ELISA 542 Prerun notes 4/99 PBS = MMBLS PBS. PBSA = PBS + 0.03% azide. PBST = PBS + 0.5% Tween 20. ----- Rinses: Putting HRP in the well (in PBST), 2 rinses eliminates all signal (Plate 7). Tap water is OK (Plate 13, student results). ----- Azide: Azide (0.03%) + Tween kills HRP (Plates 1, 2). Azide (0.001%, in Sigma's IgG at 1/100) + Tween does not inhibit (Plate 6). Azide in the absence of Tween is inhibitory (7X, Plate 3). Tween in the absence of azide is OK (Plate 2). ----- Coating: 5' is plenty (vs. overnight, 10', 20', 5' is saturated for NRS/10, Plate 5). With 5', signal is higher for NRS/1K-10K than for NRS/10! (Plates 10, which shows lower signal at dilutions >10K, Plates 9, 7, Plate 6 where I thought I must have reversed the sequence!) NRS/5K seems optimal, but it must dry! (Plates 11) Actually at 1/5K, it seems less sensitive to freshness/dryness (Plate 13). Students in lab got high signals with fresh 5' 1/5K coats! Fresh coats which haven't dried give 1/3 or less as much signal as dried coats. (Plate 11 and previous uncontrolled results, e.g. Plate 10). ----- Conjugate: HRP-GARG (A541): 1/5K for 15 min is good. 1/10K or 30K gives less signal (Plate 10). 1/5K for 5 or 10 min gives less than 15 min, so 15 min seems the minimum. Plate 11. Dilutions in PBST are stable for days (Plate 10, Plate 4). ----- Tween 20, 0.5%: Prevents conjugate from binding (test w/o bound a lot; Plate 4). Does not prevent NRS/10 from binding (Plates 7, 4). ----- Substrate: Substrate mixed for 5 days (kept in frig overnight and 2 of those days) works as well as freshly mixed on an actual NRS/HRP coat (Plate 10). 10 min is a good incubation time; color development slows down after that (Plate 4), such that at 20', color is 1.45X higher than at 10'. ----- Acid: Increased OD by about 4-fold (Plate 4). ----- Phenol red: As a marker to keep track of which wells are filled, seems not to inhibit (Plate 7, used in Plate 10 E9,11 with no evident ill effects). ----- I50: NRS 0.009%, which is 1/11K (Plate 11), or 0.006% = 1/17K (Plate 6). Plate 8 had low signal but indicated I50 a higher dilution than 10K. Plate 7 showed full inhibition at 1/400. IgG 0.35 ug/ml (Plate 6). ----- GARFc background: GARFc/10, 100, 1K, 10K gives signal when fresh, but not when dry (Plate 10, 4). Or slight (0.02) Fc/10 signal when dry (Plate 13). "NGS"/20 (A544) in PBST as 'competitor' does not interfere with NRS signal (haven't tested it on signal from 'wet' GARFc coat). A dry "NGS"/10 coat gave no signal. ----- Capture reported with GARG: N.B.: The anti-Fc in the GARG might remove some captured IgG! GARFc/10 5' DRY! + NRS/10 5' DRY! gives capture signal (vs. no NRS) of 0.38 (Plate 11), 0.25 (Plate 7). Fresh GARFc gives a background signal and no capture signal (Plate 10), or some capture signal (Plate 4). ----- Capture reported with GARFab: aFc/10 captures 0.52 while aFc/100 captures 0.35 (NRS/50, aFc was dry; Plate 13). Fresh aFc/30 gave lower capture than dry aFc/100 (Plate 13). Therefore aFc must be dry. Less NRS/150 was captured than NRS/50. NRS/50+tween on bare well gave only 0.02 background (Plate 13). Recommend aFc/10 + NRS/? (low enough to be competed by Fc).