The overall goal of this procedure is to quantify the binding of short peptides with human MHC two immune proteins using a high throughput 3 84 well plate assay. This is accomplished by first performing solution phase competition binding assays with the peptides of interest. In the second step, the equilibrated peptide MHC two complexes are captured with an anti MHC two antibody immobilized on high binding ELIZA plates.
Next, the captured complexes are incubated with the opium labeled streptavidin and then the unbound streptavidin is washed away and the captured opium is activated. Ultimately, time resolved fluorometry can be used to quantify the levels of MHC two captured control peptide. This method can provide preliminary insights into the immunogenic potential of target proteins.
It is particularly useful for validating the output of in silico predictors, Begin the procedure by adding 25 microliters of freshly diluted L 2 43 antibody solution to each well of a 3 84. Well high binding white ELIZA plate sealed the plate with a polyester film and incubate it overnight at four degrees Celsius. Then starting with a 10 millimolar stock of each test.
Peptide and DMSO make the following dilution series and citrate phosphate buffer using 3 84 well polypropylene plates. Note that a full 3 84 well polypropylene plate requires three separate EIA plates as illustrated. Next, dilute the selected MHC two stock solutions to a 101 nano molar concentration in reaction buffer using a 15 milliliter conical tube.
Then dilute the control peptide from 20 micromolar to a one to 100 ratio in the freshly prepared MHC two master mix and add 21.5 microliters of the MHC two master mix with control peptide to the positive control. Well of the 3 84 well polypropylene plate, add the MHC two master mix containing control peptide to each of the test peptide dilutions at a one-to-one ratio to create the binding reaction at this time as well. Finally, seal the binding reaction with a polyester film and incubate the plate for 12 to 24 hours in a non-carbon dioxide controlled incubator at 37 degrees Celsius without shaking.
The next day, dilute the binding reaction wells in the 3 84. Well plate at a one to one ratio with neutralization buffer. Then wash the Eliza plate three times with 60 microliters per well of PBS 0.05%between 20 next transfer, 25 microliters of each neutralized binding reaction solution from the 3 84 well plate in triplicate to the antibody coated 3 84.
Well Eliza plate then incubate the ELIZA plate covered in polyester film at four degrees Celsius overnight after the incubation. Wash the ELIZA plate three times as just demonstrated, and then add 25 microliters of freshly diluted strep din opium to each. Well incubate the plate covered with a polyester film in the dark at room temperature for one hour.
During the incubation, bring 10 milliliters of enhancement solution per plate to room temperature also in the dark after one hour, wash the ELIZA plate as demonstrated, and then add 25 microliters of enhancement solution to each well and incubate the plate covered with polyester film for 10 to 15 minutes in the dark. Finally read the fluorescence with a time resolved fluorescent plate reader with opium settings. In this representative experiment, a mature peptide sequence of enterobacter Cloe P nine nine beta-lactamase was analyzed for putative peptide binders to the MHC two allele.
DRB one fifteen oh one one hundred and seventeen non or peptides were identified with an obligatory P one anchor residue at position one that is required for MHC two binding at a 5%threshold only. Peptides with scores greater than or equal to 2.6 are likely binders. Thus at the 5%threshold only the top 11 peptides are predicted to bind MHC two DRB 1 15 0 1.
A representative panel of predicted epitopes was selected for analysis in this MHC two binding assay. Beta-lactamase peptide fragments of these 15 residues were chemically synthesized such that putative nonamer MHC two epitopes were embedded within their synthetic protein fragments. The capacity of these synthetic peptides to compete with a biotinylated myelin basic protein B control peptide for binding to MHC two DRB 1 15 0 1 was then analyzed as just demonstrated.
Competitive binding curves for the synthetic peptides are illustrated here. IC 50 values were calculated by fitting the log transform data using the one site competitive binding nonlinear fit function of prism. As demonstrated in the table as seen in the graph, the peptides naturally partitioned into three groups, strong binders with an IC 50 of less than one micromolar moderate binders with an IC 50 greater than or equal to one micromolar, but less than 100 micromolar and weak binders with an IC 50 of greater than or equal to 100 micromolar After its development.
This technique paved the way for researchers in the fields of molecular immunology and biotherapeutic design to more fully explore the key molecular recognition events that underlie anti protein immune responses in human patients.
