To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.
Prostate-specific antigen (PSA) is widely used as a diagnostic marker for the detection of prostate cancer in men. We have generated stable hybridomas producing specific monoclonal antibodies (MAbs) of the IgG class against PSA from fusions of splenocytes from immunized mice with myeloma cells. The hybridomas were adapted into serum-free media and cultured in CELLine CL-1000 bioreactors to produce milligram quantities of MAbs. Cross-reactivity study demonstrated that all the MAbs reacted did not cross-react with several other types of tumor antigens. Two of the MAbs were successfully radiolabeled by the iodogen method. The (125)I-labeled MAbs demonstrated strong binding to PSA on the surface of the LNCaP cells (Kd of 1.16 x 10(-9) M and 1.4 x 10(-9) M). Thus the (125)I-labeled MAbs retained their immunoreactivity and possessed high affinity and is potentially useful for binding to tumor cells. In conclusion, the MAbs can be used to develop radioimmunodiagnostic, radioimaging, and immunohistochemistry techniques for the early detection and treatment of prostate cancer.