Antibody profiling is a fundamental component of understanding the humoral response in a wide range of disease areas. Most currently used approaches operate by capturing antibodies onto functionalised surfaces. Such measurements of surface binding are governed by an overall antibody titre, while the two fundamental molecular parameters, antibody affinity and antibody concentration, are challenging to determine individually from such approaches. Here, by applying microfluidic diffusional sizing (MDS), we show how we can overcome this challenge and demonstrate reliable quantification of alloantibody binding affinity and concentration of alloantibodies binding to Human Leukocyte Antigens (HLA), an extensively used clinical biomarker in organ transplantation, both in buffer and in crude human serum. Capitalising on the ability to vary both serum and HLA concentrations during MDS, we show that both affinity and concentration of HLA-specific antibodies can be determined directly in serum when neither of these parameters is known. Finally, we provide proof of principle in clinical transplant patient sera that our assay enables differentiation of alloantibody reactivity against HLA proteins of highly similar structure, providing information not attainable through currently available techniques. These results outline a path towards detection and in-depth profiling of humoral immunity and may enable further insights into the clinical relevance of antibody reactivity in clinical transplantation and beyond.