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Updated: May 27, 2026
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Published on: December 2, 2010
Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and
Sherif Ibrahim1, Falko Steinbach
1Institute for Zoo and Wildlife Research (IZW), Alfred Kowalke Str. 17, 10315 Berlin, Germany.
Insights
Immunoprecipitation confirmed equine leukocyte targets for most monoclonal antibodies (mAbs). However, three mAbs against CD49d, CD163, and CD206 failed to precipitate their targets, highlighting method-specific limitations in antibody validation.
Area of Science:
- Veterinary Immunology
- Flow Cytometry
- Immunoprecipitation
Background:
- Previous studies utilized flow cytometry and immunohistochemistry to characterize equine leukocyte-specific monoclonal antibodies (mAbs).
- These methods screened for antibody reactivity and cell distribution in lymphoid tissues.
Purpose of the Study:
- To complement existing data by employing immunoprecipitation (IP) to identify the specific molecular targets of cross-reactive equine mAbs.
- To determine the molecular weight of target molecules using western blotting.
Main Methods:
- Surface molecules on primary equine peripheral blood mononuclear cells (PBMCs) and the T8888 cell line were biotinylated.
- Immunoprecipitation was performed using 24 mAbs, followed by western blotting with streptavidin-AP to detect precipitated molecules.
Main Results:
- Twenty-one out of 24 mAbs successfully precipitated molecules with molecular weights corresponding to their human orthologues.
- Validated targets included CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI, and MHCII.
- Three mAbs (anti-CD49d, anti-CD163, anti-CD206) failed IP despite prior flow cytometry identification.
Conclusions:
- Immunoprecipitation is a valuable method for validating equine leukocyte targets of mAbs.
- Discrepancies between flow cytometry and IP suggest potential issues with antibody binding or target accessibility for certain mAbs.
- Further investigation is needed for mAbs that failed IP, especially those targeting CD49d, CD163, and CD206.
Abstract:
Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules from primary equine PBMC or the equine cell line T8888 were biotinylated prior to precipitation to determine the molecular weight of the corresponding molecules in a western blot using streptavidin-AP. 21 out of 24 mAbs precipitated the molecules with a MW corresponding to its human orthologue. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI and MHCII. Three mAbs directed against CD49d, CD163, and CD206 which were unambiguously identified earlier by flow cytometry failed to immunoprecipitate the corresponding CD molecule. MAbs detecting CD molecules which are expressed internally like CD68 and mAbs of IgM class could not be included into this approach.
