IdiotypeITS103 [C5]: Mouse antibody to detect rhesus anti-SIV [ITS103] . This antibody has no therapeutic effect and is only used in vitro assays to detect the Anti-SIV [ITS103.01LS] antibody (CAT-00090).

Suggested ELISA Protocol (from Roederer's laboratory)

Notes: For measuring levels of ITS103 or ITS102, the samples must not be heat-inactivated, and steps subsequent to adding the serum/plasma samples must be performed in a biosafety cabinet.

Blocking Buffer: 5% skim milk powder in PBS + 0.05% Tween (The background is higher when a non-skim milk-based block buffer is used!)

Wash Buffer: PBS + 0.05% Tween

Day 1 

1.     Coat wells of ELISA plates (we use Thermo Nunc-Immuno Maxisorp plates, 442404) with 100µL/ well of 1µg/mL anti-Id antibody diluted in PBS at 4°C overnight.

Day 2

2.     Wash plate 6x with turn after 3 washes.

3.     Block with 200µl Block Buffer at 37^oC for 1 hour.

4.     Wash 6x with turn after 3 washes.

5.     Prepare dilution of serum/plasma in Block Buffer and setup 3 (or desired number) of half-log dilutions Add 100µL/well of diluted serum/plasma to ELISA plate in duplicate (leave the last row free for control). Incubate at 37^oC for 1 hour.

a.      The first well for the half log dilution has 1.46µL in 144.8µL, transfer 46.25µL into 100µL for 2nd dilution, transfer 46.25µL into 100µL for 3rd dilution (or a multiple of these numbers if preparing on a separate dilution plate)

b.     For later timepoints I would recommend starting with 1:100 in the first well, however for the earlier timepoints starting already at 1:316 or 1:1000 in the first well.

c.      For standard curve, take ITS103 mAb and make a 2-fold dilution starting at 0.2µg/mL (1µL of a 20ug/mL solution) in the first well. I usually do 10 dilution points (leave the last two wells with no mAb).

d.     For the control mAb, make the dilution in Block Buffer containing 1/100 of a negative plasma, as the plasma will have a slightly higher background level than the buffer alone.

e.     Prepare these dilutions and add to ELISA plates in a biosafety cabinet

6.     Wash 3x in a biosafety cabinet

7.     Add 100µl anti-monkey IgG-HRP (we use SB108A, diluted 1:5,000 in Block buffer); incubate at 37°C for 1 hour.

a.      While this is incubating warm 12mL/plate TMB substrate to room temperature (we use SureBlue TMB substrate).

8.     Wash 3x in a biosafety cabinet

9.     Add 100µL TMB substrate equilibrated to ambient temperature. Incubate at RT for 10 minutes.

10.  Add 100µL 1N H2SO4 to stop reaction.

11.   Read absorbance at 450nm on plate reader.