ELISA to measure serum protein

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sarakumar47
sarakumar47's picture
ELISA to measure serum protein

Hello friends,

I want to measure a particular protein in serum with ELISA. I have Ab to that protein. Can I go for Sandwich ELISA? If so, please give me the procedure. Otherwise, which method is more convinient to measure the protein?

sarakumar47
sarakumar47's picture
Can I follow this method?

Can I follow this method?

1. Capture with polyclonal ab
2. Block with 5% BSA
3. Wash
4. Add human serum diluted in PBS and Standards (recombinant protein) in PBS
5. Wash
6. Add monoclonal ab Diluted in 5% BSA
7. wash
8. secondary (goat anti mouse) Diluted in 5% BSA
9. substrate/stop/read

I do not have monoclonal Ab. So, Can I use the same poly clonal antibody in the step 6?

Please help me.

protoldo
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Hola unless a policlonal ab

Hola unless a policlonal ab reacts against differents epitopes, if you has imobilized the protein with an antibody, you have bound epitopes of your protein wich can´t react newly with the new add antibody or release the bound by the excess free antibody added. Why don´t you try immobilize the serum dilutions and controls, bound the primary and developping with the secondary? Good luck

sarakumar47
sarakumar47's picture
Thanks for your reply Dr.!

Thanks for your reply Dr.! Can you explain how to make serum dilutions? 1:400 is ok? I want to compare the protein levels in different groups of animals. I don't have standard for that protein. Without Standard, I cannot measure that protein. Can write the results with just comparison only but not measuring it? Is it will be acceptable? Please give me some suggestions.

protoldo
protoldo's picture
Hola, although this isnt my

Hola, although this isnt my field, you have to immibilize d100ul of different dilutions of serum from 0, 1710,    untill 10e-11,  for each aafter blocking add the primary ab, at the recomended dilution, and follow the rest of  steps. At the end and depending of the protein and antibody, you will found saturated wells in the low dilutions and other wells where colour decays with the dilution, so you can compare the response in each animal . If you don´t have the idoneous dilution of antiboy, you have to repeat the ELISA with a fix dilution of serum in each well and different dilutions of antibody.To transform this results in concentrations of protein, I cannot help you, revise the chapters about titulations of sera and antibodies. Good luck

protoldo
protoldo's picture
Hola, although this isnt my

Hola, although this isnt my field, you have to immibilize d100ul of different dilutions of serum from 0, 1/10, 1/100 untill 10e-11; for each animal.After blocking add the primary ab, at the recomended dilution, and follow the rest of;steps. At the end ,and depending of the protein and antibody, you will found saturated wells in the low dilutions and other wells where colour decays with the dilution, so you can compare the response in each animal . If you don´t have the idoneous dilution of antiboy, you have to repeat the ELISA with a fix dilution of serum in each well and different dilutions of antibody.To transform this results in concentrations of protein, I cannot help you, revise the chapters about titulations of sera and antibodies. Good luck

sarakumar47
sarakumar47's picture
Thank you so much Dr. for

Thank you so much Dr. for your timely help! I have one more question. If I add primary antibody, it will directly go and directly binds with its target only.  Then, Why should I block after immobilizing the serum? What is its role here?

protoldo
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Hola It´s to avoid

Hola It´s to avoid inespecific interactions ( The 1st ab would bound to nos specific areas, only by the excess of it ) and to occupe all the surface of the well mainly where the dilutions of serum are too high. Think, that in these wells are free surface active to bound proteins , if you don´t block, molecules of 1st antibody will bound, and you will have 1st ab bound to the protein and bound to the non uccuped surface, so the od lecture will be very high. Good luck