# Promising Novel Biomarkers in Cardiovascular Diseases
## 2 Common Detection Methods for Biomarker Analysis  
As previously mentioned, biomarkers such as sST2, H-FABP, GDF-15, and suPAR, or a combined analysis thereof, may represent a promising tool to support the clinical diagnosis of certain CVD. Therefore, the availability of detection methods to gain reproducible and reliable biomarker concentrations is crucial. Soluble proteins are secreted into the circulation where they can easily be harvested in whole blood by venous puncture. Serum or plasma samples may then be derived by centrifugation of whole blood at 2000 rpm for approximately 20 min. The samples may be measured directly, or stored at \(-80\)\({}^{\circ}\)C until further analysis to prevent the degradation of the proteins of interest. Absorbance assays, such as enzyme-linked immunosorbent assay (ELISA) belong to the main standard procedures for soluble biomarker detection. Most of the available ELISA kits for serum or plasma analyses are based on the so-called \"Sandwich\" ELISA, where a capture antibody binds the antigen in the sample. A colour reaction is achieved by the addition of an appropriate detection antibody in addition to an enzyme that is activated by a 3,3\({}^{\prime}\),5,5\({}^{\prime}\)-Tetramethylbenzidine (TMB) substrate. The optical density can then be measured with the help of an absorbance reader [62]. Although several manufacturers have attempted to develop an assay that may be used in the clinical setting, only a few ELISA kits have diagnostic grade approval. As biomarker detection using ELISA is a rather time-consuming procedure, different companies have already successfully developed some rapid tests to get reproducible biomarker level results within a couple of minutes [63]. As far as the detection of sST2 is concerned, a large range of ELISA kits from various manufacturers is available. However, most are approved for research use only. The kits from R&D Systems (Human ST2/IL-33R Quantikine ELISA Kit [9], Human ST2/IL-33R DuoSet ELISA [64]; Minneapolis, MN), and Medical & Biological Laboratories (MBL) Co, Ltd. (Woburn, MA, USA) [65; 66; 67] are most commonly used in research laboratories all over the world. To date, only two assays are available that have the certificate for clinical usage: the highly sensitive Presage Assay (Critical Diagnostics, San Diego, CA, USA) [68; 69; 70; 71; 72] and the ASPECT PLUS ST2 Quantitative Rapid Test Assay (Critical Diagnostics, San Diego, CA, USA), which is based on quantification by sandwich monoclonal lateral flow of antico Regulated blood [63; 73; 74]. For the latter, a small amount (35 \(\upmu\)L) of the patient's serum or plasma is loaded onto an analysis cassette and two drops of a provided buffer are added. The cassette is then analysed using an appropriate reader and results may be obtained within 20 min [75]. Although all available ELISA kits have been approved for diagnostic or research purposes, studies have revealed large discrepancies in serum or plasma sST2 values among the different kits [76]. Several studies have concluded that H-FABP is a promising and reliable biomarker for the diagnosis of several cardiac diseases, such as acute myocardial infarction (AMI) [77]. As a consequence thereof, several test types and assays haven been developed to determine the biomarker's levels in peripheral blood, as well as in urine. In 1995, [PERSON] et al. developed an immunosorbent technique to assess and quantify H-FABP by ELISA [78], which was the milestone for the development of further ELISA kits which are widely available nowadays: the kits from R&D Systems (Human FABP3 DuoSet ELISA [79], Minneapolis, MN) and Hycult Biotechnology (Uden, The Netherlands) [80] have great evidence to assess blood H-FABP levels in several clinical research projects. As far as the clinical diagnosis of H-FABP is concerned, the Heart FABP (FABP3) Human ELISA kit from BioVendor LaboratoryMedicine (Modrice, Czech Republic) has successfully gained clinical diagnostic grade.