(d) IgA was quantified with a two-step sandwich ILISA, demonstrating high detection sensitivity. integration into detection methods suitable for Aucubin point of care. Our results demonstrate that ILISA is a simple and versatile nanoplatform for highly sensitive and reliable detection of serological biomarkers in biomedical research and clinical applications. Keywords: iron oxide nanoparticle, immunosorbent assay, serological biomarker, quantification. Introduction Recent advances in proteomics and lipidomics have led to extensive analysis of the molecular signature of human blood 1-4. A large number of biomolecules in the blood such as antibodies, cytokines and metabolites have been identified as the markers of specific pathological conditions, since the plasma concentration of these biomarkers correlates with disease onset, disease progression as well as patient responses to therapeutic interventions 5, 6. It has been demonstrated that accurate quantification of biomolecules in human blood samples holds great promises for the detection and diagnosis of a variety of diseases including infectious disease 7-9, autoimmune disease 10, 11, cardiovascular disease 12-15 and cancer 5, 6, 16-18. Requiring only a small amount of blood, the simplicity and noninvasive nature of the blood test make it an ideal choice for point-of-care disease diagnosis with low cost. Therefore, developing highly quantitative and robust techniques for blood tests is of great significance for a broad range of clinical applications, and has the potential to shift the paradigm of disease diagnosis and treatment. Accurate characterization of disease state using blood samples often requires quantification of a panel of disease markers in a complex mixture of blood components with high specificity, sensitivity, and reliability. Current clinical blood testing methods are mostly derived from enzyme-linked immunosorbent assays (ELISA) and nephelometric assays 19-24. These assays are inherently unstable because small changes in the reagents or operating procedures can lead to large variations in enzyme catalysis or protein agglomeration. For this reason, clinical blood tests are typically performed in laboratories with well-trained technicians, special instruments Aucubin and stringent quality control, which significantly increase the cost and time required for IGF1 disease diagnosis. There is an unmet clinical need for simpler, more robust and cost-effective blood testing techniques. An alternative approach is to develop a nanocrystal-based method for biomolecule detection 25-28. Compared with the protein- or small organic molecule-based probes in conventional detection methods, the detection signal generated with a nanocrystal is associated with the whole crystal structure, which is very stable upon exposure to UV radiation, heat and oxidants. Nanocrystal-based detection probes can therefore improve the reliability of blood tests. We have previously shown that a particular type of nanocrystal, iron oxide nanoparticles (IONPs) can be used for colorimetric detection in immunosorbent assays 29. In the iron oxide nanoparticle-linked immunosorbent assay (ILISA), the IONPs bound to immobilized target molecules are dissolved into individual metal ions, which are then quantified through a stable chromogenic reaction 29. Detection sensitivity of ILISA is determined by the number of iron atoms in each IONP, i.e., the size of the iron oxide nanocrystal in a probe. In this study, we further developed the sandwich ILISA for the detection of a group of serological markers, IgA, IgG, IgM and CRP. IgA, IgG and IgM are three isotypes of antibodies that are involved during different stages or types of immune reactions. The changes in the concentration of Aucubin these molecules are indicative of many diseases. For example, lower levels of IgA may be linked to certain types of leukemia, kidney damage, and enteropathy, while higher IgA concentrations may imply multiple myeloma, autoimmune diseases, and certain infectious diseases. Further, elevated levels of IgG, IgM and CRP are associated with infectious diseases, autoimmune diseases, and inflammation. Here we show the development of a detection method with large wstite nanocrystals and a highly sensitive chromogenic iron chelator, Ferene S, which can be readily incorporated into various forms of immunosorbent assays. The performance of ILISA was thoroughly examined with sera that mimic the normal and disease states. The serum levels of total IgG and IgM in the patients with infectious diseases were also quantified with.