Production Description |
| Step 1) Build expression plasmid based on cDNA clones provided by customers |
| Step 2) Express and purify tagged (His- or GST-) recombinant proteins |
| Step 3) Immunization of 6 Balb/c mice for each peptide-KLH conjugate |
| Step 4) ELISA monitoring of anti-sera development until titers reach >10,000 |
| Step 5) Fusion of spleen cells from 1 mouse of highest ELISA titer with SP2/0 |
| Step 6) 4-5 rounds of sub-cloning and ELISA screening to establish hybridomas |
| Step 7) Identification of capture and detection antibodies for sandwich ELISA |
| Step 8) Protein A/G affinity purification of ascites from 2 paired cell lines |
Order Today To Follow This Production Schedule |
Date |
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AbMART Track |
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01-09-2009 |
4 |
Peptide Design |
Design gene cloning strategy for expression plasmid vector construction;
recombinant protein production and purification;
6 mice ready (Pre-bleed is done)
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02-13-2009 |
39 |
Protein Production |
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1st Immunization (Complete Freud Adjuvant)
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2nd Immunization (Incomplete Freud Adjuvant)
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3rd Immunization (Incomplete Freud Adjuvant)
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After 3rd Immunization, IF titers>10,000, go on to Fusion and hybridoma cloning: as early as
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4th Immunization (Incomplete Freud Adjuvant)
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04-18-2009 |
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SP2/0 fusion with spleen cells
from 1 mouse (plating in 10X 96-well plates)
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04-28-2009 |
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S1-ELISA
1-Sclone
S2-ELISA 2-Sclone
S3-ELISA 3-Sclone
S4-ELISA 4-Sclone
S5-ELISA
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4-5 rounds of sub-cloning and ELISA
screening
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06-17-2009 |
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07-01-2009 |
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Expand cell lines and make frozen
stocks
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| 07-22-2009 |
198 |
Ascites production/purification |
Protein A/G purification of ascites from 2 mice for each cell line |
| 07-29-2009 |
205 |
Antibody labeling |
Label antibodies with HRP |
| 08-03-2009 |
210 |
Sandwich ELISA Pairing |
Screen capture and detection antiboides for sandwich pairs: minimal detection sensitivity of 10-8 M (10 nM) |
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1) 2 sandwich ELISA paired hybridoma cell lines (Two 25 cm2 flasks with 2-5 x 106 cells in DMEM/high glucose/15% FBS);
2) 2-5 mg of Protein A/G purified mouse IgGs from ascites production;
3) Sandwich ELISA data;
4) Cell line isotypes
5) 1-3 mg of recombinant protein
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