Hello, I have some questios for you to clarify my thoughts:

1. What kind of ELISA

2. What system do you use (HRP, AP, fluorescence, etc)

3. You mentioned use of beta-actin for compare, so I supose that you use a reference curve?

Regards

Alejandro i am using Indirect ELISA and coating total proteins extracted from brain tissues. I am also using HRP conjugated secondary antibody. I am actually new to ELISA experiments. How to use reference curve?

Hi Hitesh, ELISA assays are only quantitative for estimating the amount of a specific protein. These assays usually "stand alone assays" because they depend on the affinities of the primary and secondary antibodies.  

Therefore, most ELISA assays cannot be compared to other measurement techniques.    

I think it should be possible to use an internal standard in ELISA, although I have never tried it. It would be important to make sure the signals for both proteins are within the dynamic range of each assay and to quantify the amount of each protein using a standard curve. Basically, this means you are doing 2 separate ELISAs, one for each protein. At the end, divide the amount of the test protein by the amount of the internal standard protein and report the ratio.

Having written this, I'm not sure I see why the internal standard is necessary. Why not use the sample protein concentration as the denominator so only one ELISA is necessary plus one protein assay?

hi sir i am agree with your answer

Hi Hitesh, ok, here is my advice, I hope you encounter usefully: if you have beta-actin as standard, you need to do a reference curve of this with know concentrations of the beta-actin, and made serial dilutions of this in one column of your ELISA plate (i.e. in the first weel you put 1mg of beta actin, to say something, and then made serial dilution, so in the next well you have 0.5 mg, and in the next, 0.25 mg, etc) I suggest you made this curve in 8 wells, then you will have standard  curve with 8 points in a graph, with this you can calculate the approximatevalue of the other points of beta actin using the equation of a line (excel does it), now, in your case the only thing you can do for calculate concentrations of the other antobodies is relative to the standard curve, so results will be like: antiboby X has two fold times concentration in relation to beta- actin. Otherwise, you will need to do a standard curve of each antibody.

Regards

Alejandro Besné @ Thanks   for information 

Don't worry I hope you encountered usefully. Regards

Standardization of the ELISA should follow the ICH guidelines (see lik below). If quantitative, use as said before a calibration with a set of at least 5 standards and a respective calibration curve. If you add two quality controls (high and low level) you can assure that the measurement is correct. The standards can be set easily either by an international standard available via NIBSC or by a "self made standard". This can be done by the definition of a positive pool which has 100 arbitrary units. You have to make sure that your assay has a dilution linearity (one sample in different dilution has to give the same final result: i.e. by using a serial dilution). Beware: in the detection of specific antibodies it’s not that easy to reach because of the fact that two properties influencing the ELISA signal: the antibody concentration and the affinity. The affinity will influence the slope of the curve. Both parameters are highly individual. So you have to make sure, that the concentration is the only parameter influencing the ELISA signal. For this purpose I suggest to use as incubation buffer: 0.01 mol/L phosphate pH 7.2, 0.3 mol/L NaCl, 3% v/v Gelafusal (digested gelatin with normally no cross reactions etc), 0.1% v/v tween 20, 0.001 g/L phenol red.

In general the calibration curve follows the Rodbard function. There are several software packages, even included in the ELISA reader internal software packages. The function is a 3 or 4 patameter exponential function which is closly related to the chemical bachground if the immune reactions at the solid phase. 

You are searching more for a normalization of the ELISA results: The normalization as term is used to get the ELISA results from varying surrounding conditions into correlation with parameters you are trying to influence. So you can use the total protein content, the concentration of housekeeping proteins (SOD, LDH, …) whatever is constant per cell number etc. It’s can be also the number of cells, positive cells (in FACS, Histology, …). In summary you need two values: The ELISSA result from your protein of interest divided by one from a housekeeping protein, cell number,,, 

http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf