13 replies to this topic

[Andrea Mica]

We're looking for a protein in blood. We know it should be there, we have monoclonals against it but we cant find it. Could it be stuck to other proteins or blood constituents?  Are there products or procedures that might split the proteins off so they can be found in an ELISA or other antibody assay?
Any labs that could undertake that work for us?

Regards,

Andrea

[K.B.]

1. Do you have standard of that protein and does the assay work with that standard?
2. Are your antibodies suitable for the assay you use? (eg. Ab's for WB may not be suitable for ELISA etc.)

[Chelo]

Andrea Mica, on 14 March 2011 - 04:09 AM, said:

We're looking for a protein in blood. We know it should be there, we have monoclonals against it but we cant find it. Could it be stuck to other proteins or blood constituents?  Are there products or procedures that might split the proteins off so they can be found in an ELISA or other antibody assay?
Any labs that could undertake that work for us?

Regards,

Andrea

If you are trying to capture your protein with a monoclonal antibody it is perfectly possible that your protein is "masked" by other proteins binding to the selected epitope. Did you test your antibody with other control preparations? It might be possible that changes in glycosilation pattern also affect the binding capacity of your MAb. I don´t know any way to get rid of this interaction unless you do a WesternBlot. However, if your antibody recognizes a particular conformation (and not a linear region) then it won´t work either.

[Andrea Mica]

K.B., on 14 March 2011 - 04:17 AM, said:

1. Do you have standard of that protein and does the assay work with that standard?
2. Are your antibodies suitable for the assay you use? (eg. Ab's for WB may not be suitable for ELISA etc.)

1.Thanks, yes we have standard protein and our assay works well with the protein in buffer or urine.
2. The antibodies worked in ELISA with buffer or urine, but looking in serum we get too much noise too little signal.

[Andrea Mica]

Thanks very much.  We might try a few other monoclonals that didnt work so well in urine buffer, but might work better in blood.

[PAO_ahac]

Why do you believe going from a cleaner matrix (serum) to WB is going to be easier?  I believe you should spend you time optimizing the serum/plasma assay to reduce noise and increase signal.  WB is a much more difficult matrix to deal with and you already have a working elisa with buffer and urine.

[newborn]

How much is your noise? the OD > 0.50?

Could you name that protein? Is there any paper that measures your protein in the blood? are you sure your protein level is higher than detected limition of your ELISA?

[Andrea Mica]

PAO_ahac, on 14 March 2011 - 10:06 AM, said:

Why do you believe going from a cleaner matrix (serum) to WB is going to be easier?  I believe you should spend you time optimizing the serum/plasma assay to reduce noise and increase signal.  WB is a much more difficult matrix to deal with and you already have a working elisa with buffer and urine.

A couple of things: someone mentioned WB above meaning western blot and others meaning Whole Blood.

We dont particularly want to look in whole blood. We are just concerned that the protein my be sticking to the cells and so in removing the cells to make serum/plasma, we'd be losing the proteins we're after.  If there are any reagents that might help release the proteins from the cells (if that's where they are) into the serum plasma that would be great.

[Andrea Mica]

newborn, on 14 March 2011 - 09:50 PM, said:

How much is your noise? the OD > 0.50?

Could you name that protein? Is there any paper that measures your protein in the blood? are you sure your protein level is higher than detected limition of your ELISA?

I'd rather not name the protein at this stage. I haven't seen any papers measuring this protein in blood (or serum)and consequently we dont know for sure that the protein level is higher than the detection limit of the ELISA - but we think it should be.

[PAO_ahac]

Some clarification needed...you indicated assay works in urine.  Did you detect your analyte in urine samples?  If you did then it is probably not sticking to cellular components.  You are using a sandwhich elisa so I am making a guess here that a model system for you might be hCG.  hCG is found in whole blood, urine, plasma, serum.  You did not give the anlyatical range of your assay but one approach would be if the level of analyte is high then dilute the blood prior to analysis.  The matrix effect (if there is any) may dissapear.  Regardless of sample type optimize the assay for serum or plasma get that to work first then try blood.  If you have the purified analyte you could also spike normal blood samples allow to equilibrate then test serum, plasma, and wb.

[PAO_ahac]

Another option...lyse the blood with water prior to analysis. the spike and recovery should indicate if the analyte sticks...

[K.B.]

Andrea Mica, on 14 March 2011 - 08:03 AM, said:

K.B., on 14 March 2011 - 04:17 AM, said:

1. Do you have standard of that protein and does the assay work with that standard?
2. Are your antibodies suitable for the assay you use? (eg. Ab's for WB may not be suitable for ELISA etc.)

1.Thanks, yes we have standard protein and our assay works well with the protein in buffer or urine.
2. The antibodies worked in ELISA with buffer or urine, but looking in serum we get too much noise too little signal.

re: 2. I was thinking about western blotting, not whole blood. Some antibodies for W-blot are not good for ELISA, but it's not the case if you can detect the standard.

Is it possible that this protein is being cleaved? Perhaps you should think about adding some protease inhibitor immediately after blood is collected or serum separated? Or maybe those are only some more or less specific interactions between proteins? In this case you may try adding a little bit of detergent eg. Tween-20.

PAO_ahac is right - you should try spiking serum with standard.

[Andrea Mica]

re: 2. I was thinking about western blotting, not whole blood. Some antibodies for W-blot are not good for ELISA, but it's not the case if you can detect the standard.

Is it possible that this protein is being cleaved? Perhaps you should think about adding some protease inhibitor immediately after blood is collected or serum separated? Or maybe those are only some more or less specific interactions between proteins? In this case you may try adding a little bit of detergent eg. Tween-20.

PAO_ahac is right - you should try spiking serum with standard.

Thanks for the answers, there are a few things for us to try.

[/quote]

[Andrea Mica]

PAO_ahac, on 17 March 2011 - 02:35 AM, said:

Another option...lyse the blood with water prior to analysis. the spike and recovery should indicate if the analyte sticks...

Thanks PAO-ahac,

I think we'll try a few of the suggestions and see what sticks (or doesnt)

Andrea