Establishment of Anti-Human ATRX Monoclonal Antibody AMab-6

Introduction

The molecular subgrouping of gliomas using mutations of isocitrate dehydrogenase (IDH), α-thalassemia/mental-retardation-syndrome-X-linked (ATRX), and TERT promoter and the status of 1p/19q more prognostically stratify patients into distinct groups compared with conventional histological classification.^(1–4) Treatment strategies can be planned with respect to molecular subtype along with the WHO grade; therefore, these molecular subtypes are clinically important. The inclusion of molecular parameters in the WHO definition of brain tumors was forwarded as the “ISN-Haarlem” consensus.⁽⁵⁾ The 2016 WHO Classification of Tumors of the Central Nervous System (2016 CNS WHO) is both a conceptual and practical advance over the 2007 CNS WHO. The 2016 CNS WHO uses molecular parameters in addition to histology to define many tumor entities; presents major restructuring of the diffuse gliomas, medulloblastomas, and other embryonal tumors; and incorporates new entities, which are defined by both histology and molecular features.⁽⁶⁾

ATRX mutation in gliomas results in the loss of ATRX protein expression, which can be diagnosed by immunohistochemistry using anti-ATRX antibodies.⁽⁷⁾ Almost all studies of ATRX have used polyclonal antibodies⁽⁸⁾ because highly sensitive monoclonal antibodies (mAbs) against human ATRX remain to be established. Herein, we report a novel anti-ATRX mAb, AMab-6, which is very useful in enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analyses.

Materials and Methods

Results and Discussion

The two major methods for identifying these subtypes of gliomas are direct DNA sequencing and immunohistochemical analyses using antibodies.⁽⁹⁾ Immunohistochemistry is a robust and widely available method, and immunohistochemical approaches with defined protocols and materials have become essential for assessing molecular genetic changes.⁽¹⁰⁾ Among many molecular parameters, the important molecules for subtype diagnosis of diffuse gliomas are mutations of IDH, TERT promoter, and ATRX and the codeletion of 1p/19q.⁽¹¹⁾ In particular, mutations of IDH⁽⁹⁾ and ATRX^(7,8) can be accurately detected by immunohistochemical analysis. We developed many mAbs against IDH mutants, including HMab-1/HMab-2 against IDH1-R132H and multispecific mAbs MsMab-1/MsMab-2 against IDH1/2 mutations.^{(7,9,12–28)} However, highly sensitive mAbs against human ATRX remain to be established.

In this study, the recombinant PA-ATRX-RAP-MAP was captured using NZ-1-Sepharose and efficiently eluted with MES buffer (pH 6.0), containing 3 M MgCl₂ (Fig. 1). Mice were then immunized with PA-ATRX-RAP-MAP to develop novel anti-ATRX mAbs. We utilized the C-terminal region of ATRX as immunogen because commercially available polyclonal antibodies (Sigma-Aldrich) against ATRX were also produced against that region.⁽⁷⁾ Using ELISA, the culture supernatants of hybridomas were screened for binding to recombinant MBP-ATRX-MAP-PA or GST-ATRX-MAP-PA that was purified from Escherichia coli. As a result, AMab-6 (mouse IgG₁, kappa) was established after limiting dilution.

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FIG. 1.

Purification of the recombinant ATRX protein. Cleared lysate (5 μL) from the transformed Escherichia coli before (input) and after (flow through) NZ-1-Sepharose capture, as well as 10 elution fractions (lanes 1–10) during column chromatography, was subjected to 5%–20% gradient SDS-PAGE under a reducing condition and stained with CBB. ATRX, α-thalassemia/mental-retardation-syndrome-X-linked; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

As shown in Figure 2, AMab-6 reacted with MBP-ATRX-MAP-PA in a dose-dependent manner. In contrast, AMab-6 did not react with MBP-IDH1. We further performed a kinetic analysis of the interaction of AMab-6 with MBP-ATRX-MAP-PA (Fig. 2). The K_D of AMab-6 was determined to be 9.7 × 10⁻¹⁰ M, indicating that the apparent binding affinity of AMab-6 is very high.

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FIG. 2.

Determination of binding affinity using enzyme-linked immunosorbent assay. Purified recombinant protein (MBP-ATRX-MAP-PA or MBP-IDH1) was immobilized at 5 μg/mL. The plates were incubated with serially diluted antibodies (26 pg/mL–10 μg/mL) followed by 1:1000-diluted peroxidase-conjugated anti-mouse IgG. The dissociation constant (K_D) was obtained by fitting the binding isotherms using the built-in one-site binding models in GraphPad Prism 6. IDH, isocitrate dehydrogenase.

Western blot analysis demonstrated that AMab-6 detected MBP-ATRX-MAP-PA, but not MBP-IDH1, indicating that AMab-6 does not detect MBP tag in Western blot analysis (Fig. 3). Immunohistochemical analyses revealed that AMab-6 reacted with the nuclei of oligodendroglioma cells without ATRX mutations (Fig. 4A). ATRX mutations in gliomas result in the loss of ATRX protein expression, which can be diagnosed using immunohistochemical analysis.⁽⁷⁾ Likewise, AMab-6 did not recognize the nuclei of diffuse astrocytoma cells, which possess ATRX mutation, but reacted with those of vascular endothelial cells without ATRX mutation (Fig. 4B). These results indicate that AMab-6 is very sensitive against ATRX, which is expressed in gliomas without ATRX mutation.

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FIG. 3.

Western blot analysis. Recombinant proteins (0.1 μg) of MBP-ATRX-MAP-PA and MBP-IDH1 were electrophoresed and transferred onto a PVDF membrane. After blocking, the membrane was incubated with AMab-6, RMab-3 (anti-IDH1), or TMab-2 (anti-MBP).

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FIG. 4.

Immunohistochemical analysis. (A) Oligodendroglioma, IDH-mutant (WHO grade II). Note the ATRX immunostaining in the nucleus of tumor cells without ATRX mutation. (B) Diffuse astrocytoma, IDH mutant (WHO grade II). Note the negative staining of ATRX in the nucleus of tumor cells with ATRX mutation, whereas positive staining was observed in the vascular endothelial cells without ATRX mutation.

In conclusion, a novel anti-ATRX mAb, AMab-6, sensitively reacted with human ATRX in ELISA, Western blot, and immunohistochemical analyses. Although many studies have used commercially available polyclonal anti-ATRX antibodies,⁽⁷⁾ we confirmed that the reactivity of AMab-6 is compatible with that of polyclonal antibodies (data not shown). Furthermore, we performed immunohistochemical analyses under many other conditions for antigen retrievals (citrate buffer, pH 6.0 or 9.0) or incubation time of AMab-6 (30 minutes, 1 hour, or overnight) and obtained positive staining results as shown in Figure 4. Altogether, AMab-6 could serve as a standard marker to determine the ATRX mutation status of gliomas during immunohistochemical analyses.