کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2088052 1545687 2015 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Phage display aided improvement of a unique prostate-specific antigen (PSA) antibody unreactive with Lys145-Lys146 internally cleaved forms
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Phage display aided improvement of a unique prostate-specific antigen (PSA) antibody unreactive with Lys145-Lys146 internally cleaved forms
چکیده انگلیسی
Prostate specific antigen (PSA) is a commonly used marker of prostate cancer. A panel of four kallikrein immunoassays has been reported to improve the prediction of prostate biopsy outcome (cancer vs benign) in men with elevated PSA in the circulation. Assay of one of the kallikrein forms, intact free PSA (fPSA-I), is based on a unique monoclonal antibody (4D4), which is specific for PSA without the internal cleavage at Lys145-Lys146. Due to high dissociation rate the 4D4 antibody is less than optimal for achieving a highly sensitive robust assay. In this study, we cloned the 4D4 Mab into a recombinant fragment (Fab) format and constructed three mutant libraries with the aim to increase its binding affinity. The libraries contained targeted mutations either in the CDR-H1, CDR-H2 or CDR-L3 region. PSA-I specific antibodies were enriched from the libraries by phage display technology. We identified fourteen unique clones with 1-5 mutated amino acids showing reduced dissociation of the PSA conjugate compared to the wt-4D4 Fab. Five of these mutant antibodies had 2-6 times higher binding affinity compared to the wt-4D4 Fab yet retaining the original specificity for PSA-I. The analytical sensitivity of fPSA-I assay with mutant L3-2 Fab was 0.12 μg/L compared to 4.46 μg/L with the original wt-4D4 Fab. In the method comparison study, the developed assay showed an excellent correlation to the existing fPSA-I assay. The high affinity and specificity of these mutant antibodies have potential to provide sensitive and robust detection of intact and nicked PSA from patient samples in different test formats.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 422, July 2015, Pages 72-79
نویسندگان
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