This is a review of secondary antibodies used in formal publications. Labome also reviews and summarizes the application of primary antibodies cited in publications, with over 60,000 publications manually curated. Antibody users can search the reviews for primary antibodies against specific genes by using the search box above. A few sample reviews are listed here: beta-actin antibody, alpha-tubulin antibody, integrin antibody, phosphotyrosine antibody, and hemagglutinin antibody.

In order to help Labome visitors identify the most suited secondary antibodies, Labome surveys literature for reagents and instruments. Table 1 and 2 indicate the publication years and major journals of the publications citing secondary antibodies.

Primary antibodies tend to be raised in goats, mice, rabbits, rats, and sheep, with mice and rabbits the most common ones. The reactive species of secondary antibodies correlate well with the host species of primary antibodies, with rabbits and mice as the dominant types.

Production of secondary antibodies requires species different from those of primary antibodies. Goats, donkeys, rabbits, sheep, and mice (for monoclonal secondary antibodies) are the hosts of choice for secondary antibodies. Table 3 lists the host species, indicating goat was the predominant species for secondary antibodies. One interesting observation during our updating of this article is that in the earlier version of this article with data from articles earlier than 2006, secondary antibodies from rabbits are quite common. However, recent articles usually cite secondary antibodies from either goat or donkey. Table 4 lists the reactive species, indicating that, no surprise here, mouse and rabbit are the two major target species.

Anti-IgG secondary antibodies are most widely used (Table 5). This is not surprising since most monoclonal and polyclonal primary antibodies are IgG class immunoglobulins. In addition, anti-IgG secondary antibodies can recognize various IgG subtypes. If the class and/or subclass of a primary antibody is unknown, an anti-IgG secondary antibody may be a good default choice.

Secondary antibodies specific to IgG subclasses (e.g., anti-IgG1, anti-IgG2, anti-IgG2a, anti-IgG2b) appear to be mainly used in double labeling experiments, including immunocytochemistry, immunohistochemistry, and flow cytometry.

Secondary antibodies tend to be against the whole IgG molecule. Those against F(ab')2 fragments or Fab fragments are used, for instance, when binding of secondary antibodies to Fc receptors on cell surfaces should be avoided, or there is endogenous immunoglobulin.

An article "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance" by Dr. Trimmer's lab at the University of California Davis tested the anti-mouse IgG subclass-specific secondary antibodies vs. general anti-mouse IgG secondary antibodies in immunoblots, immunohisto- and immunocyto-chemistry, and microplate binding assays, and found that common anti-mouse heavy and light chain secondary antibodies "have detection bias away from the IgG1 mouse monoclonal subclass, which is 65-70% of available mouse monoclonals. This leads to lower detection levels of a majority of monoclonal mouse antibodies used and decreased signal to noise ratios." The authors suggest that subclass-specific anti-mouse secondary antibodies should instead be used to increase the detection of primary antibodies significantly, and each lab should at least maintain the inventory of secondary antibodies against mouse IgG1, IgG2a and IgG2b subclasses [51]. Dr. Trimmer leads the Neuromab group at UC Davis, and Labome visitors can search for Neuromab antibodies at Labome, using the search box above.

Plasmids for recombinant anti-mouse and anti-rabbit IgG secondary nanobodies are available from Addgene (IDs 104157–104164) [52]. These nanobodies are comparable or superior to commonly used polyclonal secondary antibodies in most antibody applications, with several added benefits (besides the fact that they are recombinant and thus can be produced efficiently with large quantity and consistent quality, at the same time-saving animals and reducing costs) [52]. They can be directly expressed with a tag or a peroxidase. They can be pre-incubated with primary antibodies and thus eliminate the second incubation step common in most antibody applications. Their small size enables its utility in superresolution microscopy and other demanding applications [52].

Secondary antibodies are used in all types of immunoassays, most often in Western blot, immunohistochemistry, and immunocytochemistry, and occasionally in immunoprecipitation, ELISA, and fluorescent in situ hybridization (Table 6). Basic research, clinical analysis, and disease diagnosis use secondary antibodies in ELISA and flow cytometry assays.

Secondary antibodies serve two purposes, one is to amplify any signal, and another one is to enable easy detection. Without secondary antibodies, the primary antibodies would be conjugated. Most secondary antibodies in the survey were conjugated. The common conjugates of secondary antibodies were horseradish peroxidase, Alexa Fluor 488, and Cy3. Table 7 lists all the conjugates in the survey.

