C3a ELISA study for substrate hemocompatibility

Purpose: Study the hemocompatibility of different substrates using the concentration of C3a as the indicator.

Background: C3a is a protein produced during immune response. Directly from Wikipedia: The classical pathway of complement activation is initiated when the C1 complex, made up of C1r and C1s serine proteases, recognizes the Fc region of IgM or IgG antibodies [1]. The lectin pathway is activated when pattern-recognition receptors, like mannan-binding lectin or ficolins, recognize and bind to pathogen-associated molecular patterns on the antigen, including sugars [2].

 

Blood incubation on substrates

We defined a specific substrate area to be tested by placing a hole-punched PDMS block on top of the substrate. The punched holes are each 10mm in diameter. See below:

the substrates are: glass with 10 nM fMLP (n = 3), glass(n = 3), teflon (n = 3), cellulose membrane (n = 3), PDMS(n = 3), lift-off membrane (n = 3), nanoporous nitride membrane (n = 3). NOTE: The 10 nM fMLP in theory serve as a positive control as fMLP should be recognized by the pattern-recognition receptors to initiate the Lectin Pathway. The cellulose sample is actually a circular sheet of cellulose (9 mm diameter) that was placed on top of teflon in the well. Teflon was chosen as the hosting background, under the assumption that teflon minimally activate the immune response/C3a production. PDMS is also chosen as a testing substrate to assess if the PDMS block itself also trigger C3a production.

250 μL of blood was placed in each well. All samples are kept in petridish and placed in an incubator (37°C; 5% CO₂; 80% humidity) for 2 hr. Kimwipes that were prewetted with DiH₂O were placed in each petridish to help reduce sample evaporation.  NOTE : To avoid possible low-level complement activation, the blood were drawn into a ETDA tube (lavender topped) instead of heparin tube (green topped) in accordance to the ELISA kit recommendation.

 

Sample processing (plasma extraction and precipitation)

It is necessary to precipitate out proteins in the sample since proteins compete with complements in the ELISA assay.

After the 2 hr incubation, the samples were collected into 1.5 mL microfuge tube and spun at 2000 rcf for 15 min to collected cell-free plasma (the yellow portion on top). Whole proteins were then precipitated from the plasma via the following: Cell-free plasma (70 μL) was combined with of the complement reagent A (70 μL) and vortexed thoroughly to mix. 10N HCL (15.5 μL) was then added to the mixture and vortexed thoroughly to mix and let sit for 1 hr. The mixture were then spun at 10,000 RPM at the standard benchtop centrifuge for 5 min. 55 μL of the supernatant was then combined with 9N NaOH (6 μL) and vortexed thoroughly to mix. Complement reagent B (187 μL) was added the mixture and vortexed thoroughly to mix. 3.33 μL of Assay Buffer was then added to the mixture to bring the sample to a 1:10 dilution. The sample was then further diluted 1:50 in Assay Buffer. The 1:500 diluted samples should be within the detection range of the ELISA kit.

NOTE: Interestingly, for both time that I ran the the C3a ELISA experiment there always seems to be red blood cell lysis for the cellulose sample (the right sample below). Tejas said he observed the same thing when he ran his ELISA assay.

NOTE: these are samples with 70 μL of the supernatant removed.

 

The ELISA assay

The ELISA is ran in accordance to manufacturer protocol: ab133037-complement-c3a-des-arg-human-elisa-kit-v4-website

 

Result

• The error bar represents the standard error of mean.
• n = 3 for each group

raw data: with PDMS

full subtraction of PDMS from all:

 

 

NOTE:

1. Since PDMS block is used to define the surface area to volume ratio of the system, we also included PDMS as an internal reference.  [C3a] of the PDMS x the surface area of the wall/total surface area is calculated as the background correction, and is subtracted from the [C3a] measured for all samples.

2. The first time that I ran the C3a ELISA I ran a 1:10 diluted sample against standards that range from 1000 ng/mL to 0.1 ng/mL. The [C3a] measured for the different samples are all around the 1000 ng/mL range which is almost outside the range of the standards. I ran the C3a ELISA for a 2nd time with the 1:500 diluted sample against standards that range from 500 ng/mL to 0.4 ng/mL. It is comforting to see that I hit the predicted order of magnitude on the 2nd try (1:500 of 1000 ng/mL is ~2 ng/mL, similar to the average of 2.5 ng/mL observed here). This somewhat suggest repeatability. Interestingly, cellulose, which seem to trigger red blood cell lysis, did not produce high level of C3a as saw in the paper by the Roy group (https://trace-bmps.org/wp-content/uploads/2016/10/muthusubramaniam-roy-annal-of-biomedical-engineering-2011.pdf).

 

References

1. Arlaud, G. J., Gaboriaud, C., Thielens, N. M., Rossi, V., Bersch, B., Hernandez, J. F., & Fontecilla‐Camps, J. C. (2001). Structural biology of C1: dissection of a complex molecular machinery. Immunological reviews, 180(1), 136-145.
2. Dunkelberger, J. R., & Song, W. C. (2010). Complement and its role in innate and adaptive immune responses. Cell research, 20(1), 34-50.

 

Post Hoc hypothesis test (Fisher’s LSD)

Except for glass, all other substrate types were different from the fMLP control. No differences were seen for all other pairwise comparisons, other than the comparison between NPN and glass.