Enhancing red blood cell compatibility : in vitro hemagglutination prevention using a trispecific triabody as a blocking fragment for blood group antigens
Hafeez, Saleha and Asghar, Muhammad LU
(2026)
In Journal of Biological Engineering
20(1).
- Abstract
Background: Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To address this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs. Results: In this study, two triabody configurations differing in the placement of the anti-Rh(D) variable domain were generated, producing closed (C1) and open (O1) formats. The selected triabody-C1 was expressed in Escherichia coli BL21(DE3) and purified... (More)
Background: Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To address this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs. Results: In this study, two triabody configurations differing in the placement of the anti-Rh(D) variable domain were generated, producing closed (C1) and open (O1) formats. The selected triabody-C1 was expressed in Escherichia coli BL21(DE3) and purified into two fractions, AE3-B1 and AE3-B2. Hemagglutination assays demonstrated that AE3-B2 did not induce hemagglutination, whereas AE3-B1 showed mixed-field hemagglutination under standard conditions and complete hemagglutination under potentiator-enhanced hemagglutination conditions. ELISA-based binding assays indicated that the triabody’s monomers functioned independently with free antigens, while RBC-bound antigen assays revealed altered binding behavior upon sequential antigen engagement. Blood incompatibility related hemagglutination assays using monoclonal antibodies and incompatible O-negative blood plasma demonstrated complete prevention of hemagglutination by AE3-B2 triabody-coated RBCs, confirming effective antigen blocking. Conclusions: The trispecific triabody efficiently prevents hemagglutination by blocking A, B, and Rh(D) antigens on RBCs, enabling them to exhibit enhanced compatibility and hemagglutinating patterns similar to O-negative cells. These findings provide a promising strategy to increase the pool of compatible blood for transfusion, particularly in emergency and resource-limited settings, while emphasizing that future in vivo investigations are needed to confirm efficacy and safety.
(Less)
- author
- Hafeez, Saleha
and Asghar, Muhammad
LU
- organization
- publishing date
- 2026-12
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Antigen blocking, Binding cooperativity, ELISA, Hemagglutination, Transfusion compatibility, Trispecific triabody, Universal red blood cells
- in
- Journal of Biological Engineering
- volume
- 20
- issue
- 1
- article number
- 70
- publisher
- BioMed Central (BMC)
- external identifiers
-
- scopus:105037021910
- ISSN
- 1754-1611
- DOI
- 10.1186/s13036-026-00661-w
- language
- English
- LU publication?
- yes
- id
- 6fe4ba76-2eb2-4ab1-9863-35d80951e7e2
- date added to LUP
- 2026-05-26 12:21:42
- date last changed
- 2026-05-29 13:17:58
@article{6fe4ba76-2eb2-4ab1-9863-35d80951e7e2,
abstract = {{<p>Background: Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To address this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs. Results: In this study, two triabody configurations differing in the placement of the anti-Rh(D) variable domain were generated, producing closed (C1) and open (O1) formats. The selected triabody-C1 was expressed in Escherichia coli BL21(DE3) and purified into two fractions, AE3-B1 and AE3-B2. Hemagglutination assays demonstrated that AE3-B2 did not induce hemagglutination, whereas AE3-B1 showed mixed-field hemagglutination under standard conditions and complete hemagglutination under potentiator-enhanced hemagglutination conditions. ELISA-based binding assays indicated that the triabody’s monomers functioned independently with free antigens, while RBC-bound antigen assays revealed altered binding behavior upon sequential antigen engagement. Blood incompatibility related hemagglutination assays using monoclonal antibodies and incompatible O-negative blood plasma demonstrated complete prevention of hemagglutination by AE3-B2 triabody-coated RBCs, confirming effective antigen blocking. Conclusions: The trispecific triabody efficiently prevents hemagglutination by blocking A, B, and Rh(D) antigens on RBCs, enabling them to exhibit enhanced compatibility and hemagglutinating patterns similar to O-negative cells. These findings provide a promising strategy to increase the pool of compatible blood for transfusion, particularly in emergency and resource-limited settings, while emphasizing that future in vivo investigations are needed to confirm efficacy and safety.</p>}},
author = {{Hafeez, Saleha and Asghar, Muhammad}},
issn = {{1754-1611}},
keywords = {{Antigen blocking; Binding cooperativity; ELISA; Hemagglutination; Transfusion compatibility; Trispecific triabody; Universal red blood cells}},
language = {{eng}},
number = {{1}},
publisher = {{BioMed Central (BMC)}},
series = {{Journal of Biological Engineering}},
title = {{Enhancing red blood cell compatibility : in vitro hemagglutination prevention using a trispecific triabody as a blocking fragment for blood group antigens}},
url = {{http://dx.doi.org/10.1186/s13036-026-00661-w}},
doi = {{10.1186/s13036-026-00661-w}},
volume = {{20}},
year = {{2026}},
}