment 3
[0889] Example 3: Expression of Rabbit Antibody
[0890] PCR amplification of V domains
[0891] Total RNA was prepared from B cell lysates (resuspended in RLT buffer, Qiagen, cat. no. 79216) using the NucleoSpin 8 / 96 RNA kit (Macherey & Nagel; 740709.4, 740698) according to the manufacturer's protocol. RNA was eluted with 60 μl of RNase-free water. cDNA was generated by reverse transcriptase reaction using 6 μl RNA using Superscript III first strand synthesis SuperMix (Invitrogen 18080-400) and oligo dT primers according to the manufacturer's instructions. Implement all steps on the Hamilton ML Star system. The primers rbHC.up and rbHC.do for heavy chain and rbLC.up and rbLC.do for light chain (Table 3) were amplified with AccuPrime Super Mix (Invitrogen 12344-040) using 4 μl cDNA in a final volume of 50 μl. Increased immunoglobulin heavy and light chain variable regions (VH and VL). All forward primers were specific for the signal peptide (VH and VL, respectively), whi...
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