ZBiotech’s Neu5Ac and Neu5Gc N-glycan microarray helps researchers identify a carbohydrate-based biomarker for cancer detection.

Shewell and colleagues developed a new precise assay for detecting the presence and the levels of N-glycolylneuraminic acid (Neu5Gc)-containing glycans, which are stably present in cancer tissues. An enzyme called cytidine monophosphate N-acetylneuraminic acid (Neu5Ac) hydroxylase, which converts Neu5Ac to Neu5Gc, is inactive in normal human tissues, but consistently active in cancerous tissues. Glycan-based antigens have been the source of cancer biomarkers. The levels of cancer antigen 15-3 (CA15-3) are higher in most women with metastatic breast cancer, and therefore CA15-3 is considered a biomarker to monitor disease progression. Another cancer antigen, called CA125, is present in ovarian cancer, and the CA125 test has been used to screen for ovarian cancer. However, these markers are also present in other physiological conditions, like pregnancy or heart disease, and therefore not cancer-specific. Neu5Gc is exclusively present in cancer tissues and could overcome non-specific limitations to serve as a robust biomarker for early cancer detection and monitoring responses to the treatment.

In this study, the researchers developed a surface plasmon resonance–based method for efficiently detecting Neu5Gc in patient serum samples. The detection is based on a binding reaction between Neu5Gc and a recombinant lectin called SubB2M. SubB2M is engineered from the B-subunit of the subtilase cytotoxin from E. coli, which detects the α2,3 and α2,6 Neu5Gc linkages to substituent sugars. A non-binding version of SubB2M, SubBA12, was implemented in the method to control any non-specific binding to SubB2M.

Researchers proved the specificity and efficacy of SubB2M binding to Neu5Gc using our Neu5Ac/Neu5Gc N-glycan microarray, created with 42 different glycans terminated with Neu5Ac or Neu5Gc. The negative control, SubBA12, exhibited no binding to Neu5Ac or Neu5Gc, compared to SubB2M, as shown by the glycan array assay. Also, it was important to prove that SubB2M does not bind to any other sugar molecules as it would diminish the sensitivity and preciseness of the assay. Using our glycan microarray products, the researchers further showed that SubB2M does not bind any other sugar molecules except Neu5Gc, demonstrating the feasibility of SubB2M usage.

The researchers tested the method using a serum cohort including samples from stage I, II, III, and IV breast cancer patients and comparable samples from cancer-free individuals. The analysis showed the presence of Neu5Gc in all stages of breast cancer but not in cancer-free samples. The assay was 98.76% sensitive and 100% precise. In another set of samples taken every 6 months during cancer treatment, tracked Neu5Gc levels were consistently dropping. Therefore, the SubB2M-based detection method can help monitor breast cancer from the developmental to the post-treatment stage.

Our Neu5Ac and Neu5Gc N-glycan microarray is a valuable tool, and it can help researchers to identify the specificity of the lectin (protein) reaction to the glycan. The use of the Neu5Ac and Neu5Gc N-glycan microarray quickly and precisely determines to which exact glycans the lectin can bind and how specific the reaction is. Our microarray was critical in developing the highly-sensitive carbohydrate biomarker-based assays in this case.