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EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

Antibody information [CD229 (Lymphocyte Antigen 9, LY9)]

OTI2H4

Clone name OTI2H4
Description Mouse monoclonal
Antigen used Human LY9 protein aa 48-454
Epitope Unknown
Isotype IgG1
Confirmed species reactivity Human
Ab used OriGene (Cat. TA811828) 1 mg/ml

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	1 μg/ml	Working	Odyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)	2 μg/ml	Working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	1.25 μg/ml	Working	BOND-MAX automated immunohistochemistry
Immunoflourescence (IF)	3.3 μg/ml	Working	Immunofluorescence staining
Flow cytometry (FC)	2.3 μg/ml	Not working	Flow cytometry
IHC-P Species	1.25 μg/ml	Not working	BOND-MAX automated immunohistochemistry

Ab ID Card application validation / characterisation

WB Validation

Over-expression/cross reactivity

WB Validation: Over-expression/cross reactivity — Western blotting (WB) of the OTI2H4 mAb using cell extracts of HEK293T hCD229/LY9 (GFP-tagged) as positive control and HEK293T-GCET2 as negative control. Bands were observed only in the HEK293T hCD229/LY9 transfectants.Anti-GAPDH was used as loading control.

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Gene inactivation

WB Validation: Gene inactivation — The specificity of the OTI2H4 mAb for the endogenous CD229/LY9 protein was confirmed by WB using two different cell extracts: U266 cells before and after LY9 gene inactivation (U266 KO LY9) using CRISPR-Cas9 technology. Three bands around 75-110kDa were detected in the U266 WT (corresponding with the three LY9 isoforms) while no bands were found in U266 KO LY9. Anti-GAPDH was used as loading control.

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WB Characterisation

Endogenous expression

WB Characterisation: Endogenous expression — The detection of endogenous expression of CD229/LY9 protein using the OTI2H4 mAb was confirmed by WB in the DAUDI, SUDHL4 and L363 cell lines and human tissues (tonsil and liver). DAUDI, SUDHL4, L-363 and human tonsil showed two bands corresponding to the CD229/LY9 isoforms. No bands were detected in human liver. Anti-GAPDH was used as loading control.

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ICC / ICC-P Validation

Over-expression/cross reactivity

ICC Validation: Over-expression/cross reactivity — Immunocytochemistry (ICC) of the OTI2H4 mAb was performed on frozen cytospin preparations of GFP-tagged human CD229/LY9 and MYC-tagged murine PDL1 proteins expressed in HEK293T cells. Labelling with the OT12H4 mAb was present in the HEK-hLY9-GFP transfectants but not in the negative control HEK-mPDL1-MYC cells. Labelling with anti-MYC and anti-GFP was used to confirm the efficiency of transfection.

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Gene inactivation

ICC Validation: Gene inactivation — The specificity of the OTI2H4 mAb for the endogenous CD229/LY9 protein was confirmed by ICC in cytospin preparations of the U266 cell line before (U266 WT) and after LY9 gene inactivation (U266 KO Ly9) using CRISPR-Cas9 technology. LY9 membrane expression was observed in the U266 WT cells, while no staining was observed in the U266 KO LY9 cells.

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IHC-P Characterisation

Endogenous expression

IHC-P Characterisation: Endogenous expression — Immunohistochemical labelling with the OT12H4 mAb showed CD229/LY9 was strongly expressed in haematopietic tissues. A) In tonsil, strong expression was detected in B- and T-cells in the interfollicular areas and in plasma cells in the subepithelium outside the follicles as well as in the mantle zone. B) Mature T-cells in the thymic medulla were strongly positive with weaker expression in the thymic cortex. C) Strong expression was also detected in mantle and marginal zones as well as some T-cells within the white-pulp area of spleen. D) Some scattered cells were CD229/LY9-positive in bone marrow.

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IF Characterisation

Endogenous expression

IF Characterisation: Endogenous expression — Triple immunofluorescence of formalin fixed human tonsil with OTI2H4 mAb (green), anti-CD8 (white) and anti-CD20 (red).

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FC Validation

Over-expression/cross reactivity

Not working

Gene inactivation

Not working

FC Characterisation

Endogenous expression

Not working

IHC-P Domestic species

Not working

IHC-P Wild species

Not working

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