Team:Aboa/Demonstrate
Demonstrate
Here we show that our thing actually works. This page is mostly just repeating our result, but with focus on showing that our idea actually worked.
p-Azido-L-phenylalanine incorporation
P-azido-L-phelynalanine (pAzF) was succesfully incorporated into protein as can be seen from Figure 1. DBCO Cy5.5 label did not bound unspecifically into proteins that do not contain pAzF (Fab0).
Figure 1. (A) 1 µg of Ni-NTA purified pAzF modified and unmodified Fabs. (B) SEC purified unmodified Fab. (C) The labeling of our produced Fab with DBCO-Cy5.5 visualized using Li-COR Odyssey at 700 nm bandwidth. A clear labeling of the C-terminal pAzF (amino acid residue 460) can be observed from this picture. No unspecific labeling of Fab0 can be observed. Copper free click-chemistry displays a very specific way of labeling irrespective of the purity of the sample. The other variants (191, 108) were not labeled very efficiently. This could be due to poor solvent accessibility of the pAzF residue or degradation of the protein. Unbound label can be seen at 1 kDa.
Capture antibody immobilization & function
Antibodies were immobilized into DBCO coated magnetic beads and they were able to bind Digoxigenin. This can be seen from Figure 2. Cy5 signal measured from coated beads were constantly higer than from passively immobilized and more constant than from randomly immobilized.
Figure 2. (A) The size (Forward scatter, FSC-area) and shape (Side scatter, SSC-area) distribution of randomly and site-specifically immobilized beads. This shows how a site specifically labeled antibody with only one reactive site does not cross-link the beads in the same manner as a randomly conjugated antibody. (B) A histogram of fluorescence intensity of Cy5 at 700nm. the total population 100,000 beads. Uncoated beads in red. Passively adsorbed beads in blue, random conjugation in orange and site specific conjugation using pAzF in green. A much larger variance is achieved when using random conjugation because of the crosslinking of beads. Site specifically coated beads are more uniform. (C) when monomeric beads are measured, a much more uniform coating is once more observed with virtually no unspecific binding or empty beads with a huge difference in variance. Variance of the assay is a critical factor for the sensitivity and repeatability of any test.
Test performance and error
As can be seen from figure 3. site specific immobilization improves test signal-to-background ratio and reduces test variance, thus improving test sensitivity.
Figure 3. A) and B) show the assay performance as signal to background ratio and the error as a robust coefficent of variation. The test error is already, without any optimization at a clinically relevant level