Optimize immunodetection with recombinant secondary antibodies

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Sensitive immunodetection in cells and tissues requires high-affinity antibodies that exhibit minimal background staining. Invitrogen™ Superclonal™ secondary antibodies represent a breakthrough in recombinant antibody technology, providing sensitive binding to their targets and very low levels of nonspecific staining. Because of their consistent performance from lot to lot, these next-generation secondary antibodies are becoming the reagent of choice in both research and clinical applications [1–4]. Superclonal secondary antibodies are available unconjugated, as well as conjugated to biotin, HRP, and the brightly fluorescent Alexa Fluor dyes.

Advantages of Superclonal recombinant antibodies over traditional polyclonals

Superclonal recombinant secondary antibodies enable the accurate and precise detection of mouse, rabbit, and goat IgG antibodies in cell imaging (Figure 1), flow cytometry, western blot, and ELISA applications, with little to no cross-reactivity to IgGs from other species. Their recombinant origin helps ensure consistency between lots, minimizing the need to optimize each lot before using them in an immunoassay. By comparison, typical polyclonal secondary antibodies are affinity purified from the serum of immunized animals, resulting in a large, undefined pool of antibodies with unknown epitope-binding characteristics. Although broad epitope coverage is a benefit of traditionally produced polyclonal secondary antibodies, poor lot-to-lot consistency due to animal variability and purification processes can lead to significant cross-reactivity and high background signals. With extensive validation using a panel of primary antibodies, we have demonstrated that Superclonal secondary antibodies show high affinity to their target IgG species with minimal cross-species reactivity, providing consistently high signal-to-noise ratios across lots and in a variety of immunodetection protocols.

Multicolor immunocytochemical analysis using fluorescent Superclonal secondary antibodies

Figure 1. Multicolor immunocytochemical analysis using fluorescent Superclonal secondary antibodies. U2OS (human osteocarcinoma) cells were labeled with rabbit anti–acetyl-histone H3 (Lys9) primary antibody (ABfinity™ Rabbit Oligoclonal, clone 17HCLC) followed by the Alexa Fluor™ 647 conjugate of Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody, red), and with mouse anti–α-tubulin primary antibody, clone B-5-1-2, followed by the Alexa Fluor™ 488 conjugate of Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody (green).

Producing high-quality recombinant antibodies

To produce Superclonal secondary antibodies, we employ recombinant technology followed by a multifaceted clonal selection process that involves several phenotypic screens (Figure 2). First we construct a cDNA library of IgG light chain and heavy chain genes from goat and rabbit peripheral blood mononuclear cells (PBMCs), and then reverse engineer these genes for heterologous co-expression [5]. Library construction is followed by thorough screening and selection of the monoclonal antibodies. Throughout the screening process, we use highly cross-adsorbed polyclonals as a benchmark to eliminate clones with similar or higher cross-reactivity to IgGs from closely related species. Finally, monoclonals are selected based on their high affinity to the target IgG species along with specificity equal to or better than that of the polyclonal benchmarks across a variety of immunodetection protocols, including cell imaging, flow cytometry, western blot, and ELISA applications.

Selected monoclonal antibodies with high sensitivity and specificity are then sequenced and pooled to simulate the diversity of a polyclonal antibody. Several iterations of this pool are tested to find the combination that yields the highest signal-to-noise ratio in various immunoassays. The resulting “superclonal” mixture of recombinant goat or rabbit secondary antibodies binds with the epitope-specific precision of monoclonal antibodies, while also achieving the multi-epitope coverage (e.g., toward both heavy and light chains of target IgGs) and signal amplification of polyclonal antibodies. These well-characterized Superclonal secondary antibodies are then conjugated to brightly fluorescent Invitrogen™ Alexa Fluor™ dyes (Figure 3), or to horseradish peroxidase (HRP) or biotin, and subjected to stringent testing to confirm their performance across detection platforms. In direct ELISAs, the Superclonal secondary antibodies show little or no cross-reactivity with IgGs from other species, whereas the highly cross-adsorbed polyclonals show dose-dependent cross-reactivity to IgGs from closely related species (Figure 4). Furthermore, Superclonal secondary antibodies exhibit much lower background signals in immunocytochemical assays (Figure 5). To date we have validated the Superclonal secondary antibody conjugates with more than 300 primary antibodies for immunodetection applications.

