The overall goal of this experiment is to establish a cell-free assay quantifying the neutralizing capacity of G-M-C-S-F auto antibody. We start by obtaining transgenic silkworms. Bring the human SGMR alpha gene to produce recombinant SGMR alpha and proceed to label the recombinant human G-M-C-S-F through bioTE.
Next we evaluate the correlation between the cell-free system and a regular bioassay with a study of inhibition of G-M-C-S-F binding to SGMR alpha by G-M-C-S-F autoantibodies. Results obtained demonstrate a significant correlation between growth inhibition of the bioassay and binding inhibition of the cell-free system by serum IgG fractions and purify G-M-C-S-F autoantibodies. The main advantage of this technique over existing methods like TF one bioassay is that this system then as a high analysis with good reproducibility to measure neutralizing capacity of GM CSF auto antibody.
Since GM CSF auto antibody causes autoimmune primary overall prognosis, the implications of this technique extend toward diagnosis. It can also be applied to competitive tib binding assays using recombinant cytokine receptors such as receptors to BEGF and TNFA Demonstrating the procedure will be presented by ano a PhD student from my laboratory. He first developed this pH free system.
This video shows the silkworm used for the production of recombinant SGMR alpha. The first step is to amplify CDNA for SGMR Alpha from a human placenta CD NA library by polymerase chain reaction with the resulting PCR amplicon. Perform a second PCR to add a 50 base five prime UTR sequence of ULA virus poly heatron to the five prime end.
And to tag an R RRGs his tag to the three prime end clone the amplified PCR R product into the plasmid P MSG one one mg co inject the construct PS GMR slash M1 one mg with the helper vector PHA three pig into eggs of silkworm PNDW one strain. Rear the hatched G zero larvae to moths at 25 degrees Celsius. Then screen G one embryos either by mating among siblings or with PNDW one by MG FP expression in the eyes indicative of transgenic silkworms bearing.
The SGMR alpha gene proceed to extract recombinant SGMR alpha from the cocoons of transgenic silkworms with phosphate buffer containing 500 millimolar sodium chloride and stir at four degrees Celsius for 24 hours. Centrifuge the samples at 20, 000 GS for 15 minutes. To remove precipitates, apply the supernatant fluid to a nickel affinity column.
Wash the column and dilute proteins with a linear gradient of ole from 10 millimolar to 250 millimolar. Pool the fractions containing the purified SGMR alpha and dialyze against phosphate puffed saline. First dialyze samples with PBS.
To remove sorbitol from the G-M-C-S-F preparation, add one milliliter of 20 millimolar cold sodium meta date solution and incubate the sample for 30 minutes on ice in the dark. Now add glycerol to a final concentration of 15 millimolar and dialyze against 100 millimolar sodium acetate buffer. To continue add biotin azide to the solution at a final concentration of five millimolar and mix for two hours at room temperature.
Finally, remove the non-reactive material by dialysis against PBS coat a microtiter plate with 50 microliters of monoclonal anti polyhis antibody overnight at four degrees Celsius. Wash five times with PBST. Then add 100 microliters of a blocking solution and incubate for three hours at four degrees Celsius.
Again, wash five times with PBST. Add 50 microliters of SGMR alpha in the blocking solution and incubate overnight at four degrees Celsius after five washes with PBST. Add 25 microliters of the samples containing G-M-C-S-F auto antibody and then 25 microliters of biotinylated G-M-C-S-F incubate for one hour at four degrees Celsius.
To form the SGMR alpha G-M-C-S-F complex. Continue to wash five times with PBST and then add 50 microliters of alkaline phosphatase streptavidin for one hour at four degrees Celsius to detect the biotinylated G-M-C-S-F bound SGMR alpha wash five times with PBST at 50 microliters of a chemiluminescence substrate to the solution incubate for one hour at room temperature and determine chemiluminescence activity using a plate reader. In SDS page analysis, the silkworm derived recombinant SDMR alpha is present in both monomeric and DME forms under non reducing conditions, but only the monomeric form under reducing conditions.
This recombinant mixture of monomers and dis sulfide linked dimers of SGMR alpha is functional in a competitive binding assay. The complex of biotinylated G-M-C-S-F with SGMR alpha displays specificity as it can be dissociated by adding various concentrations of neutralizing antibody but not non neutralizing antibody. Furthermore, increasing concentrations of G-M-C-S-F neutralizing antibodies correlate positively with increased percent binding inhibition in a dose dependent manner.
As expected, the binding inhibition of G-M-C-S-F non neutralizing antibodies was not dose dependent. This cell-free system demonstrates close correlation of the inhibition of G-M-C-S-F binding to SGMR alpha by G-M-C-S-F autoantibodies with calculated values of r equals 0.988 and P value of 0.002. The binding inhibition increased in a dose dependent manner by G-M-C-S-F autoantibodies.
Similarly, the binding inhibition significantly correlated with the growth inhibition of import. This assay has utility and analysis of the serum IgG fractions from patients with autoimmune PAP as demonstrated here by the relationship between the IC 50 for percent binding inhibition and the percent growth inhibition by the serum IgG fractions. Furthermore, the coefficient of variation between experiments indicates an advantage for the cell-free system over the growth inhibition bioassay.
Once masta, this cell-free GM GSF receptor ligand binding assay can be successfully completed in 38 hours. It's important to remember to keep the reacting, temperature and incubation time. This technique paved the way for researchers in the field of screening for the bio activities of noblet antibody drugs instead of conventional bioassays.
