AtaGenix Empowers Research on Shrimp Plasma MANF's Anti-Inflammatory Role via Conserved Receptor Tyrosine Phosphatase

The evolutionary mechanisms of inflammation regulation in metazoans remain unclear. This study uses shrimp as a model, identifying MANF as an LPS-induced plasma protein through plasma proteomics. MANF suppresses ERK phosphorylation and Dorsal expression in shrimp hemocytes, exerting anti-inflammatory effects.

The related study was published in The Journal of Immunology on February 11, 2022, DOI: 10.4049/jimmunol.2100595.

This study aims to validate the anti-inflammatory function and mechanism of shrimp MANF, including assessing changes in plasma MANF levels under LPS induction, the effects of recombinant MANF on ROS production and inflammation-related gene expression in shrimp hemocytes, and the role of MANF in regulating the ERK pathway via RPTP.

Producing high-affinity MANF monoclonal antibodies requires addressing specificity and affinity issues in phage library screening. ELISA detection must ensure sensitivity and reproducibility of capture and detection antibody pairing. Western blot and co-immunoprecipitation experiments need optimized controls to validate protein interactions.

• Antibody/Protein Development Strategy: Recombinant MANF is used as an immunogen, with monoclonal antibodies prepared via phage library screening and purified using Protein A, yielding high-affinity mAbs such as R4P1-C3 and R4P1-H5.
• Experimental Workflow: Western blot uses R4P1-H5 to detect MANF levels, with dilution optimized based on a standard curve. ELISA employs R4P1-C3 for capture and HRP-conjugated R4P1-H5 for detection, suitable for quantitative plasma sample analysis.
• Validation Methods: Recombinant MANF serves as a positive control, with rEGFP as a negative control. RPTP knockdown validates MANF’s dependency on ERK suppression. Immunofluorescence co-localization confirms RPTP’s cell membrane localization.
• Additional Support: SDS-PAGE purity reports and ELISA standard curves are provided to ensure batch consistency. It is recommended to use an LPS-induced model to validate antibody specificity during replication.

Monoclonal antibodies prepared via phage library screening confirmed shrimp MANF as an LPS-induced plasma protein. Recombinant MANF treatment suppressed ROS production and inflammatory gene expression in shrimp hemocytes. MANF’s interaction with RPTP inhibits ERK phosphorylation, providing insights into MANF’s role in regulating immunity and metabolism in metazoans, with implications for understanding inflammation evolution.

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