M1000 tr fret instrument

Example 5

This example demonstrates the ability of LAG-11 and LAG-56 to inhibit the interaction between human LAG3 and FGL1. The peptides were tested using a human LAG3/FGL1 TR-FRET Binding Assay (BPS Bioscience) carried out according to the manufacturer's instructions.

Peptide stocks of LAG3-11, LAG3-42, LAG3-48, LAG3-51, LAG3-54, LAG3-56, LAG3-60 and OVA were prepared at 1 mM followed by a 1:5 dilution in plate reactions. LAG3-11 and OVA were tested at 200, 50, 13, 3, 0.8, 0.2, 0.05, and 0.01 μM. The other peptides were tested at 200, 50, 13, and 3 μM. A neutralizing anti-human LAG3 antibody (BPS Bioscience Cat. #71219) was used as a positive control.

Reaction mixes were incubated 1 hr at RT before development. After development, plate was read in a Tecan M1000 TR-FRET instrument. Percent activity was calculated as follows:
% Activity=100×[(sample−minimum)/(maximum−minimum)]

The results are shown in FIG. 11.

Example 2

This example tested the ability of peptides to inhibit the interaction between human LAG3 and FGL1. The peptides were tested using a Human LAG3/FGL1 TR-FRET Binding Assay (BPS Bioscience) carried out according to the manufacturer's instructions.

Peptides 6, 41, 68, 69, 115, 146, 154, and 155 were tested at 100, 33, and 11 μM. Peptide LG56 (HIQNWSYWLNQDMMNQQVWKS; SEQ ID NO:14) served as a peptide positive control. Peptide 93 (SEQ ID NO:15) served as a negative or low activating peptide control. A neutralizing anti-human LAG3 antibody (BPS Bioscience Cat. #71219) was used as an antibody positive control.

Reaction mixes were incubated 1 hr at RT before development. After development, plate was read in a Tecan M1000 TR-FRET instrument. The results are shown in FIG. 1, and the determined IC₅₀ values are shown in Table 3, below.