Systematic evaluation of monoclonal antibodies and immunoassays for the detection of Interferon-γ and Interleukin-2 in old and new world non-human primates

Abstract

Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species. A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-γ and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-γ and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset). Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC). Pan-reactive mAb pairs for IFN-γ well as IL-2 were identified and used in ELISA to measure IFN-γ and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-γ and IL-2 responses using cynomolgus PBMC. Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC. The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.

Keywords: Antibody; ELISA; ELISpot; Interferon-γ; Interleukin-2; Non-human primates.

Fig. 1

ELISA analysis of individual monoclonal antibodies (mAbs) to human (HUM) IFN-γ for cross-reactivity with non-human primate (NHP) IFN-γ. Anti-HUM IFN-γ mAbs were coated in ELISA plates and allowed to bind serial dilutions of HUM and NHP IFN-γ. The ability of the mAbs to capture the different IFN-γ variants was detected using a mAb to the peptide tag BAM in the N terminus of IFN-γ. The symbols correspond to IFN-γ from HUM, rhesus (RHE), cynomolgus (CYN) and pigtail macaque (PTM; all three macaques have identical IFN-γ sequences) as well as African green monkey (AGM), sooty mangabey (SM), olive baboon (BAB), Ma’s night monkey (AOT), common marmoset (MAR) and squirrel monkey (SAI). All samples were tested in duplicates and shown is the absorbance value. The experiment was repeated with reproducible results.

Fig. 2

ELISA analysis of individual monoclonal antibodies (mAbs) to human (HUM) IL-2 for cross-reactivity with non-human primate (NHP) IL-2. Anti-HUM IL-2 mAbs were coated in ELISA plates and allowed to bind serial dilutions of HUM and NHP IL-2. The ability of the mAbs to capture the different IL-2 variants was detected using a mAb to the peptide tag BAM in the C terminus of IL-2. The symbols correspond to IL-2 from HUM, rhesus (RHE) and pigtail macaque (PTM; RHE and PTM have identical IL-2 sequences) as well as cynomolgus macaque (CYN), sooty mangabey (SM), olive baboon (BAB), Ma’s night monkey (AOT), common marmoset (MAR) and squirrel monkey (SAI). All samples were tested in duplicates and shown is the absorbance value. The experiment was repeated with reproducible results.

Fig. 3

Flow cytometry analysis of the functionality of monoclonal antibodies (mAb) to human (HUM) IFN-γ and IL-2 with HEK cells secreting non-human primate (NHP) IFN-γ and IL-2. A) HEK cells expressing HUM and NHP IFN-γ were stained with anti-HUM mAb MT126L followed by goat anti-rat IgG-PE and the fluorescence intensity is shown on the Y-axis. The graphs show staining with HEK cells transfected with plasmids encoding IFN-γ from HUM, rhesus (RHE), cynomolgus (CYN) and pigtail macaque (PTM; all three macaques have identical IFN-γ sequences) as well as African green monkey (AGM), sooty mangabey (SM), olive baboon (BAB), Ma’s night monkey (AOT), squirrel monkey (SAI), and common marmoset (MAR). The degree of transfection efficiency is indicated by the green fluorescence protein (GFP) reporter shown on the X-axis. Staining with isotype controls was only positive in the GFP channel (data not shown). B) HEK cells expressing HUM and NHP IL-2 were stained with anti-HUM mAb MT8G10 followed by goat anti-mouse IgG-PE and the fluorescence intensity is shown on the Y-axis. The degree of transfection efficiency is indicated by the GFP reporter and is shown on the X-axis. The graphs show staining with HEK cells transfected with plasmids encoding IL-2 from HUM, RHE and PTM (RHE and PTM have identical IL-2 sequences) as well as CYN, SM, BAB, AOT, SAI and MAR. Staining with isotype controls was only positive in the GFP channel (data not shown). C) HUM and CYN peripheral blood mononuclear cells, either unstimulated or stimulated with PMA/Ionomycin, were stained with MT126L (IFN-γ) and MT8G10-biotin (IL-2) followed by goat anti-rat IgG-Alexa Fluor® 488 and streptavidin-PE. Fluorescence intensity for staining of IFN-γ is shown on the X-axis and staining of IL-2 is shown on the Y-axis. The experiments were repeated with reproducible results.

