1) anti-PSA IgY antibody displayed the ability to bind both free and complexed PSA at nearly the same molar ratio (Fig. ELISA (IBL International, Hamburg, Germany). == Results == LoB, LoD, and LoQ, were 0.061, 0.083, and 0.100 ng/mL, respectively, and linearity range was 0.1003.375 ng/mL. tPSA concentrations from our IgY-based ELISA strongly correlated with those from the commercial assays. == Conclusions == Our IgY-based ELISA is an efficient equivalent to the above commercial assays. The use of IgY as the detecting agent could reduce the risk of false positive results, as well as decrease the overall cost of analysis. Keywords:Prostate specific antigen,In vitrodiagnostics, Prostate cancer, Immunoglobulin Y, Sandwich ELISA, Performance == INTRODUCTION == Prostate cancer screening and early diagnosis is based on digital rectal examination Mouse monoclonal to SORL1 and prostate specific antigen (PSA) serological testing [1]. If necessary, transrectal ultrasound-guided prostate biopsy is used; however, this is an invasive method that requires local anesthesia and antibiotic cover or multiparametric magnetic resonance imaging [2]. In either case, proper interpretation of the results, extensive experience, and close collaboration with urologists is mandatory, which contributes to the overall difficulty of both diagnostic approaches [3]. In 1986, the U.S. Food and Drug Administration (FDA) approved the Hybritech Tandem PSA assay (Hybritech Inc., San Diego, CA, USA) to measure serum PSA concentrations [4]. Initially, the assay was introduced for monitoring the course of disease; however, eight years later, it was accepted for screening potential prostate cancer development with a cut-off value of 4 ng/mL of total PSA (tPSA) in serum [5]. However, the reference cut-off value for prostate cancer screening remains the subject of continuous debate, and several attempts have been made to find a compromise between the specificity and sensitivity of the PSA assay, including setting age- and race-specific reference ranges [6]. PSA is a serine protease belonging to the kallikrein family. It is synthesized as preproPSA that is sequentially cleaved AZD5363 to form the proPSA zymogene and then the mature enzyme [7,8]. Additionally, PSA can be hydrolyzed at alternative positions, leading to the formation of catalytically inactive nicked forms [7,9,10]. The diagnostic approach is further complicated by the fact that PSA is present in the serum not only as free PSA (fPSA), but also in complexes with AZD5363 serpins: -1-antichymotrypsin (PSA-ACT) or -2-macroglobulin (PSA-2M; a form that escapes immunological detection) [9,10]. Nicked PSA does not form complexes with ACT and 2M and therefore participates in the fPSA fraction [7]. Assays used to measure tPSA need to detect not only the fPSA sub-fractions but also AZD5363 the PSA-ACT complexes, which constitute 7090% of tPSA in human serum [11]. However, the concentration and ratio of various PSA sub-fractions provide valuable information on health status and are considered as a supplement to tPSA measurements [12,13,14,15]. Thus, although not ideal, PSA remains one of the most tested, validated, and commonly used prostate cancer biomarkers. We propose the application of a recently developed PSA-specific chicken immunoglobulin Y (IgY) antibody as an alternative to currently used mammalian IgG antibodies [16]. The interaction between the Fcregion of IgG with rheumatoid factor (RF), the presence of human anti-mouse antibodies (HAMA) in serum, as well as the activation of the complement system might result in the interferences observed in immunoassays based on mammalian antibodies [17]. Since IgY antibodies do not participate in the above described interactions, their application leads to a reduction in these types of cross-reactions in serological assays [18,19,20]. The isolation method of IgY antibody from egg yolks is simple, efficient, and non-invasive. There are also significant economic and ethical aspects involved in the utilization of antigen-specific IgY antibodies as an alternative to IgG that need to be considered [21]. We describe the application of an anti-PSA IgY antibody in a sandwich ELISA and evaluate its diagnostic potential compared with the assay on Cobas 6000 analyzer (Roche Diagnostics, Warszawa, Poland) and PSA total ELISA (IBL International, Hamburg, Germany) used to measure serum PSA concentration. == METHODS == == IgY antibodies == PSA-specific IgY antibodies were obtained by immunizing 22-week-old white leghorn laying hens with fPSA (Fitzgerald Industries International, Acton, MA, USA) as the antigen [16,22]. Egg yolk antibody isolates were subjected to affinity chromatography purification with PSA conjugated to CNBr-sepharose (Thermo Fisher Scientific, Gdask, Poland), following the manufacturer’s protocol. The next steps.