The choice of conjugates of secondary antibodies depends on the specific application. For Western blot, the conjugate was likely to be horseradish peroxidase (HRP), alkaline phosphatase (AP), and fluorescent dyes like IRDye (e.g., IRDye 680, IRDye 800) and Alexa Fluor dye (e.g., Alexa 680 ) series (Table 8). For example, Chopra S et al incubated western blots with 1:5,000 HRP-conjugated anti-rabbit secondary antibody ( G-21234) from Thermo Fischer before detecting with SuperSignal West Pico and Femto chemiluminescent substrates from Thermo Fisher and a ProteinSimple FlourChemE instrument [31]. A comparison with our survey result including data before 2006 indicates that Alexa Fluor 680 and IR dyes are gaining popularity among Western blot applications. HRP conjugated secondary antibodies from GE-Healthcare (Amersham Biosciences), Jackson ImmunoResearch Laboratories, Santa Cruz Biotechnology, Thermo Fisher (Invitrogen, Zymed Laboratories, Pierce, Molecular Probes) were often used in Western blot, and Alexa Fluor 488 conjugated secondary antibodies were from Thermo Fisher and Jackson ImmunoResearch Laboratories.

For immunofluorescence (immunocytochemistry and immunohistochemistry), Alexa Fluor dye series of conjugates (e.g., Alexa Fluor 488, 546, 568, 594, 596, 647, 633), Cyanine dye series of conjugates (e.g., Cy2, Cy3, and Cy5), FITC, and rhodamine red are likely used (Table 9), with Thermo Fisher, Jackson Immunoresearch Laboratories as the leading suppliers. For example, Duncan A et al stained mouse brain sections with a mouse monoclonal anti-TCF7L2 antibody in 10% normal donkey serum (NDS), 0.5% Triton X-100 PBS, and then incubated with Alexa 488-conjugated donkey anti-mouse secondary antibody ( A-21202) from Thermo Fisher at 1:2000 dilution in PBS-T containing 2% NDS [4]. Chopra S et al used 3.75 μg/ml cyanine 2-conjugated donkey anti-rabbit immunoglobulin G (catalog 711-225-152) and 3.75 μg/ml cyanine 3-conjugated donkey anti-rat IgG (catalog 712-165-150), both from Jackson Immunoresearch, to examine the Cox-2 and CD45 expression in mouse paw cryosections [31]. AP or HRP conjugated with polymers along with secondary antibodies, for example, rabbit Mach-2 HRP-Polymer from BioCare or rabbit Polink-1 HRP or mouse Polink-2 AP from GBI Labs [58], are also commonly used for IHC experiments.

Secondary antibodies conjugated with FITC, PE, or Alexa Fluor 647 were used in flow cytometry; those conjugated with HRP, AP, and biotin were used in ELISA assays; and unconjugated secondary antibodies were used for protein isolation in immunoprecipitation.

Many high-quality reagent providers supply secondary antibodies or complete kits to both the research and diagnostics communities. The relatively small number of publications in this survey precludes an extensive sampling of suppliers. Thermo Fisher / Invitrogen (including Molecular Probes, Biosource International, and Zymed Laboratories), Jackson ImmunoResearch Laboratories, BD, Santa Cruz Biotechnology, GE Healthcare Bio-Science (including Amersham Biosciences), MilliporeSigma, were the most cited suppliers (Table 10). For example, Meertens L et al used Alexa Fluor 488-conjugated goat anti-rabbit IgG ( A11034) from Invitrogen, Alexa Fluor-647-conjugated goat anti-chicken IgG ( ab150175 from Abcam, Alexa Fluor 488-conjugated goat anti-mouse IgG ( 115-545-003 from Jackson ImmunoResearch, Alexa Fluor 647-conjugated goat anti-mouse IgG ( 115-606-062 from Jackson ImmunoResearch, peroxydase-conjugated donkey anti-rabbit IgG ( 711-035-152 from Jackson ImmunoResearch and anti-mouse/HRP (P0260) from DAKO Cytomotion [6]. Samir P et al used HRP-conjugated secondary anti-rabbit ( 111-035-047), anti-mouse ( 315-035-047), or anti-goat ( 705-035-003) antibodies from Jackson Immuno Research Laboratories in Western blotting and Alexa Fluor 568-conjugated anti-rabbit IgG ( A11036), Alexa Fluor 568-conjugated anti-rat IgG ( A11077) and Alexa Fluor 488-conjugated anti-rabbit IgG ( A11034) from Life Technologies in immunocytochemistry [59]. DiTroia SP et al stained mouse ovarian sections with an ImmPRESS reagent kit from Vector Laboratories after the incubation of primary antibodies [60]. van de Kooij B et al conducted western blot analysis with LI-COR goat-anti-mouse, anti-rabbit, and anti-rat secondary antibodies labeled with either IRDye 680 or IRDye 800 (all at 1:10,000) [56]. Fanning S et al used secondary antibodies from Rockland, Sigma, American Qualex and GE Healthcare [61].

