Colleagues,

I think I might have found the reason why my anti-protease activity measured negative. It depended on the concentration and amount  of  trypsin that was added to the sera. The trypsin concentration in solution  was about  800 mg/l and at the same time the amount added to the sera was double the sera amount. I performed the assay again and used equal amounts of sera and trypsin (maintaining the same concentration) and the results were all positive. The assayed results ranged between 30% to 50% inhibition. .

Hi Emmanuel,

can you provide more details about your assay and results?

Dear Emmanuel Delwin Abarike

Pls. find the attached files

http://www.sigmaaldrich.com/life-science/learning-center/life-science-video/universal-protease.html

http://jgp.rupress.org/content/26/4/405

http://www.jove.com/video/899/sigma-s-non-specific-protease-activity-assay-casein-as-a-substrate

http://www.atsjournals.org/doi/abs/10.1164/ajrccm.152.5.7582300#.V-9QVzZWEt4

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106377/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785526/

http://jgp.rupress.org/content/26/4/405

http://www.sciencedirect.com/science/article/pii/S0022202X15423310

http://jb.asm.org/content/189/22/8130.full

http://medind.nic.in/iaf/t04/i2/iaft04i2p132.pdf

http://www.atsjournals.org/doi/abs/10.1164/rccm.200302-203OC#.V-9QVjZWEt4

http://link.springer.com/article/10.1007/s10126-010-9349-0

http://ac.els-cdn.com/S0022202X15423310/1-s2.0-S0022202X15423310-main.pdf?_tid=c2653930-879a-11e6-b89b-00000aab0f27&acdnat=1475301123_28f899990d6d158bf28012fd69b2715f

https://www.ncbi.nlm.nih.gov/pubmed/20225660

http://www.hoajonline.com/vetmedanimsci/2054-3425/1/2

Hoping this will be helpful            

Regards

Prof. Houda Kawas

Dear Carlo Zambonelli, below are the assay results:

well1 well2 well3 well4 well5

Trypsin blank 0.048 0.049 0.047 0.048 0.048

Sample 1 0.048 0.049 0.049 0.040 0.051

sample 2 0.052 0.052 0.052 0.050 0.051

sample3 0.05 0.051 0.040 0.05 0.048

sample 4 0.052 0.052 0.053 0.056 0.056

sample 5 0.049 0.048 0.05 0.05 0.052

The formula I used to compute the percent trypsin inhibition is = ((trypsion blank OD- sample OD)/trypsin blank OD x 100

it seems like well 1, well2..... well5 are just replicates. if it is the case then just find standard deviation first. then go for the calculation. you will definately get good  interpretable results