Quantifying the Terminal Complement Complex (C5b-9) in Plasma

September 08, 2014

Duke University Medical Center
Associate Professor

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Company:

BD Biosciences

Product Name:

Human C5b-9 OptEIA ELISA Set

Catalog Number:

558315

Activation of any of the three complement pathways (classical, alternative, or lectin) results in the formation of the terminal complement complex, also called the membrane attack complex, or MAC. The MAC is composed of the complement proteins C5b, C6, C7, C8, and multiple copies of C9 (C5b-9). The MAC can be found embedded in the plasma membrane of cells and as a soluble complex in the blood. The presence of the MAC in blood may be associated with active infections, cancer, autoimmune diseases, or other pathological conditions. Since the formation of the MAC exposes a cryptic epitope in C9, the complex can be detected with antibodies recognizing this epitope. We were interested in determining if a specific autoantibody found in the blood of some cancer patients was causing increased complement activation and, hence, an increase in blood C5b-9. Although soluble C5b-9 ELISA kits are available from several vendors, the OptEIA set from BD Biosciences was by far the most economical. While most vendors offer pre-prepared plates and reagents, the OptEIA set includes only the reagents, with plate set-up left to the researcher.

Experimental Design and Results Summary

Application

Measuring C5b-9 levels in plasma.

Starting Material

Human anti-coagulated blood; cells spun out to yield plasma.

Protocol Overview

This is a typical capture ELISA format. Ninety-six well plates are coated with the supplied C5b-9 capture antibody and incubated overnight at 4°C. The plates are then washed, blocked for 1 h, and standards and experimental samples added and incubated for 2 h at room temperature. The plates are washed, and the secondary antibody and detection reagent added. After 1 h incubation, the plates are washed and the chromogenic substrate added. The amount of color is then quantified by absorbance.

Tips

The instructions included with the kit are well-written and easy to follow. As suggested, plasma should be used since the C5b-9 levels in serum are artificially elevated due to effects of the clotting reaction. Also, the plasma should be analyzed fresh or frozen at -80°C prior to analysis. Avoid freeze-thaws as this can also increase C5b-9 levels.

Results Summary

Since our laboratory has a large collection of serum samples, I tried the ELISA on these first. As cautioned in the instructions, C5b-9 levels of all samples exceeded the highest standard level and could not be used. I then used the ELISA on plasma samples that had been stored at -80°C and never thawed. All levels fell within the standard curve when the plasma samples were diluted 1:25.

Additional Notes

Although the instructions recommend TMB as the chromogen substrate, I was able to substitute ABTS without compromising the sensitivity. Also, I used PBS/0.1% (v/v) Tween-20 (PBST) as the blocking agent and for all dilutions and washes. We have found that PBST works as well as, if not better than, blocking agents containing milk, serum, or albumin.

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Summary

The Good

Easy to follow instructions and very economical.

The Bad

No suggestions for plasma dilutions are given. I chose 1:25 and that worked well. Also, the C5b-9 standard is supplied as a lyophillized powder in 5 sealed vials. Once the protein in a vial is resolubilized, it must be used immediately. Hence, the kit is designed to be used on entire 96-well plates. A method to perform the assay in partial plates would be useful.

The Bottom Line

Overall, this is an excellent, high-quality product. In addition, I found it to be quite adaptable to our lab-specific protocols. Since the kit does not include pre-coated ELISA plates, the cost is kept very reasonable.

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