Table 2.
Fusion results
| Desired specificity | Immunogen | Antigen | Antigen class | Immunogen load (μg per mouse) priming injectionsf/final booster | Bleedout serum IgG titre on antigena | # Clones screened/total | # Positive clones/# screened | Number of clones carriedb |
|---|---|---|---|---|---|---|---|---|
| Native SARS CoV virus | Gradient purified SARS-CoV (lysate and pure virus) | Same | B → Ac | 175 SC (lysate)/5 IP (pure virus) | >5000 | 2874/2874 | 172/2874 | 17 |
| Neisseria meningitidis capsular polysaccahride, type specific | Synthetic capsular polysaccharide on a protein carrier | Purified capsular polysaccahride (no carrier) | C → Ad | 75 SC/5 IP | >5000 | 1132/1132 | 12/1132 | 12 |
| Anthrax toxin | Purified, recombinant Bacillus anthracis protective antigen (PA) | Same | A | 85 SC/5 IP | >5000 | 472/>1000 | 14/472 | 9 |
| Native mycoplasma bacterium (subspecies specificity) | Whole Mycoplasma mycoides subsp. mycoides SC organism | Same | A | 100 SC/5 IP | >3000 | 400/>1000 | 25/400 | 8 |
| Native FMD virus (cross-serotype recognition) | Purified, recombinant O-VP2 protein | FMD virus, three serotypes | A → Be | 65 SC/2 IP | >1000 on FMD virus | 2577/>3100 | 3/2577 | 3 |
| Native FMD virus (type specific) | VP1-peptide (-KLH) | FMD virus, Type C | A → Ce | 200 SC/10 IP | >1000 type C FMDV | 574/>1000 | 2/574 | 2 |
SC, subcutaneous; IP, intra-peritoneal.
Reciprocal dilution.
Only the best clones are kept. This is empirically determined for each antigen and depends upon properties of both the antigen and the hybridoma clone; including isotype (as IgG are predominantly kept), level of expression, antigen coating. The screening ELISA parameters we use are O.D.s at 405nm greater than 0.8 at 1 h in greater than or equal to a 1/8 dilution of supernatant.
Positive influencing factors including final booster and screening performed with gradient purified virions; these factors shifted the rating of this antigen from B to A.
Positive influencing factors including highly purified synthetic carbohydrate (CHO), attached to a T-cell epitope rich carrier protein, and an unconjugated CHO used as antigen in screening ELISA to remove mAbs to carrier protein; these factors shifted the rating of this antigen from C to A as CHO are usually the most difficult of all antigens to have to produce mAbs against.
Negative influencing factors including such as generation of mAbs to peptide/rec. protein and screening for cross-reactivity on native antigen/organism shifts these antigen scale ratings from A to B or even C.
Sum of all but the final injection with a total of 4–5 injections.