In both enzymes, the primary (D176) and secondary (G199) specificity sites are conserved, while the tertiary specificity site (position 210) in LM-TL is a Gly residue, while in Rhombeobin it is an Ala residue.
The secondary antibody specificity was confirmed on cryostat sections done without primary antibody incubation (figure 4C).
A serial section is also treated without any primary antibody, as a control for the secondary antibodies specificity.
On the same slide, a serial section was treated without the primary antibody as a control for secondary antibody specificity.
To test for secondary antibody specificity, sections were incubated with either only DCC, UNC5H, or netrin-1 primary antibody and, subsequently, with combinations of anti-rabbit, anti-mouse and anti-chicken Alexa Fluor-conjugated antibodies.
Using a heuristic algorithm particle swarm optimization (PSO), all the designed primers were computationally validated to fit the common primer design constraints, such as the melting temperature, primer length and GC content, PCR product length, secondary structure, specificity, and terminal limitation.
To confirm secondary antibody specificity, negative controls were included in which the primary antibodies were omitted.
The prediction model used amino acid atom potentials and torsion angle distribution to assess the change in solvent accessibility and secondary structure specificity at the mutation site.
In addition, secondary antibody specificity for mouse IgA was confirmed by testing against plates coated with mouse IgA, IgG1, IgG2a, IgG2b; as expected, secondary antibodies were specific for mouse IgA at the dilution used in the ELISA.
It is also possible that anti-topo I ELISA positives in SLE in some studies reflect detection of low-affinity antibodies or antibodies other than IgG class because of secondary antibody specificity.
