A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXβ, and ATXγ) and Novel Autotaxins (ATXδ and ATXε)

Abstract

Background: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXβ, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated.

Methods: Anti-ATXβ and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXβ and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of "classical ATX" (ATXα, ATXβ, and ATXγ) and "novel ATX" (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples.

Results: The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups.

Conclusions: We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Schematic representation of “classical ATX” (ATXα, β, γ) and “novel ATX” (ATXδ, ε).

The amino acid numbers corresponding to human ATXβ and ATXδ are shown, respectively. “Novel ATX” contains a 4-amino acid deletion in the L2 linker.

Fig 2. Specificity and cross reactivity of ATX isoform specific mAbs.

(A) Purified hisATXβ and hisATXδ recombinant proteins (200 ng/lane) expressed using the baculovirus system were subjected to a western blot analysis with anti-classical ATX mAb (R4+4 mAb, left) and anti-novel ATX mAb (R4-127 mAb, right). (B) Human, mouse, rat, rabbit, dog and bovine serum (1 μL/lane) were subjected to a western blot analysis with anti-classical ATX mAb (R4+4 mAb, left) and anti-novel ATX mAb (R4-127 mAb, right).

Fig 3. Development of the automated immunoassay for quantitative determination of ATX isoforms.

(A, B) The calibration curves of the classical (A) and novel (B) ATX assays. Rate means the production per second of 4-Methylumbelliferone from 4-methylumbelliferyl phosphate by alkaline phosphatase. (C, D) Dilution linearity of the serum ATX isoform assays. Three different pooled serum samples (samples 1, 2, and 3) were diluted and measured using the classical (C) and novel (D) ATX assays.

Fig 4. Concentration of ATX isoform in sera from healthy subjects.

The concentration of total ATX (A), classical ATX (B), and novel ATX (C) antigen and the content ratio of classical ATX (D) and novel ATX (E) relative to total ATX was examined using the sera of healthy subjects (24 men and 33 women). *Statistically significant, as determined using the Mann-Whitney U test (P < 0.01).