Secondary Antibody Review
Mary Johnson (mary at labome dot com)
Synatom Research, Princeton, New Jersey, United States
last modified : 2015-11-11; original version : 2012-10-24
Cite as
MATER METHODS 2012;2:132

A systematic review of secondary antibodies cited in literature, based on two surveys conducted by Labome.

  • Goats and donkeys are the predominant host species for secondary antibodies.
  • Rabbit and mouse are the two major target species.
  • Anti-IgG secondary antibodies are commonly cited; a recent study suggests researchers should start to use subsclass-specific anti-mouse secondary antibodies such as anti-IgG1, or anti-IgG2a etc., to increase the detection signals.
  • Western blot, immunocyto- and immunohisto-chemistry are the most common applications for secondary antibodies.
  • Horseradish peroxidase-conjugated secondary antibodies are commonly used in Western blots; while Alexa Fluor 488-conjugated secondary antibodies are in immunochemistry. PE conjugated secondary antibodies are seldomly cited in more recent publications.
  • Invitrogen/Thermo (including its associated brands) and Jackson Immuno Research are the dominant providers.

This is a review about secondary antibodies used in formal publications. Labome also reviews and summarizes the application of primary antibodies cited in publications, with over 10,000 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.

2008 and earlier8
Table 1. The number (num) of publications per year in the secondary antibody survey.

In order to help Labome visitors identify the most suited secondary antibodies, Labome has conducted two surveys over the years. In the ongoing survey (as of November 10, 2015), information about 1049 instances of secondary antibodies from 422 publications has been collected and organized. Table 1 and 2 indicate the publication years and major journals of these 351 publications.

Journal nameNum
PLoS Biol103
Mol Cell Biol10
J Virol8
Proc Natl Acad Sci U S A7
J Exp Med6
Mol Biol Cell5
J Clin Invest4
J Immunol3
PLoS Genet3
Hum Mol Genet3
Table 2. The number (num) of publications for some of journals in the survery. Only journals with more than 2 articles are listed. Other journals have 1 or 2 articles each.

In an earlier 2008 survey, information about secondary antibodies from 99 publications in the journals Science and Journal of Biological Chemistry were collected and cataloged. The publications were randomly selected. Their authors came from more than 80 universities or research institutes in 16 countries. Curated data included host species, reactive species, Ig class, Ig subclass, conjugation, experimental methods, antibody dilution, and suppliers. Quite often, the relevant information were not available in these articles. In such cases, the corresponding authors were contacted and their clarifications if any were incorporated. For the list of the publications and detailed information about individual secondary antibody collected in this survey, please see the data table.

rabbit 133 [1-28]
mouse 126 [1-6, 9, 11, 12, 17, 20, 23-26, 29-33]
rat 26 [20, 23, 24, 32, 34-36]
donkey 99
rabbit 35 [25, 36-41]
mouse 25 [10, 14, 25, 33, 38, 39]
goat 19 [24, 37, 39]
Table 3. Host and reactive species of secondary antibodies and their numbers (num) of instances among the analyzed publications.
Survey Results
Host and reactive species

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.

mouse320 [1-6, 9-12, 14, 17, 20, 23-26, 29-34, 38-40, 42-60]
rabbit319 [1-28, 36-43, 45, 47, 50-53, 55, 56, 59-73]
rat70 [10, 20, 23, 24, 32, 34-36, 43, 44, 47, 51, 54, 55, 67, 69]
goat52 [24, 37, 39, 45, 47, 50, 56, 57, 65, 74, 75]
guinea pig18 [24, 36, 41, 44, 66-69]
Table 4. Reactive species of secondary antibodies and their numbers (num) of instances among the analyzed publications.

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 earlier verion 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.

IgA5 [29, 76-79]
IgD12 [78, 80-87]
IgE2 [88, 89]
IgG187 [1, 5, 8, 10, 11, 14, 17-20, 24, 25, 28, 29, 35, 38, 43, 47, 49, 54, 56, 59, 61-63, 65, 70, 75, 90-96]
IgG123 [83, 89, 91, 92, 97-104]
IgG2a7 [88, 89, 92, 97, 104-106]
IgG2b7 [97-100, 105, 107, 108]
IgG33 [77, 91, 100]
IgM25 [8, 79, 99, 103, 109-111]
Table 5. Immunoglobulin classes and their numbers of instances among the analyzed publications. The number in IgG row includes the numbers from IgG1, 2a, 2b, 3 and unclassified.
Ig class, subclass, and secondary antibody form

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.

Western Blot157
Table 6. Types of applications and their instances among the analyzed publications.

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 agasint F(ab')2 fragments or Fab fragment are used, for instances, when binding of secondary antibodies to Fc receptors on cell surfaces should be avoided, or there is endogenous immunoglobulin.

