We are trying to activate complement protein in plasma from human sample (collected in sodium heparin anticoagulant) using cobra venom factor (CVF). Since, EDTA/Heparin block the complement activation we are looking to reverse or purify plasma before using CVF for activation. I have few queries on this.
a) Is there any method to reverse or purify EDTA/Heparin from plasma sample?
b) How can we test if activation is successful or not?
Can you give me your advice?
Thanks,
Ashok
Dear Ashok Silwal ,
Regarding the EDTA, simply adding sufficient CaCl to the plasma will allow you to saturate the EDTA in your sample, after which you can use the sample for complement activity assays if there are no additional inhibitors present. This is also referred to as re-calcified plasma. Note that you need to add additional CaCl and MgCl besides the amount needed to saturate the EDTA, as complement requires Ca and Mg ions for its activity. Also, upon adding a surplus of CaCl, the coagulation system will also be activated if there is no additional coagulation inhibitor present, similar to normal collected blood. This wil result in the formation of a clot. Thus after adding CaCl, let the clot form for ~1 hour and centrifuge your sample to separate the clot from the re-calcified plasma. I've sometimes experienced some additional small clot formation in re-calcified plasma during assays, even after removing the initial large clot.
Regarding the heparin, which also prevents the formation of a clot, you can try to add heparinase to your sample to cleave the heparin. I don't know if there is another method to remove heparin. There are several enzymes described in literature, see what is easily available for you.
Keep in mind that complement activity in re-calcified/heparinase-treated plasma might not be optimal. The additional preparation steps might affect the overall complement activity of your sample as to compared the activity when using serum, i.e. the activity might be lower in re-calcified plasma due to the treatment.
Hope this helps,
Richard
Dear Ashok Silwal,
I have done complement activity measurements in EDTA-plasma and that works, once the sample is properly diluted to complement activating buffer (Tris+Ca/Mg or Veronal Ca/Mg). Heparin works via different modality, mainly inhibiting the charge based interactions of various initiators and complexes. Also some complement factors have heparin binding domains, such as factor H. So simple dilution of heparin might still skew the results.
If I would have a similar need, I would make a pilot study with fresh donors where you can control all the variables. Draw serum, EDTA-plasma and heparin plasma from a single donor, prepare the sera and plasma, store at -80C in aliquotes. Then thaw the sample and dilute appropriately to assay the activity of the sample or perform the in vitro activation it with CFV/Zymosan/Inulin. Test all three sample types side-by-side to get an idea what happens to the overall activity. My gut feeling is that with heparin you might need to dilute the sample more than is applicable to assaying activity.
Last, the remainder activity in your sample may be dependent how the samples have been prepared and stored. Prolonged storage at above -80C will for sure lead to degradation of activity and generation of complement activation fragments.
Dear Richard Pouw ,
Thank you for the great insight. Do you have any experience in activating complement with Veronal buffer?
Dear Juha Kotimaa ,
Thank you for your answer. We are trying to get rid of EDTA/heparin by buffer exchange using VBS-Mg EGTA and then activating it by CVF. since we have limited sample, we are not sure about pilot study with zymosan/insulin.
Can you please share your experience with activation study and how was your result?
Hi Ashok Silwal,
I am afraid my experience is limited to diluting EDTA-plasma to activation buffer and the triggering the complement cascade on ELISA. In your case you would need to recosntitute complement favourable condition and activate it in VBS/MgEGTA in some lower dilution, followed by further dilution and analysis.
Buffer exchange may be tricky to get to work consistently with sera, you will have dilution effect at least, probably some autoactivation as well.
Dear Juha Kotimaa ,
Thank you! I will definitely consider your suggestions.
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