Indirect Antiglobulin Test: Paper-based Assay Development and Antibody-Antigen Interaction Characterization

In recent years, paper-based diagnostic assays for blood typing have advanced rapidly for the typing of ABO and Rh(D) blood groups. Most of these paper-based assays were based on the direct agglutination mechanism using immunoglobulin M (IgM) antibodies treated on paper. The attempt to directly agglutinate red blood cells (RBCs) on paper using IgG antibodies has consistently failed. This was expected due to the ‘non- agglutinating’ nature of IgG antibodies. The indirect antiglobulin test (IAT), which is a 2- stage test including the sensitisation of red blood cells (RBCs) with specific IgG antibodies and the bridging of IgG-sensitised RBCs with anti-IgG, was developed to detect IgG antibodies and IgG-sensitised RBCs. The IAT is routinely used in RBC antigen phenotyping, antibody detection and identification, and cross-matching donor and recipient for blood transfusion. Standard methods for the IAT such as the traditional tube test, gel card column agglutination technology and solid phase assay are cumbersome, time consuming and requires modern laboratory equipment handled by skilled personnel. An easy-to-use paper diagnostic for IAT would be invaluable for any blood analysis involving the IgG antibodies. IgG antibodies can cross the human placenta, potentially resulting in haemolytic disease of the fetus and newborn; their detection is crucial. <br> In this thesis, two paper-based IAT assays were developed as proofs of concept. Both assays involved the treatment of paper with anti-IgG that could capture IgG-sensitised RBCs. The first assay utilised the filtration ability of paper to trap agglutinated RBCs within the fibre network while allowing non-agglutinated RBCs to be filtered out through the pores between fibres. Instead, the second assay was based on the principle of paper chromatography. Once again, RBC agglutinates are trapped between the fibre networks, but non-agglutinated RBCs are eluted away from the sample loading point with the capillary force of a buffer solution wicking the inter-fibril capillaries of paper. These assays do not require specific laboratory equipment nor skills. They are simple to use and results are easy to read, eliminating human error. Given the different mechanisms on which both assays rely upon, the different needs and testing conditions required by end-users can be suited by selecting the proper test. <br> Whilst the blood typing principle and current technologies are well established, the fundamentals of antibody – antigen interactions occurring within a blood typing system are poorly quantified. Blood typing specific antibody – antigen interactions have always been reported as affinity constants and relative binding units. The interaction force and energies have never been reported. In particular, the interactions between the anti-IgG and RBC surface antigen bound IgG antibodies have never been rigorously quantified. RBC surface antigen distribution have always been observed and indirectly evaluated using labelled antibodies coupled with scanning electron microscopy or flow cytometry techniques. These techniques involve over-manipulation of the RBCs and results are unspecific and general. <br> The atomic force microscope (AFM) is a versatile instrument for biological imaging which can resolve features from the micro scale down to the nano scale. It also allows imaging not only in air but also in liquid, enabling imaging of biomolecules in physiological conditions. The unique force mapping mode of the AFM allows the simultaneous measurement of interaction forces and localization of sites where specific interactions have occurred. Using the AFM force mapping mode, we have quantified for the first time the interaction energies between anti-IgG – IgG on RBC and IgM – RBC surface antigen while simultaneously mapping the location and quantifying the density of the antigen sites. This was achieved by functionalizing the AFM cantilever tip with anti-IgG and IgM antibodies, respectively, in two separate studies. The antigen of interest in both studies was kept as the D antigen. For the first time, the distribution of antigens on individual RBCs as mapped by anti-IgG and the IgM-functionalized AFM tips was measured and compared under physiological conditions and found to be similar. However, heterogeneity observed in the distribution of antigens among RBCs isolated from a single donor and on RBCs isolated from multiple donors bearing the same blood group emphasized the importance of mapping antigen distribution on single cells rather than relying on an ensemble set of information. This technique has allowed the investigation of antigen distribution in real-time, in situ and label free. It is anticipated that the developed technique can be expanded to the use in the development of selective and personalized medical treatment