An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas

Figures & data

Figure 1. Schematic representation of the immuno-wall device. (A) Immuno-wall chips with 40 microchannels (each 1 mm in width, 40 μm in height and 8.5 mm in length) in a cyclic olefin polymer substrate were constructed using photolithography. (B) The channels were filled with 6% azide-unit pendant water-soluble photopolymer (AWP) and 10 mg ml^–1 streptavidin. UV light (313 nm, 20 mW cm^–2) through slits in a photomask was used to immobilize the photoreactive polymer in the center of the channels, before the uncured polymer was washed with PBS. (C, D) A biotinylated anti-R132H-IDH1 antibody (HMab-2), an anti-wild-type IDH1 antibody (RcMab-1), and a fluorescent DyLight650-conjugated goat anti-rat IgG antibody were used to label IDH1-R132H. Note that RcMab-1 is interacted with multiple numbers of DyLight 650-conjugated anti-rat IgGs.

Figure 2. Sensitivity and specificity of the immuno-wall assays. (A) Western blotting confirmed that the anti-IDH1-R132H antibody, HMab-2, was specific to the lysate from NHA-IDH1-R132H and U87-IDH1-R132H cells, whereas the anti-IDH1 antibody, RcMab-1, recognized both wild-type IDH1 and IDH1-R132H in NHA and U87 cells. (B) U87 and NHA cells expressing wild-type IDH1 did not show fluorescence, while both types of cells expressing IDH1-R132H displayed strong fluorescence.

Figure 3. Effect of mutated IDH1/wild-type IDH1 ratio on the detection of mutated IDH1. When mutated IDH1 comprised more than 10% of the cell lysate, strong fluorescence was observed. However, when the proportion of mutated cells was below 5%, fluorescence was difficult to distinguish from the background.

Table 1. Patient characteristics and summarized results.

Case no.	Age	Sex	Pathological diagnosis	Sample weight (mg)	Protein lysate concentration (mg ml^–1)	Amount of total protein (μg)	DNA sequencing	Immuno-wall	Mutant allele frequencies (%)
1	30	F	Anaplastic astrocytoma (grade III glioma)	11	2.71	542.4	Mut	Mut	31.5
2	53	M	Gliomatosis cerebri (grade III glioma)	7	2.89	578.4	WT	WT	0
3	50	F	Diffuse astrocytoma (grade II glioma)	10	3.47	694	Mut	Mut	45.8
4	78	F	Glioblastoma (grade IV glioma)	11	2.69	538	WT	WT	0
5	19	F	Dysembryoplastic neuroepithelial tumor	9	3.21	642.2	WT	WT	0
			(DNT, non-glioma)
6	58	F	Pineal parenchyma tumor	5	1.86	371.6	WT	WT	0
			of intermediate differentiation (PPTID, non-glioma)
7	59	M	Glioblastoma (grade IV glioma)	10	2.56	511.4	WT	WT	0
8	25	F	Anaplastic astrocytoma (grade III glioma)	12	2.89	578.6	Mut	Mut	34.7
9	47	M	Glioblastoma (grade IV glioma)	14	3.56	712.8	WT	WT	0
10	48	M	Diffuse astrocytoma (grade II glioma)	7	2.35	470.2	Mut	Mut	27.1

Figure 4. Effect of the mutant allele frequency in clinical samples on the detection of mutated IDH1. The lysate from a tumor sample with a mutant allele frequency of 40% was mixed with the lysate from a tumor sample with wild-type IDH1 such that the mutant allele frequency decreased stepwise from 40% to 20, 10, 5, 2, and 1%. The immuno-wall assay IDH1-R132H detected the mutant allele frequency in samples containing as low as 10% mutant IDH1.

Figure 5. Tumor boundary detected using the immuno-wall assay. During tumor removal surgery in a patient, two specimens were collected. (A1, A2) A specimen from the center of the tumor tested positive in the immuno-wall assay. (B1, B2) In contrast, a specimen from the margins of the tumor, which appeared normal, tested negative. (A3, A4, B3, B4) The IDH1 status of these samples was confirmed using immunohistochemistry and DNA sequencing.