Shrimp MANF: Anti-Inflammatory Discovery via Phage Display & Custom ELISA | AtaGenix

Based on: Luo K, Chen Y, Wang F, J. Immunology, 2022 (DOI: 10.4049/jimmunol.2100595)

Why This Study Matters

Inflammation is a double-edged sword: too little leaves the host vulnerable to infection; too much causes self-damage. In mammals, this balance is modulated by a complex network of cytokines, receptors, and intracellular signaling cascades. But which of these regulatory mechanisms are truly ancient and conserved — and which are evolutionary add-ons? Invertebrates like shrimp offer a stripped-down model system where the core immune logic is easier to dissect.

This study, using the Pacific white shrimp (Litopenaeus vannamei), discovered that MANF — previously known only as a vertebrate neurotrophic factor — functions as an LPS-induced anti-inflammatory plasma protein in invertebrates. MANF binds RPTP on hemocytes (the shrimp equivalent of macrophages), dephosphorylates ERK, and suppresses NF-κB (Dorsal) activation. The finding that this mechanism also operates when shrimp RPTP is expressed in human 293T cells suggests deep evolutionary conservation — with potential implications for neuroprotection and inflammation research in mammals.

Figure 1. MANF is conserved from invertebrates (shrimp) to vertebrates (human). Phylogenetic analysis and sequence alignment demonstrate high structural conservation, suggesting that MANF's anti-inflammatory function predates the vertebrate immune system.

The Challenge

Proving that MANF is a secreted anti-inflammatory factor — not just an intracellular stress-response protein — required quantifying its levels in shrimp plasma before and after LPS challenge. No commercial ELISA kits exist for shrimp MANF. The research team needed two critical custom reagents: (1) highly specific monoclonal antibodies against shrimp MANF for sandwich ELISA construction, and (2) recombinant MANF protein for functional studies (dose-response experiments on hemocyte ERK/NF-κB signaling).

Both reagents presented unique difficulties. Shrimp MANF had not been previously characterized as a protein, so no validated immunogens or expression constructs existed. The antibodies had to be specific enough to detect MANF in the complex background of shrimp hemolymph plasma, and the recombinant protein had to retain biological activity for cell-based signaling assays.

AtaGenix's Approach

• Monoclonal Antibody Discovery: AtaGenix used its phage display platform to screen for anti-shrimp MANF antibodies. Phage display was chosen over hybridoma because the target was a novel, uncharacterized invertebrate protein with no prior antibody reagents — phage libraries offer broader epitope coverage for de novo targets. Selected clones were validated for specificity and paired for sandwich ELISA configuration (capture + detection).
• Recombinant MANF Expression: Shrimp MANF was expressed in AtaGenix's CHO transient system to produce protein with proper folding and disulfide bonds (MANF contains a conserved CXXC motif critical for function). The CHO-expressed protein was used directly in hemocyte stimulation experiments to test dose-dependent effects on ERK phosphorylation and NF-κB expression.
• Sandwich ELISA Development: AtaGenix's matched antibody pair was used to build a quantitative sandwich ELISA for measuring MANF levels in shrimp plasma — the pivotal assay that demonstrated LPS-induced MANF secretion in vivo.

Key Findings

1. MANF Is Secreted Into Plasma After LPS Challenge

Using the AtaGenix-developed sandwich ELISA, the team showed that MANF levels in shrimp plasma increased significantly after LPS injection — the first evidence that MANF functions as a secreted immune mediator in any invertebrate species.

2. MANF Suppresses ERK/NF-κB via RPTP

Recombinant MANF treatment of hemocytes reduced ERK phosphorylation and NF-κB (Dorsal) expression in a dose-dependent manner. RNA interference knockdown of RPTP abolished this effect, proving that MANF acts through RPTP-mediated dephosphorylation of ERK — a previously unknown signaling axis.

Figure 2. AtaGenix-produced recombinant MANF suppresses ERK phosphorylation and NF-κB (Dorsal) expression in shrimp hemocytes. RPTP knockdown abolishes the anti-inflammatory effect, confirming the MANF–RPTP–ERK signaling axis.

3. Cross-Species Conservation

When shrimp RPTP was overexpressed in human 293T cells, MANF's effect on ERK switched from activation (via endogenous mammalian receptors) to inhibition (via RPTP) — demonstrating that the MANF–RPTP anti-inflammatory axis is functionally conserved across 600 million years of evolution.

Figure 3. AtaGenix's workflow: phage display screening identified matched monoclonal antibody pairs, which were used to construct a sandwich ELISA for quantifying MANF levels in shrimp plasma. This assay provided the critical in vivo evidence that MANF is secreted as an anti-inflammatory factor after immune challenge.

Reference

Luo K, Chen Y, Wang F. Shrimp Plasma MANF Works as an Invertebrate Anti-Inflammatory Factor via a Conserved Receptor Tyrosine Phosphatase. The Journal of Immunology. 2022. DOI: 10.4049/jimmunol.2100595

This case study is based on a published research collaboration. Results may vary depending on target protein, antibody specificity requirements, and assay format. All proprietary client information is subject to NDA.