If one looks at scientific blogs (for example ResearchGate, IHC World, Protocol Online), various threads are discussing the re-use of antibodies, both primary and secondary.

The main reason that researchers are interested in re-using antibodies is to save money and stretch tight research budgets as far as possible.

Most of the discussions are in the context of Western blotting, though there are a few discussions about antibody re-use in immunohistochemical applications. While there is much valuable information to be gleaned from such blogs, it should always be remembered that the talks represent the experiences of individual researchers with specific antibodies. However, some general themes and recommendations emerge, as discussed below.

There is one peer-reviewed article that systematically looked at the potential for reusing antibodies (both primary and secondary) in Western blotting applications [62]. The authors investigated the effect of storing the antibodies in 5% non-fat milk at either 4°C (no freeze-thaw cycles) or -20°C (involving a freeze-thaw cycle between each re-use). The buffering conditions (e.g., PBS or TBS) were not stated. The secondary antibodies (anti-mouse-HRP conjugate and anti-rabbit-HRP conjugate) were both obtained from MilliporeSigma. Regardless of storage conditions, these secondary antibodies could be re-used at least four times to detect 0.2μg of a target protein by ECL/film with a gradual loss of signal. It is not clear whether the loss of signal strength was due to gradual antibody depletion and/or loss of antibody reactivity.

Interestingly, the four primary antibodies tested showed much higher variability. Some could be re-used at least four times while others could only be used twice. The critical point is that some antibodies are more robust than others when it comes to repeated re-use. This is consistent with the discussion threads on the various scientific blogs. Some investigators have good experiences of re-using secondary antibodies while others have had bad experiences; not all antibodies behave the same. It is essential to establish what works for your antibody and application.

The choice of storage buffer is an important consideration. In the paper discussed above the authors looked only at the use of non-fat milk as the blocking agent. Although cheap, its use can be problematic due to microbial growth unless an antimicrobial agent is added (though sodium azide cannot be used with HRP-conjugated antibodies as it inhibits peroxidase activity). Boonrod et al [62] reported that after being used three times antibody solutions containing 5% non-fat milk and stored at 4°C “turned yellowish with an unpleasant odor” even in the presence of 0.02% sodium azide. Other researchers have made similar observations and recommend diluting the antibody with Tris-buffered saline containing Tween and/or BSA, especially if the recycled antibody solution is to be stored at 4°C. If the secondary antibody is conjugated to a fluorescent dye, it is also essential to store the antibody in the dark.

Brelje et al [63] state that they routinely re-use secondary antibodies in laser scanning confocal microscopy. They report that with fluorescent dye-conjugated antibodies the non-specific background fluorescence decreases with repeated use (the authors do not state how many times they typically re-use their secondary antibodies). These authors found that antibodies conjugated to fluorescent dyes are particularly susceptible to aggregation during freeze-thaw. If this is generally applicable then researchers re-using fluorescently labeled antibodies (for whatever application) should, perhaps, consider storage at 4°C.

It is clear that in many cases it is possible to re-use secondary antibodies several times in both Western blotting and immunohistochemical applications. However, not all antibodies cope equally well with being re-used, and the researcher needs to verify that their particular secondary antibody can be re-used multiple times. It is also important to decide whether it is cost-effective to re-use secondary antibodies. This will be determined by several factors such as the cost of the antibody (relatively cheap HRP-conjugated or more expensive fluorescent dye-conjugated) and the antibody consumption of each particular laboratory. Furthermore, a failed experiment with a precious sample due to the failure of a re-used secondary antibody may outweigh any financial benefits.