My final thesis is about meassuring the OD of IgG From sera which reacted with antigen of malaria vector (An.sundaicus) by using ELISA, but I got problem when the control negative (without antigen) give me the number of OD, if anybody here know how it can be, please tell me. Thank you so much before :)
check the blocking step:
The ELISA plate (polystyrene) attached with any subject that is protein in nature so the igG may be attached to the place even in the absence of the antigen
It may happen due to some issues:
- Blocking step, as well pointed out above; you may consider change the blocking agent (0.5% Tween 20 in PBS, Casein, FBS, etc) or time/condition of incubation (usually longer times or incubation at higher temperatures improve the blocking)
- Secondary antibody. It may require some standardization as higher concentration of 2nd Ab may increase the signal but also the background. You may consider trying to dilute your secondary Ab (and titration of this Ab may vary according to the manufacturer and even between different lots). If blocking is OK, it should not happen unless if you're using a high concentration of Ab, which is binding to the plate. Please note that volume of secondary should not exceed the volume used for blocking step.
- Washing step. You may try to check whether your washing step should be modified (increase the number of washing). No need for washing step after blocking.
Thank you so much for All of your advise Abdelrazak and Prof. Fujiwara. I ve been made a trial well (without antigen, without primer n secondary antibody, without TMB and without H2SO4) I only filled up with PBS-T and the result give the same number with the control (without antigen) the OD is about 0,049. T.T
0.049 is actually nothing (maybe the absorbance of the solid phase and liquid only). Usually you could use this well as control and subtract this value from the readings of each well...
How if my samples O.D are not really high about 0.121-0.988 ? And than could I making the value of control OD "0" with the consequence I need to cut down the samples OD with the control OD ?
You should do what Dr Fujiwara suggested: a complete examination of all components of the test and their concentrations. Your samples should also be diluted in a blocking solution. "Control" all "Blank" or "0" should not be blocking only but contain a negative sample. For example: if your test is for an antibody in serum or plasma, the control should contain the same volume of negative serum or plasma.
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