Available antibody that I have are- Rabbit Anti EPO Ab (Polyclonal) as primary Antibody. Also have three secondary antibody- Rabbit Anti Rat IgG+IgM+IgA HRP, Rabbit Anti Rat IgG HRP and Rabbit Anti Rat IgM HRP. To develop standard curve I just coat a recombinant human EPO in ELISA plate and then applied primary antibody and then applied all three secondary antibodies separately. Only for Rabbit Anti Rat IgM HRP case observed UV values but no difference in high to low concentration all values almost similar. And for Rabbit Anti Rat IgG+IgM+IgA HRP case a low signal found.
Can anybody help me figure out whats happened over there? And also nice if anybody can suggest me about suitable primary and secondary antibody to detect anti EPO Ab of rat?
Hello Md.
It is best to get a ELISA kit containing all the reagents for standard curve. As you already have 1st, 2nd antibodies and antigens available, and your test animal's serum is already fixed, I would like to give comments on the basis what you have and what you should have more for decent ELISA assay.
For standard curve, you need 1st Rat anti-EPO antibody (but not your collected rat anti-serum against hEPO), followed by 2nd Rabbit anti-rat antibody-HRP (adjusting the Ig isotypes) from your lists. As you would like to see positive reaction in your collected rat anti-serum against hEPO.
For test assay using a similar combination of antibodies, therefore, 1st antibody would be your collected rat anti-serum against hEPO after coating hEPO. 2nd antibody would be Rabbit anti-rat antibody-HRP (adjusting the Ig isotypes) from your lists.
In addition to those antibodies, you need to use normal rabbit serum (10% NRS in buffer for dilution of 1st and 2nd antibodies) to avoid non-specific binding of your antibody reactions.
This is a method for indirect ELISA, and it's a relatively precise assay since you gave a hint that you are going to use indirect ELISA.
Best wishes.
There are in principle two ways to detect in serum samples antibodies against a protein:
- You can bind the target protein (EPO in your case) to the plate (directly or via a sandwiching antibody), incubate with the serum, wash and then determine bound antibodies with a detection antibody. That would be a polyclonal antibody against immunoglobulins from, in your case, rat (say, rabbit anti total rat Ig). If you want to distinguish the class of your antibody, you would use more specific, anti IgG or anti IgM, detection antibodies. At any rate, the detection antibody has to be labelled, for example with an enzyme like HRP. So the topology would be Plate - EPO - rat anti-EPO antibody - detection antibody with label
- The second possibility is to bind serum proteins to the plate and then detect binding of labelled EPO. Again, label could be an enzyme, or for example a fluorescent dye or a radioactive marker. The topology would be plate - rat anti-EPO - EPO with label.
Btw, I found PVDF-membranes much handier than ELISA plates for such assays. Binding of the first protein is almost quantitative, compared to a few % for plates. Hence spot-blots are at least an order of magnitude more sensitive than ELISAs. In addition, later incubation and washing steps are easier to perform (unless, of course, you have a robot to do it for you).
For the standard curve you need purified rat anti-EPO antibodies. In that way, you would coat the plate with human EPO, subsequently adding your standard or samples (all containing rat anti-human EPO antibodies). As secondary antibodies, you need conjugated antibodies recognizing the rat isotypes you want to detect. Are you interested in the IgG response or in every isotype?
Thanks for your descriptive suggestion Dr. Sang Ho Lee. But so far I couldn't find any manufacturer who can provide Rat Anti EPO Ab (polyclonal), but Rabbit Anti EPO Ab (Polyclonal) is there which I have also. Therefore I think about to shift from rat to mice, but again there is a problem Mouse Anti EPO Ab (polyclonal) is not available but monoclonal is there. If I ignore standard curve preparation then from experimental condition there is chance to get positive or negative result but I will not quantify that. So do you have any idea how can I quantify if I get any positive result.
Dear Gertrudis Rojas thanks for your comments. Do you have any idea from where I can purchase purified rat anti-EPO antibodies? Initially I want to focus on IgM and IgG response later I want to do isotype detection also.
Hi Md,
If you change your coating EPO source into mouse, then you could find available combinations. There are Rat anti-mEPO antibodies from Novus. Subsequently, all your antibody combinations, too.
But those are monoclonal, will it be ok to use monoclonal Ab as primary antibody?
Hi, Md.
If you are not planning to isolate rat anti-hEPO antibody from your rat anti-hEPO antiserum, followed by creating of more specific fragments of Ig to have more specificity, it will be a remaining choice to use that Mcab.
Suppose you coat hEPO antigens in both standard curve and test assay, followed by the recommended Rat anti-mEPO antibody and Rat anti-hEPO antibody, respectively, and then layered by Rabbit anti-rat antibody-HRP. You will have readings.
One more thing you should do is to compare the baseline reactivity between the two 1st antibodies. The differences in the readings could be compensated by looking the graphical presentations.
Best of luck.
If you want to use a standard, it has to be of the same species as your sera samples. Otherwise, the secondary antibody for the standards and the samples would have to be different, so the standard will not be a real standard detected in the same way as your samples. If your samples are from rat, you have to use rat antibodies to make the standard curve. A monoclonal antibody could be used as long as it comes from the appropriate species.
Alternatively, you could use titers (lowest positive dilutions) to evaluate your samples, instead of quantifying the antibody content according to a standard curve.
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