We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19–9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy (161 words).

In the era of proteomics, high-throughput screening of posttranslational modification states of proteins, especially protein phosphorylation, is considered of utmost importance. However, current protein phosphorylation detection methods depend on either the combination of proteolysis and mass spectrometry, or time-consuming immunoassay that requires inevitable washing processes. As a way to rapidly assay protein phosphorylation events, here we propose the use of Open Sandwich immunoassay that detects antigen-dependent stabilization of antibody variable region (Fv). As a model system, the heavy and light chain variable regions (V(H)/V(L)) of anti-phosphotyrosine antibody PY20 were used to evaluate its performance. When V(H)/V(L) interaction was first estimated by phage ELISA, wild-type Fv showed a modest phosphotyrosine (PY)-dependent increase in signal. However, after screening of mutants at an interface residue, one with weak V(H)/V(L) interaction (HQ39R) showed markedly improved (>200%) antigen-dependent signals. Using this mutant, two fusion proteins in which each variable region fragment was tethered to a GFP color variant were made (V(H)-eYFP/V(L)-eCFP) to monitor PY-induced fluorescence resonance energy transfer (FRET) between them. The results showed significant PY- or tyrosine phosphorylated peptide-induced enhancement in FRET in homogeneous solutions, indicating applicability of the method for rapid screening of tyrosine phosphorylation in vitro or in situ and possibly in vivo.

Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and molecular/cellular biotechnology. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addition, frequently used bacterial alkaline phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here we tried to express the fusion proteins of antibody variable region(s) and secreted human placental alkaline phosphatase (SEAP) using mammalian cell culture. As a result, functional V(H)-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid production of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent V(H)/V(L) interaction for antigen quantitation.

Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75°C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.

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