Protein lysates were identified as inner cortex using microscopic analysis and grouped into non-rejection (= 11), borderline-TCMR (= 6), TCMR (T-cell mediated rejection, = 9) and AMR (antibody-mediated rejection, = 18). (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies. Keywords: biomarkers, antibody-mediated rejection (AMR), neural net analysis, CXCL9 (MIG), aptamer, antibody, aptamer-antibody-hybrid lateral circulation assay (hybrid-LFA) 1. Introduction Kidney transplantation (Ktx) is the most frequent transplantation worldwide, but transplant survival is limited by a high incidence of late antibody-mediated rejection (AMR) [1,2]. KTRs are regularly and continuously monitored by detection of donor-specific antibodies (DSAs) and transplant biopsies. Mechanisms and causes of AMR have been investigated by plasma cell and biopsy analyses [1,3,4]. Identification of DSAs along with several biopsy-based morphological and molecular criteria are relevant for AMR diagnosis according to the current BANFF-classification criteria [5]. Although DSAs are important, they are not an unambiguous, diagnostic criterion for AMR [5]. Specifically, detection of de novo DSAs after Ktx can lead to perform protocol biopsies [6]. However, it has been shown that circulating DSAs in KTRs with an inconspicuous clinical course do not necessarily indicate ongoing rejection of the graft, and CMPD-1 in some cases with a histological obtaining suspicious for AMR, single antigen HLA-DSA cannot be recognized by solid phase assays (SPA) and therefore new methods for HLA antibody screening were built up [7,8]. This has also been exhibited in a recent cross-sectional analysis of stable recipients with DSA-positivity 6 months post Ktx, of which only 51% were diagnosed with biopsy-proven AMR [9]. Despite detailed guidelines for the practical use of DSA detection with the modern sensitive but costly SPA methods, interpretation of the results is complex and the diagnostic value as post-transplant monitoring is considered to be limited [10,11]. C4d deposition of peritubular capillaries in transplant biopsies were also not consistently associated with the full histopathological obtaining of AMR [12,13]. Thus, an alternative easy and cheap diagnostic CMPD-1 for acknowledgement of AMR is usually urgently needed based upon biomarkers with high specificity for AMR [14,15]. Consequently, numerous methods for non-invasive, non-DSA-based diagnostic strategies have been explored in recent years. Among the diverse immunological biomarkers that could be relevant, many non-invasive biomarkers have shown potential in preliminary studies. The chemokines CCL3, CCL4, CXCL9, CXCL10 and CXCL11 have recently been identified as markers for inflammation of the microvascular blood vessels [16,17,18,19,20,21,22] and chemokines play a role as early markers in renal allograft rejection [15,23,24]. They act as a signaling system between allogen-presenting cells such as macrophages and immunocytes. In addition, cytokines can be produced by activated graft endothelium as part of the organ-related rejection process [25,26] with low-level activation of T-cells, natural killer (NK) cells and CMPD-1 myeloid cells. Cellular margination and activation including monocytes or macrophages and NK cells was explained in AMR in parallel to an altered proinflammatory gene expression profile, also in biopsies with a classification to be unsuspicious [27]. Translation of these biomarkers to the medical center requires strong validation studies and standardized, commercially available assays that need to be paralleled, but not solely depending on increased serum creatinine levels [28], which may also indicate AMR. Test platforms for reliable detection of appropriate rejection markers usually require expertise due to their complex handling and interpretation, including additional gear, and, thus, are not relevant for POCT. Hence, an LFA might serve as an alternative approach, because the patient is able to perform the test at home and to identify the test result by vision within minutes. Such sandwich assays usually apply antibodies, but new test setups are being sought in the field of LFAs as the use of antibodies has some disadvantages and limitations: generally generated in animals or cells, batch-to-batch variations, limited shelf life, hard to label at a specific location and sensitive to temperature, possibly even irreversible denatured. In comparison, aptamers, which are single-stranded DNAs or RNAs, can be chemically synthesized [29], modified with functional groups, in most cases without loss of affinity [30]. Furthermore, they are thermally stable, cheap to produce, can detect small molecules and provide a constant production quality. Initial CMPD-1 aptamer-based sandwich assessments have already been explained [31,32,33,34,35] and various LFAs have been reported using a hybrid of aptamer and antibodies for the PIK3CG composition of the sandwich [36,37,38]. It was the aim.