In general, in antibody detection assay both, the affinity and the concentrations generates the signal. Therefore, standardization is always difficult. The easy way: create your own “arbitrary units”. The best way is to compare affinities (but it is no common to do this)

1. you can use a defined monoclonal with a known concentration to compare your samples with. Dilute this in different steps so that you can calculate the concentration. This will be your calibration curve. There are several models to calculate this curve so that you can transfer via this curve the OD's into a concentration. 

Pro: easy to use

Con: a monoclonal has one affinity, the polyclonal has many and you will detect an “average”

2. you can purify from a polyclonal Antiserum via positive affinity chromatography the specific antibodies. They can be quantifies in µg/mL … . Those should be used as a primary standard to quantify a secondary standard (e.i. antiserum, etc.).

Pro: a lot of work

Con: you will lose the low affinity antibodies (they would not bind to the affinity column) and the high affinity as well (they could not eluted). And again, the polyclonal samples has many and you will detect an “average”

3. you can work with different species, a murine monoclonal and a human antiserum. The specific antibodies can compete for the binding. You need to have only species specific labelled antibodies which are able to differentiate between both species.

Pro: a lot of work

Con: you will lose the low affinity antibodies (they would not bind to the affinity column) and the high affinity as well (they could not eluted). And again, the polyclonal samples has many and you will detect an “average”

4. You can make your assay more “independent” from affinities. To get this, use for the sample incubation a buffer with higher salt concentrations (0,3 mol/L … 0,45 mol/L NaCl)

5. An easy estimation of the affinity is the incubation of constant dilutions with increasing salt concentrations (0.15 mol/L up to 3 mol/L NaCl, Urea up to 7 mol/L, ….) Low affinity antibodies will lose their binding to the antigen at lower concentration, high affinity antibodies much later.

Comments: The solid phase antigen is difficult to quantify. The binding is realized via hydrophobic interactions (hydrophobic amino acids à polystyrene rings) at concentrations between 1 – 10 µg/mL. The average bound is at 100 - 200 ng/cm². The antigen-antibody reaction is never 1:1. You cannot compare the antigen-antibody reaction in liquid phases (Heidelberger precipitation curve) with solid phase reactions. The antigen at the solid phase in present in Excess.

In general, in antibody detection assay both, the affinity and the concentrations generates the signal. Therefore, standardization is always difficult. The easy way: create your own “arbitrary units”. The best way is to compare affinities (but it is no common to do this)

1. you can use a defined monoclonal with a known concentration to compare your samples with. Dilute this in different steps so that you can calculate the concentration. This will be your calibration curve. There are several models to calculate this curve so that you can transfer via this curve the OD's into a concentration. 

Pro: easy to use

Con: a monoclonal has one affinity, the polyclonal has many and you will detect an “average”

2. you can purify from a polyclonal Antiserum via positive affinity chromatography the specific antibodies. They can be quantifies in µg/mL … . Those should be used as a primary standard to quantify a secondary standard (e.i. antiserum, etc.).

Pro: a lot of work

Con: you will lose the low affinity antibodies (they would not bind to the affinity column) and the high affinity as well (they could not eluted). And again, the polyclonal samples has many and you will detect an “average”

3. you can work with different species, a murine monoclonal and a human antiserum. The specific antibodies can compete for the binding. You need to have only species specific labelled antibodies which are able to differentiate between both species.

Pro: a lot of work

Con: you will lose the low affinity antibodies (they would not bind to the affinity column) and the high affinity as well (they could not eluted). And again, the polyclonal samples has many and you will detect an “average”

4. You can make your assay more “independent” from affinities. To get this, use for the sample incubation a buffer with higher salt concentrations (0,3 mol/L … 0,45 mol/L NaCl)

5. An easy estimation of the affinity is the incubation of constant dilutions with increasing salt concentrations (0.15 mol/L up to 3 mol/L NaCl, Urea up to 7 mol/L, ….) Low affinity antibodies will lose their binding to the antigen at lower concentration, high affinity antibodies much later.

Comments: The solid phase antigen is difficult to quantify. The binding is realized via hydrophobic interactions (hydrophobic amino acids à polystyrene rings) at concentrations between 1 – 10 µg/mL. The average bound is at 100 - 200 ng/cm². The antigen-antibody reaction is never 1:1. You cannot compare the antigen-antibody reaction in liquid phases (Heidelberger precipitation curve) with solid phase reactions. The antigen at the solid phase in present in Excess.

Provide details of your enzyme immune-assay procedure. Nonetheless, coat 100 ul of 10 ug/ml concentration overnight at 40 C. After washes and blocking with 1% skimmed milk powder in BSA, dispense different dilutions of your antibody, then develop the reaction with conjugate and substrate.You should consider lowest concentration of antigen giving the OD value of 1.0 with highest dilution of your primary antibody. Therefore you are required to determine concentration of antigen to be coated and then determine ab titre by checker-board method. Include blank control in every plate/assay.