Development of a Cell Marker ELISA for the Detection of Goose T Cell Surface CD8α Molecules

Abstract

CD8 molecule is a key marker on T cell surface and is connected with the antigen recognition and activation of T lymphocytes. In order to provide a detection method for quantifying goose CD8α expression, this study raised the protein and antibody for goose CD8α and developed a feasible cell marker enzyme-linked immunoabsorbent assay (ELISA) method. Recombinant protein of the extracellular region gene of goCD8α was expressed in prokaryotic expression system, and specific polyclonal antibodies for goCD8α were raised and purified, which was further confirmed by Western-blot, immunofluorescence assay (IFA), and immunohistochemistry (IHC). A cell marker ELISA was established and optimized to detect the change of goCD8α expression between goose parvovirus (GPV)-infected and mock-infected goose peripheral blood mononuclear cells (PBMCs), which is consistent with our previously results of real-time quantitative PCR (qPCR). Cell marker ELISA can provide a new method to detect goCD8α in protein level and in a sensitive, specific, and simple way. This may provide a convenient and novel method for the detection of goCD8α expression.

Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

“…Previously, CD8α antibody has been reported in duck (Kothlow et al 2005), chicken (Luhtala 1995), and turkey (Powell et al 2009). In our previous studies (Cheng et al 2016), we used goose CD8α to develop a cellular ELISA (2.5×10 4 cells per 100 μL) for the detection of goose CD8α, but it cannot rapidly detect goose CD8α in clinical diagnosis. Therefore, we established a method for the specific and rapid detection of goCD8α.…”

“…Initially, the extracellular region without the goCD8α signal peptide gene was amplified with the specific primers and then cloned into a pET-32a(+) vector to obtain goCD8αexc-pET-32a. To establish an efficient DAS-ELISA method, the recombinant protein (goCD8α-exc) (Cheng et al 2016) was purified and used as the detective antigen during the DAS-ELISA. The titer of the coated antibody (mouse an-ti-his-goCD8α-exc PAb) was 1:12 800 (Cheng et al 2016) and was diluted in a bicarbonate buffer (0.05 mol L -1 , pH=9.6) before being used to coat 96-well microplates (100 μL per well).…”

“…To establish an efficient DAS-ELISA method, the recombinant protein (goCD8α-exc) (Cheng et al 2016) was purified and used as the detective antigen during the DAS-ELISA. The titer of the coated antibody (mouse an-ti-his-goCD8α-exc PAb) was 1:12 800 (Cheng et al 2016) and was diluted in a bicarbonate buffer (0.05 mol L -1 , pH=9.6) before being used to coat 96-well microplates (100 μL per well). In the same way, non-immunogenic serum was used as the negative control.…”

“…Subsequently, the recombinant protein (goCD8α-exc) was added and incubated. Then, the antibody titer of the capture antibody (rabbit anti-his-goCD8α-exc PAb, 1:51 200) (Cheng et al 2016) was added to each well after three washes and was incubated. Thereafter, the wells were washed three times with PBST and incubated with 100 μL of horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (Sangon Biotech, Shanghai).…”

New Tool for Rapid and Accurate Detection of Interleukin-2 and Soluble Interleukin-2 Receptor α in Cancer Diagnosis Using a Bioresponsive Microgel and Multivalent Protein Binding

“…These results imply that the measurement of IL-2 and IL-2/sIL-2Rα in human blood is highly necessary in clinical practice. Currently, enzyme-linked immunosorbent assay (ELISA) has been considered the gold standard for the detection of biomarkers , in the blood including IL-2 and sIL-2Rα. , However, ELISA has the limitations of requiring complicated procedures and being time-consuming, limiting its use in rapid diagnosis of diseases on site . Alternatively, real-time polymerase chain reaction (RT-PCR) has been shown to be highly sensitive for analyzing alterations of a target protein at the gene level, but, like ELISA, RT-PCR also requires many hours to a day for the final result.…”