Fig. 4

Western blot analysis of mAb reactivity with human (HUM) and non-human primate (NHP) IFN-γ and IL-2. Supernatants from HEK cells expressing HUM and NHP IFN-γ (approximately 15 ng/lane) were separated on a SDS-PAGE gel and transferred to a Western blot membrane. The cytokines were detected using mAbs to the respective cytokines. A) Detection of IFN-γ using mAb 111W. The image shows detection of IFN-γ derived from HUM, rhesus (RHE), cynomolgus (CYN) and pigtail macaque (PTM; all three macaques have identical IFN-γ sequences) as well as African green monkey (AGM), sooty mangabey (SM), olive baboon (BAB), Ma’s night monkey (AOT), squirrel monkey (SAI), and common marmoset (MAR). The molecular weight of fully glycosylated dimers (50 kD) as well as monomers (25 kD) are indicated by arrows. Below these bands are bands corresponding to dimers and monomers, respectively, with incomplete glycosylation. B) Detection of IL-2 using mAb 13D9. The image shows detection of IL-2 derived from HUM, RHE and PTM (RHE and PTM have identical IL-2 sequences) as well as CYN, SM, BAB, AOT, SAI and MAR. The molecular weight of IL-2 (16 kD) is indicated by an arrow. The experiments were repeated with reproducible results.

Fig. 5

Reactivity with non-human primate (NHP) IFN-γ and IL-2 displayed by capture ELISA systems based on monoclonal antibodies (mAb) to human (HUM) IFN-γ and IL-2. A) For IFN-γ, three different capture mAbs, 1-D1K, GZ-4 and MT126L, were evaluated in combination with the biotinylated detection mAb 7-B6-1 for reactivity with serial dilutions of HUM and NHP IFN-γ. IFN-γ variants tested were from HUM, rhesus (RHE), cynomolgus (CYN) and pigtail macaque (PTM; all three macaques have identical IFN-γ sequences) as well as African green monkey (AGM), sooty mangabey (SM), olive baboon (BAB), Ma’s night monkey (AOT), squirrel monkey (SAI), and common marmoset (MAR). B) For IL-2, two different capture mAbs, MT2A91 and MT2C95, were used alone or as a mixture and in combination with the biotinylated detection mAb MT8G10 for reactivity with serial dilutions of HUM and NHP IL-2. IL-2 variants tested were from HUM, RHE and PTM (RHE and PTM have identical IL-2 sequences) as well as CYN, SM, BAB, AOT, SAI and MAR. All samples were tested in duplicates and shown is the absorbance value. The experiment was repeated with reproducible results.

Fig. 6

Reactivity of capture ELISAs with IFN-γ and IL-2 from stimulated peripheral blood mononuclear cells (PBMC) from human (HUM) and non-human primates (NHP). Supernatants from PBMC stimulated with SEA/B were analyzed by capture ELISAs for IFN-γ (A) and IL-2 (B). A) IFN-γ was measured using mAb MT126L/7-B6-1-biotin in PBMC supernatants from human (HUM), rhesus macaques (RHE; identical IFN-γ sequence with pigtail (PTM) and cynomolgus (CYN) macaque), African green monkey (AGM), baboon (BAB*; yellow and olive baboon hybrid), common marmoset (MAR) and squirrel monkey (SAI). B) IL-2 was measured using mAb MT2A91 + MT2C95/MT8G10-biotin in PBMC supernatants from HUM, RHE (identical IL-2 sequence with PTM), CYN, AGM, BAB*, MAR and SAI. Supernatants were diluted 5, 50 and 500 times before analysis. All samples were tested in duplicates and shown is the absorbance value. The results of the IFN-γ ELISA was confirmed with other PBMC supernatants from the same NHP whereas the IL-2 ELISA was performed once.

Fig. 7

ELISpot and FluoroSpot analysis of IFN-γ and IL-2 secreted by peripheral blood mononuclear cells (PBMC) from cynomolgus macaque. PBMC were incubated overnight in medium only or stimulated with rhesus cytomegalovirus pp65 or anti-CD3 mAb. A) ELISpot analysis of IFN-γ responses using the mAb combination MT126L and 7-B6-1-biotin and IL-2 using the mAb combination MTA91/MT2C95 and MT8G10-biotin. B) FluoroSpot analysis of IFN-γ and IL-2 using the same antibodies as in ELISpot but in the same well. Images shown represent IFN-γ responses analyzed with a reader filter for fluorophores absorbing and emitting light at 490/550 and IL-2 responses analyzed with a reader filter for 550/570 nm. Shown is also a computerized overlay analysis of the IFN-γ and IL-2 images (IFN-γ + IL-2) where double-producing cells are visualized as yellow spots. The experiments were repeated with reproducible results.