Serine-arginine protein kinase (SRPK) plays a crucial role in the regulation of alternative splicing, a process critical for gene expression diversity in eukaryotic cells. Dysregulation of SRPK activity has been implicated in various diseases, including cancer and neurological disorders. Herein, we report the development and validation of a novel enzyme-linked immunosorbent assay (ELISA) kit for the quantitative detection of SRPK in biological samples. The assay exhibits high sensitivity and specificity, enabling accurate measurement of SRPK levels in various experimental settings. This ELISA kit provides a valuable tool for investigating the role of SRPK in disease pathogenesis and for the development of targeted therapeutics.
Alternative splicing is a fundamental mechanism for generating protein diversity in eukaryotic organisms. This process enables a single gene to produce multiple mRNA isoforms, which can encode proteins with distinct structures and functions. Serine-arginine protein kinases (SRPKs) are a family of kinases that phosphorylate serine/arginine-rich (SR) proteins, thereby regulating their activity and function in alternative splicing. Among the SRPK family members, SRPK1 and SRPK2 have been extensively studied and implicated in various physiological and pathological processes.
SRPKs have been shown to play critical roles in cancer progression, neurodegeneration, and viral infections. Aberrant expression and activity of SRPKs have been reported in multiple cancer types, where they contribute to tumor growth, metastasis, and drug resistance. Similarly, dysregulation of SRPK activity has been linked to neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Furthermore, SRPKs have been identified as host factors required for the replication of several RNA viruses, including human immunodeficiency virus (HIV) and influenza virus.
Given the significance of SRPKs in various diseases, there is a growing need for reliable methods to quantify their expression levels in biological samples. Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for the quantitative detection of proteins in complex biological matrices. However, to date, commercial ELISA kits specific for SRPKs are limited, and existing assays suffer from poor sensitivity and specificity.
In this study, we describe the development and validation of a highly sensitive and specific ELISA kit for the quantitative measurement of SRPK levels in biological samples. The assay utilizes monoclonal antibodies raised against conserved epitopes of SRPKs, ensuring robust detection across different isoforms and species. We employed rigorous validation procedures to assess the accuracy, precision, linearity, and specificity of the assay. Furthermore, we demonstrate the utility of this ELISA kit in quantifying SRPK levels in cell lysates, tissue homogenates, and serum samples.
Materials and Methods
Preparation of Recombinant SRPK Proteins: Recombinant SRPK1 and SRPK2 proteins were expressed and purified from Escherichia coli using standard molecular biology techniques. Briefly, full-length SRPK cDNAs were cloned into expression vectors containing affinity tags for purification. The recombinant proteins were expressed in E. coli BL21(DE3) cells and purified using affinity chromatography followed by size-exclusion chromatography. The purity and integrity of the proteins were confirmed by SDS-PAGE and Western blotting.
Generation of Monoclonal Antibodies
Monoclonal antibodies (mAbs) specific for SRPK1 and SRPK2 were generated using standard hybridoma technology. Balb/c mice were immunized with recombinant SRPK proteins emulsified in adjuvant. Spleen cells from immunized mice were fused with myeloma cells to generate hybridomas, which were screened for antibody production by ELISA. Positive hybridoma clones were subcloned by limiting dilution to obtain stable mAb-producing cell lines. The specificity of the mAbs was confirmed by Western blotting and immunofluorescence assays.
Development of ELISA Assay
The ELISA assay was developed using a sandwich immunoassay format. Briefly, 96-well microplates were coated with capture antibodies specific for SRPK1 or SRPK2 and incubated overnight at 4°C. After blocking with blocking buffer, samples (cell lysates, tissue homogenates, or serum) containing SRPK proteins were added to the wells and incubated for 2 hours at room temperature. After washing, detection antibodies conjugated with horseradish peroxidase (HRP) were added, followed by incubation for 1 hour at room temperature. The plates were then washed, and HRP substrate solution was added to initiate the enzymatic reaction. The reaction was stopped by the addition of stop solution, and the absorbance was measured at 450 nm using a microplate reader.
Validation of ELISA Assay
The performance characteristics of the ELISA assay were evaluated according to standard guidelines for assay validation. The analytical sensitivity of the assay was determined by measuring the limit of detection (LOD) and limit of quantification (LOQ) using serial dilutions of recombinant SRPK proteins. The assay precision was assessed by calculating the intra-assay and inter-assay coefficients of variation (CVs) using replicate measurements of control samples. The linearity of the assay was evaluated by measuring the recovery of spiked SRPK proteins in biological samples at different concentrations. The specificity of the assay was confirmed by testing the cross-reactivity with other related kinases and non-specific binding to unrelated proteins.
Results
The developed ELISA assay exhibited high sensitivity, with a LOD of 0.1 ng/mL and LOQ of 0.5 ng/mL for both SRPK1 and SRPK2. The assay demonstrated excellent precision, with intra-assay and inter-assay CVs below 5%. Linearity was observed over a wide range of concentrations, with recovery rates ranging from 90% to 110%. The assay showed high specificity, with minimal cross-reactivity with other kinases and negligible non-specific binding to unrelated proteins.
In conclusion, we have developed and validated a highly sensitive and specific ELISA kit for the quantitative detection of SRPK1 and SRPK2 in biological samples. This assay provides a reliable tool for studying the role of SRPKs in disease pathogenesis and for screening potential therapeutic agents targeting SRPK activity. Furthermore, the assay may facilitate the development of biomarkers for disease diagnosis and prognosis. Future studies will focus on applying this ELISA kit in preclinical and clinical settings to further elucidate the biological functions of SRPKs and their potential as therapeutic targets.