Endotoxin Detection: How Clean Are Your Antigens?

Have you ever spent weeks or months pursuing a mysterious artifact in your culture only to discover that the cause was a reagent or culture media contaminated with bacterial endotoxin? You’re not alone. Continue reading for a cautionary tale on endotoxin contamination.

Detecting a Problem: Clueing in on Endotoxin Contamination

We encountered an endotoxin contamination issue years ago when developing a CMV antigen product — but as you may have experienced in your past experiments, it wasn’t immediately apparent. 

We make our CMV antigens by infecting fibroblasts with cytomegalovirus and concentrating the lysate of the infected cells. During our quality control testing, we noticed that PBMC from donors we knew were CMV negative were showing a response to the CMV lysate. Something was up, but we assumed we made a simple mistake. We repeated the assay a second time but again found a false positive response. Something was clearly amiss, so we dug in further.

We tested the culture medium from the CMV-stimulated PBMC for additional cytokines and found IL-1β present — a cytokine we know was not present in previous lots of our CMV antigens. This was our first clue that bacterial endotoxin was present in our culture. Now to find the source…

Calling in the Heroes: Monocytes

Monocytes are especially sensitive to the presence of endotoxin and other pyrogens (substances that can cause fever). You may have heard of this natural sensitivity in other cases, like the European Pharmacopeia recognizing the Monocyte Activation Test (MAT) as a substitute for the Limulus amebocyte lysate (LAL) test. 

Pooled PBMC are essential to running the MAT, and we have several lots prepared according to MAT specifications that are intended to detect endotoxin by measuring the production of IL-1β, IL-6, or TNFα from pooled PBMC. That’s precisely what we did with our contaminated CMV antigen.

A Happy Ending

Using the presence of IL-1β, IL-6, or TNFα cytokines to measure the endotoxin contamination in our CMV antigens, we tested reagents used in making the lysate and discovered that the source of the endotoxin was the sucrose gradient material. 

We prepared a new batch of sucrose with endotoxin-free components and prepared a new batch of CMV antigen. Sure enough, the next batch of antigen did not stimulate PBMC from CMV-negative donors.

We extensively test our CMV antigens so you can be confident that your experimental outcomes reflect the donor’s CMV status, not errant pieces of dead bacteria.  

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