Figure 9: The efficacy of the enzyme-labeled antibody was tested by direct Dot-ELISA. Human IgG were coated on to four nitrocellulose strips that were labeled 1:1000, 1:2000, 1:4000 and 1:8000. After air drying, the unadsorbed sites on the membrane were blocked with blocking buffer. After blocking, the membranes were agitated in different dilutions of the HRP conjugate (1:1000, 1:2000, 1:4000 and 1:8000). Excess HRP conjugate was removed by washing the membrane. The membrane was then agitated in the substrate solution, tetra methyl benzidine–H2O2 (TMB/H2O2) till blue/grey colored spot developed.