Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies

Abstract

The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms.

Large‐scale blood group genotyping – clinical implications

“…There is no doubt however that, for the foreseeable future, serological methods will remain a normal procedure among transfusion medicine specialist, as the primary method for detecting blood group‐specific antibodies in patient serum. Recombinant blood group antigens may offer opportunities to replace reagent (panel) red cells and methods demonstrating the effectiveness of this approach for FY (Sheffield et al , 2006; Ridgwell et al , 2007), Gerbich (Jaskiewicz et al , 2002; Schawalder et al , 2004) and Lutheran and Kell (Ridgwell et al , 2007) have been published. However, recombinant Rh antigens pose the biggest challenge for such antibody detection technology as anti‐Rh are very clinically significant, and any array‐based system must include this on the platform to have any useful application in transfusion medicine.…”

Silencing and overexpression of human blood group antigens in transfusion: Paving the way for the next steps

“…In addition, the complexity of some blood systems whose antigens may interact closely with other proteins, such as in the Rh complex [14,15], makes the choice of cell line crucial since some are incapable of forming the studied protein network [8,13,15]. Thus, for study or production of human blood group antigen, many reports describe the use of heterologous systems including transfected human embryonic kidney epithelial cells 293 [16], HeLa cells [17], A431 cells derived from human epidermoid carcinoma [18], CHO hamster cells [19], COS monkey cells [13,20], Mouse Erythroleukemic Line (MEL) [15,21], and NS-0 line from myeloma [22], Xenopus laevis oocytes [23], yeast cells [23,24], and Escherichia coli [25]. With regard to these models, it should also be noted that the expression of certain antigens is not limited to erythroid cells so that the absence of native expression may be determinant factor in choosing human heterologous cell lines [13].…”

“…Recombinant proteins are used for their capacity to inhibiting hemagglutination assay indicating incompatibility reaction or to trap the antibodies or ligands associated with these proteins in ELISA-type testing [20,24,[64][65][66][67].…”

Recombinant blood group proteins in clinical practice – from puzzling to binary antibody testing

“…They were designed and validated to express the most relevant polymorphic and Table 1 Diagnostic potential of recombinant blood group antigens Technological advantages One antigen/protein per well approach allows for: Direct antibody identification Antibody detection and identification in a single step Better standardization of assays (antigen quality and quantity) Advantages over conventional RBC antibody tests Higher accuracy and diagnostic power Easy, rapid and improved detection and identification of antibodies to high-prevalence and low-prevalence antigens [4-8] Improved resolution of antibody mixtures (multiple common antibodies, underlying antibodies in the presence of antibodies to highfrequency antigens) [8] Higher sensitivity (increased antigen density/accessibility) [9,10] Neutralization of clinically insignificant antibodies to high-prevalence antibodies in compatibility testing (soluble rBGPs) [8]. Clinical advantages Faster and safer blood supply to immunized patients [2,8] Suitable for routine laboratory applications Facilitates automated antibody identification [11].…”