In this graph various dilution of mouse anti rabbit Ig G antibodies were used to determined concentration of rabbit Ig G. the most effective dilution will be between 1/4000 to 1/8000, as in rest of the cases, at lower concentration of Ig G there is no linearity with increasing concentration of rabbit IgG (like in case of 1/2000). At1/2000 dilution concentration of mouse anti-rabbit Ig G is too high compared to rabbit IgG and hence there is no linier relation. Similarly at more than 1/8000 dilution concentration of anti rabbit IgG becomes to less compared to concentration of rabbit Ig G. hence there will not be any anti rabbit Ig G available to bind with rabbit Ig G at higher concentration. In given scenario the optimum dilution for mouse anti- rabbit IgG will be 1/6000 which gives linear correlation with increase in concentration of rabbit IgG.
Here in case of goat anti rabbit Ig G the over all binding and absorption profiles restricts it use for estimation of rabbit IgG. There are two different scenario 1) In case of lower dilution (1/2000) there is lots of non specific binding resulted in to very high absorption at lower concentration of rabbit Ig G and get saturated at slightly higher concentrations of rabbit Ig G.
2) In case of higher dilution (1/4000 and above) there is very less binding due to non specificity of polyclonal anti body.