Superclonal secondary antibodies

Figure 2. Superclonal secondary antibodies—a defined pool of well-characterized monoclonal antibodies. The development of a Superclonal™ secondary antibody entails the construction of IgG light chain (LC) and IgG heavy chain (HC) cDNA libraries using goat and rabbit peripheral blood mononuclear cells (PBMCs), followed by combinatorial screens to produce multiple monoclonal antibody candidates (mAbs). The mAbs are then analyzed for affinity as well as performance in several immunodetection protocols (ELISA, western blotting, and immunocytochemical (ICC) assays); the corresponding LC and HC cDNAs are also sequenced to confirm diversity among the individual mAbs. The mAbs that exhibit superior performance in the immunoassays and show little or no cross-reactivity are pooled to produce a Superclonal secondary antibody.
 

Multiplex immunocytochemical analysis using fluorescent Superclonal secondary antibodies

Figure 3. Multiplex immunocytochemical analysis using fluorescent Superclonal secondary antibodies. SH-SY5Y (human neuroblastoma) cells were labeled with mouse anti-Aβ40 primary antibody followed by the Alexa Fluor™ 488 conjugate of Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody (green), and with rabbit anti-DISC1 ABfinity™ Rabbit Oligoclonal Antibody, followed by the Alexa Fluor™ 555 conjugate of Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody (red). Nuclei were stained with SlowFade™ Gold Antifade Mountant with DAPI (blue). Images were captured at 60x magnification.

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Figure 4. Comparison of IgG species cross-reactivity. To compare the cross-reactivity of Superclonal™ secondary antibodies to highly-cross adsorbed polyclonal secondary antibodies, colorimetric ELISAs using purified goat, guinea pig, bovine, and mouse IgGs were performed. (A) When comparing rabbit anti–goat IgG secondary antibodies (RAG), the RAG polyclonal antibody showed a dose-dependent cross-reactivity to bovine IgG, whereas the RAG Superclonal antibody did not cross-react with any of the species tested. (B) When comparing rabbit anti–mouse IgG secondary antibodies (RAM), the two polyclonal antibodies tested cross-reacted with rat IgG (polyclonal 1) or both goat and rat IgG (polyclonal 2), whereas the RAM Superclonal antibody displayed minimal cross-reactivity.
 

Figure 5. Reduction in nonspecific staining with Superclonal secondary antibodies. Nucleoli of HeLa (human cervical carcinoma) cells were labeled with mouse anti-nucleostemin primary antibody, which was then detected with the Alexa Fluor™ 488 conjugate (green) of (A) highly cross-adsorbed goat anti–mouse IgG polyclonal antibody or (B) Goat Anti–Mouse IgG (H+L) Superclonal™ Secondary Antibody. Similar intensity of staining in the nucleolus is observed with both secondary antibodies, but the Superclonal antibody exhibits significantly less cytoplasmic staining, indicating enhanced specificity.

Consistent performance across lots

To ensure reproducible performance of each of the Superclonal antibodies, we run quantitative analyses for every unconjugated and conjugated antibody. Figure 6 shows the performance of five different lots of unconjugated Goat Anti–Rabbit IgG (H+L) Superclonal Secondary Antibody on western blots. We routinely compare the performance of newly manufactured lots of a Superclonal antibody to previous lots using several different immunoassay formats (cell imaging, flow cytometry, western blot, ELISA) and quantify the results with standard densitometry or fluorescence-based algorithms. In all cases, Superclonal secondary antibodies show consistent performance across lots, unlike currently available polyclonal secondary antibodies, which require optimization by the researcher for each new lot due to variability that can arise from the animal sources and purification procedures. The lot-to-lot consistency of Superclonal antibodies minimizes the need to optimize each lot before using it in an immunodetection protocol, not only saving time but producing results that are reproducible from day to day and from experiment to experiment.

Comparison of Superclonal secondary antibody performance across multiple lots
Figure 6. Comparison of Superclonal secondary antibody performance across multiple lots. Western blot analysis was performed on whole cell extracts (20 μg lysate) of HeLa (human cervical carcinoma) cells for detection of (A) endogenous α-Akt (~60 kDa) and (B) endogenous α-Traff (~63 kDa), using the corresponding rabbit primary antibodies. Each lane on the blots was then incubated with one of five different lots of Goat Anti–Rabbit IgG (H+L) Superclonal™ Secondary Antibody (Cat. No. A27033), followed by the HRP conjugate of Rabbit Anti–Goat IgG (H+L) Superclonal™ Secondary Antibody (Cat. No. A27014); the last lane was incubated with primary antibody and the benchmark polyclonal secondary antibody (pAb). Endogenous tubulin, detected with mouse anti-tubulin primary antibody and anti– mouse IgG secondary antibody, was used as a control for loading.

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