A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.
This study reports a rapid and user friendly paper blood typing assay based on the Indirect Antiglobulin Test (IAT) principle. Red blood cells (RBCs) were incubated with IgG anti-D and supernatant was removed postincubation. The remaining RBCs were spotted on paper pre-treated with anti-IgG. A few drops of washing saline solution were deposited onto the blood spot on paper to flow through all non-agglutinated RBCs, leaving a clear white paper; this indicates a negative sample. Agglutinated RBCs resist saline solution flushing and form a bright red spot on paper signalling a positive reaction to the antibody tested. The IAT on paper successfully identified RBCs sensitised by IgG anti-D. Positive samples were clearly distinguished from negative samples. The critical variables were optimised to maximise the sensitivity and reproducibility of the paper diagnostic. This paper-based IAT assay further simplifies the current methods for IgG antibody-based RBC antigen typing. Its rapid and easy-to-read characteristics enable automated high-throughput analytical systems or a simple manual test for operators with minimum skills required.
Current blood typing methods rely on the agglutination of red blood cells (RBCs) to macroscopically indicate a positive result. An indirect agglutination mechanism is required when blood typing with IgG forms of antibodies. To date, the interaction forces between anti-IgG and IgG antibodies have been poorly quantified, and blood group related antigens have never been quantified with the atomic force microscope (AFM). Instead, the total intensity resulting from fluorescent-tagged antibodies adsorbed on RBC has been measured to calculate an average antigen density on a series of RBCs. In this study we mapped specific antibody interaction forces on the RBC surface. AFM cantilever tips functionalized with anti-IgG were used to probe RBCs incubated with specific IgG antibodies. This work provides unique insight into antibody-antigen interactions in their native cell-bound location, and crucially, on a per-cell basis rather than an ensemble average set of properties. Force profiles obtained from the AFM directly provide not only the anti-IgG – IgG antibody interaction force, but also the spatial distribution and density of antigens over a single cell. This new understanding might be translated into the development of very selective and quantitative interactions that underpin the action of drugs in the treatment of frontier illnesses.
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