Alexa Fluor 488139 [1-4, 7, 9, 10, 13, 14, 17, 19, 20, 23, 24, 26, 30, 32-34, 36-39, 41, 44, 47-51, 54, 57, 59-61, 63-65, 68, 74, 112, 113]
Alexa Fluor 546 14 [21, 59]
Alexa Fluor 555 22 [3, 4, 23, 35, 36, 39, 50, 58, 114]
Alexa Fluor 568 30 [1, 7, 9, 10, 13, 37, 44, 47, 51, 57, 60, 74, 115]
Alexa Fluor 594 35 [24, 48, 112]
Alexa Fluor 633 6 [1, 107, 113, 116-118]
Alexa Fluor 647 31 [1, 6, 12-14, 23, 24, 26, 36, 37, 39, 51, 59]
Alexa Fluor 680 14 [1, 17, 27, 119, 120]
alkaline phosphatase 8 [44, 89, 107, 121-125]
bead 13 [17, 54, 70]
biotin28 [1, 20, 65]
Cy2 8 [62, 68, 110, 126-130]
Cy346 [1, 14, 19, 25, 38, 43, 60, 62, 68, 72, 131]
Cy5 22 [20, 60, 61]
DyLight 6496 [12, 41, 57, 62, 132, 133]
FITC36 [8, 18, 20, 25, 31, 43]
horseradish peroxidase139 [1, 4, 5, 8, 9, 11, 12, 14, 15, 19, 20, 22, 24, 26, 31, 32, 34, 39, 42, 44, 45, 50, 52, 57, 59, 64, 73, 74, 94, 134]
PE 20 [135]
IR Dye 68014 [1, 17, 27, 52, 119]
IR Dye 80011 [1, 17, 52, 119]
gold8 [13, 16, 136-141]
rhodamine7 [2, 142-147]
TRITIC 1 [148]
125I 2 [149, 150]
Table 7. Types of conjugates and their instances among the analyzed publications

An article "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance" by Dr. Trimmer's lab at University of California Davis tested the anti-mouse IgG subclass-specific secondary antibodies vs. general anti-mouse IgG seconday 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 the monoclonal mouse antibodies used and decreased signal to noise ratios." The authors suggest that subsclass-specific anti-mouse secondary antibodies should instead be used to signficantly increase the detection of primary antibodies, and each lab should at least maintain the inventory of secondary antibodies against mouse IgG1, IgG2a and IgG2b subclasses [151]. Dr. Trimmer leads the Neuromab group at UC Davis, and Labome visitors can search for Neuromab antibodies at Labome, using the search box above.

Alexa Fluor 4883
Alexa Fluor 5683
Alexa Fluor 68014
alkaline phosphatase 3
horseradish peroxidase110
IR Dye 68014
IR Dye 80011
Table 8. Common conjugates of secondary antibodies used in western blot.
Secondary antibody applications

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 antiobodies in ELISA and flow cytometry assays.

Alexa Fluor 488126
Alexa Fluor 54613
Alexa Fluor 55520
Alexa Fluor 56826
Alexa Fluor 59423
Alexa Fluor 64726
horseradish peroxidase12
Table 9. Common conjugates of secondary antibodies used in immunocytochemistry or immunohistochemistry.

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 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). A comparison with our survey result including data prior to 2006 indicats 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, Invitrogen (Zymed Laboratories, Pierce, Molecular Probes ) were often used in Western blot, and Alexa Fluor 488 conjugated secondary antibodies were from Invitrogen (Zymed Laboratories, Pierce, Molecular Probes ) and Jackson ImmunoResearch Laboratories.

Invitrogen / Molecular Probes / Life Tech / Zymed / Pierce / Biosource402
Jackson Immuno Research193
GE Healthcare / Amersham46
Santa Cruz Biotechnology44
Sigma Chemical Co35
Li-Cor Bioscience24
Southern Biotech23
Bio-Rad / AbDSerotec22
Vector Laboratories17
Cell Signaling12
R & D Systems8
Calbiochem / EMD Millipore7
Table 10. Suppliers and their numbers of instances among the analyzed publications.

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 Invitrogen, Jackson Immunoresearch Laboratories as the main suppliers.

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.

Secondary antibody suppliers

Many high quality reagent providers supply secondary antibodies to both the research and diagnostics communities. The relatively small number of publications in this survey precludes an extensive sampling of suppliers. Invitrogen (including Molecular Probes, Biosource International, and Zymed Laboratories), Jackson ImmunoResearch Laboratories, BD, Santa Cruz Biotechnology, GE Healthcare Bio-Science (including Amersham Biosciences), Sigma, were the most cited suppliers (Table 10).

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