Kunz Pascal B, Tulin Ea Kristine Clarisse, Mehta Akul Y, Jia Tianwei, Heimburg-Molinaro Jamie, Otto Vivianne I, Stowell Sean R, Cummings Richard D
Department of Surgery, Beth Israel Deaconess Medical Center, National Center for Functional Glycomics, Harvard Medical School, CLS 11087-3 Blackfan Circle, Boston, Massachusetts 02115, United States.
Institute of Pharmaceutical Sciences, Vladimir-Prelog-Weg 1-5/10, Zurich ETH, Zurich 8093, Switzerland.
Glycobiology. 2025 Nov 6;35(11). doi: 10.1093/glycob/cwaf043.
A major challenge in the glycosciences is the scarcity of sensitive and specific glycan-binding reagents, such as monoclonal antibodies, for detecting and isolating glycans. Here we report the development and characterization of new monoclonal antibodies (mAbs) that bind carbohydrate-based red blood cell (RBC) antigens including the ABO(H) antigens. This approach exploits the immune system of the sea lamprey (Petromyzon marinus), which strongly responds to human glycans to enable the generation of high affinity antibodies. To develop these mAbs, we immunized the lamprey with RBCs and designed a targeted antibody enrichment and screening process using intact RBCs and a custom microarray displaying blood group antigens. Through multiple rounds of enrichment and testing we identified two mAbs; A_25 and A_39. Glycan binding analysis of the mAbs using glycan microarrays, the Luminex platform and western blot analysis revealed their binding to H antigens and terminal N-acetyllactosamine Galβ1-4GlcNAc (LacNAc, a type 2 sequence). Mechanistic insights into antigen specificity were gained through glycan inhibition assays, sequence homology analysis, and nanomolar-range affinity measurements. mAb binding to RBCs was determined using flow cytometry. Both mAbs bound RBCs of all ABO blood groups, whereas strongest binding was observed for blood group O RBCs. Our findings highlight the efficacy of the lamprey system to develop glycan-specific mAbs. These reagents allow investigation of expression of the H antigen and LacNAc-containing glycans in human tissues. In the future, they could also be modified using molecular engineering techniques to generate mAbs specific to other understudied blood group antigens.
糖科学领域的一个主要挑战是缺乏用于检测和分离聚糖的灵敏且特异的聚糖结合试剂，如单克隆抗体。在此，我们报告了新型单克隆抗体（mAb）的开发与特性鉴定，这些抗体可结合基于碳水化合物的红细胞（RBC）抗原，包括ABO（H）抗原。该方法利用了海七鳃鳗（Petromyzon marinus）的免疫系统，它对人类聚糖有强烈反应，从而能够产生高亲和力抗体。为了开发这些单克隆抗体，我们用红细胞免疫七鳃鳗，并设计了一种靶向抗体富集和筛选过程，使用完整的红细胞和展示血型抗原的定制微阵列。通过多轮富集和测试，我们鉴定出两种单克隆抗体：A_25和A_39。使用聚糖微阵列、Luminex平台和蛋白质免疫印迹分析对这些单克隆抗体进行聚糖结合分析，结果显示它们与H抗原和末端N - 乙酰乳糖胺Galβ1 - 4GlcNAc（LacNAc，一种2型序列）结合。通过聚糖抑制试验、序列同源性分析和纳摩尔级亲和力测量，获得了对抗原特异性的机制性见解。使用流式细胞术测定单克隆抗体与红细胞的结合。两种单克隆抗体均能结合所有ABO血型的红细胞，而与O型血红细胞的结合最强。我们的研究结果突出了七鳃鳗系统开发聚糖特异性单克隆抗体的有效性。这些试剂可用于研究人类组织中H抗原和含LacNAc聚糖的表达。未来，它们还可以使用分子工程技术进行修饰，以产生针对其他研究较少的血型抗原的单克隆抗体。
