Benefits and pitfalls of secondary antibodies: why choosing the right secondary is of primary importance

Abstract

Simultaneous labeling of multiple targets in a single sample, or multiplexing, is a powerful approach to directly compare the amount, localization and/or molecular properties of different targets in the same sample. Here we highlight the robust reliability of the simultaneous use of multiple mouse monoclonal antibodies (mAbs) of different immunoglobulin G (IgG) subclasses in a wide variety of multiplexing applications employing anti-mouse IgG subclass-specific secondary antibodies (2°Abs). We also describe the unexpected finding that IgG subclass-specific 2°Abs are superior to general anti-mouse IgG 2 °Abs in every tested application in which mouse mAbs were used. This was due to a detection bias of general anti-mouse IgG-specific 2°Abs against mAbs of the most common mouse IgG subclass, IgG1, and to a lesser extent IgG2b mAbs. Thus, when using any of numerous mouse mAbs available through commercial and non-profit sources, for cleaner and more robust results each mAb should be detected with its respective IgG subclass-specific 2°Ab and not a general anti-mouse IgG-specific 2°Ab.

Figure 1. SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections.

(A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.

Figure 2. Simultaneous triple labeling with different combinations of three mAbs in rat and mouse brain.

Sections from rat (A–D) and mouse (E–H) were simultaneously labeled with three different mAbs, and SCS 2°Abs. (A) Ankyrin-G (green), Caspr/Paranodin (red), and Kv1.2 (blue), in a region of rat hindbrain white matter with myelinated axons containing nodes of Ranvier. (B) BK channel (green), GFAP (red), and Kv1.2 (blue), in a region of rat cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). (C) Ankyrin-G (green), Kv2.1 (red), and Kv1.2 (blue), in rat hippocampal dentate gyrus. Labels mark the middle molecular layer (MML), inner molecular layer (IML), granule cell layer (GCL), and hilus (HI). (D) Caspr/Paranodin (green), Ankyrin-G (red), and Kv1.2 (blue), in rat hippocampal dentate gyrus, labels are as in Panel C. (E) mAbs and 2°Abs as in A, but in mouse hindbrain white matter with myelinated axons containing nodes of Ranvier. (F) Kv1.2 (green), Ankyrin-G (red), and GFAP (blue), in a region of rat cerebellar cortex. Labels as in B. (G) Ankyrin-G (green), KChIP1 (red), and Kv1.2 (blue), in mouse cerebral cortex. (H) Kv2.1 (green), Kv1.2 (red), and PSD-95 (blue), in mouse hippocampal dentate gyrus. Labels as in D. Scale bars: A, E, F = 10 µm; B = 20 µm; C, D, G, H = 50 µm.

Figure 3. HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses.

Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.

Figure 4. HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications.

(A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a>IgG2b>IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods. Changes in red∶green tint reflect bias of HL for (more red) IgG2a>IgG2b>IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

Figure 5. HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations.

(A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

Figure 6. HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates.

(A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C, and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′)₂ fragment of HL (e.g., L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard & Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

Figure 7. Analysis of knockout mouse tissue reveals increased background of HL 2°Abs.

Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.