Secondary Antibody Review
  1. Mary Johnson Ph. D.
    mary at labome dot com
    Synatom Research, Princeton, New Jersey, United States
last modified : 2014-03-17; 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.

This is a review about secondary antibodies used in peer-reviewed publications. Labome also reviews and summarizes primary antibodies cited in publications, with over 10,000 publications manually curated. The 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, alpha tubulin, integrin antibody, phosphotyrosine antibody, and hemagglutinin antibody.

Table 1. The number (num) of publications for each year in the latest secondary antibody survey

In order to help visitors identify the most suited secondary antibodies, Labome conducted two surveys over the years. In the 2012 survey, information about 163 instances of secondary antibodies from 105 publications from 21 journals (mainly the journal Science) were collected and organized. Table 1 and 2 indicate the publication years and major journals of these 105 publications.

Journal nameNum
Mol Cell Biol6
J Virol4
Proc Natl Acad Sci U S A2
J Cell Biol2
Mol Biol Cell2
Table 2. The number (num) of publications for some of journals in the 2012 survery. Only journals with more than 1 articles are listed, other journals have 1 article each.

In an earlier 2008 survey, information about secondary antibodies from 99 recent 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. 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 secondary antibodies used, please see here.

mouse 13 [1] [2] [3] [4] [5] [6] [7] [7] [7] [8] [1] [1] [9]
rabbit 12 [1] [2] [10] [4] [11] [12] [7] [7] [7] [13] [9] [14]
sheep 3
mouse 2 [15] [16]
rabbit 1 [15]
donkey 2
rabbit 1 [1]
rat 1 [1]
rabbit 2
goat 1 [17]
rat 1 [18]
Table 3. Host and reactive species of secondary antibodies and their numbers (num) of instances among the analyzed publications.
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.

mouse38 [19] [20] [18] [21] [22] [1] [23] [2] [24] [10] [25] [26] [27] [3] [4] [28] [29] [15] [30] [5] [6] [31] [7] [7] [7] [16] [32] [33] [34] [8] [35] [1] [1] [36] [37] [9] [38] [39]
rabbit30 [28] [21] [22] [1] [20] [2] [14] [10] [25] [26] [27] [4] [15] [11] [12] [7] [7] [7] [18] [40] [13] [33] [41] [35] [1] [36] [37] [9] [14] [42]
rat5 [18] [43] [44] [18] [1]
goat5 [21] [20] [17] [33] [45]
guinea pig 1 [21]
human1 [46]
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. Among the 163 instances of secondary antibodies, 33 of them indicated the host species of the secondary antibodies. Table 3 lists the host species, indicating goat had been the predominant species for secondary antibodies. There were 79 instances where the reactive species were indicated, Table 4 lists the reactive species, indicating that, no surprise here, mouse and rabbit are the two major target species.

IgG9 [47] [20] [48] [26] [27] [4] [28] [49] (IgG1), [3] (Fc gamma fragment)
IgM2 [49] [50]
IgA1 [51]
Table 5. Immunoglobulin classes and their numbers of instances among the analyzed publications.
Ig class, subclass, and secondary antibody form

Anti-IgG secondary antibodies are most widely used. Among the 12 instances where an Ig class is indicated, 9 cases were IgG (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.

immunocytochemistry 42
immunohistochemistry 45
Western Blot 61
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 488 24 [45] [21] [40] [30] [52] [53] [33] [54] [55] [56] [22] [57] [18] [58] [59] [27] [60] [61] [7] [4] [62] [63] [1] [11]
Alexa Fluor 546 2 [4] [59]
Alexa Fluor 555 5 [21] [53] [18] [63] [7]
Alexa Fluor 568 6 [33] [58] [27] [61] [23] [14]
Alexa Fluor 594 4 [64] [22] [57] [60]
Alexa Fluor 633 2 [45] [65] [66] [67]
Alexa Fluor 647 2 [58] [7]
Alexa Fluor 680 1 [34]
alkaline phosphatase 2 [53] [68]
bead 1 [44]
biotin 9 [52] [49] [13] [18] [69] [70] [71] [72] [18]
Cy2 3 [21] [73] [10]
Cy3 11 [56] [24] [52] [21] [1] [1] [73] [30] [55] [74] [10]
Cy5 6 [74] [1] [56] [52] [24] [6]
DyLight 649 1 [3]
FITC 5 [75] [41] [76] [51] [77]
horseradish peroxidase 39 [78] [79] [2] [25] [80] [81] [82] [83] [84] [42] [19] [15] [15] [14] [30] [85] [86] [38] [28] [46] [31] [35] [8] [12] [17] [44] [23] [87] [57] [88] [89] [90] [91] [33] [92] [54] [68] [39] [48]
PE 2 [43] [45]
IR Dye 680 2 [93] [26]
IR Dye 800 4 [93] [34] [20] [26]
nanogold 1 [94]
rhodamine 3 [29] [77] [1]
TRITIC 1 [75]
125I 1 [92]
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 in 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 [95]. Dr. Trimmer leads the Neuromab group in UC Davis, and Labome visitors can search for Neuromab antibodies at Labome, using the search box above.

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 488 2
Alexa Fluor 568 2
Alexa Fluor 594 1
Alexa Fluor 680 1
alkaline phosphatase 2
Cy3 1
Cy5 2
horseradish peroxidase 38
IR Dye 680 2
IR Dye 800 4
rhodamine 1
Table 8. Common conjugates of secondary antibodies used in western blot.

Secondary antibodies serve two purposes, one is to amplify the 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). HRP conjugated secondary antibodies from Amersham Biosciences, Bio-Rad, Cell Signaling, GE Healthcare Bio-Science, Jackson ImmunoResearch Laboratories, LI-COR Biotechnology, Pierce Biotechnology, Santa Cruz Biotechnology, Sigma, and Invitrogen(Zymed Laboratories) were often used in Western blot, and AP conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Sigma, Bio-Rad, and DAKO.

Alexa Fluor 488 25
Alexa Fluor 546 1
Alexa Fluor 555 6
Alexa Fluor 568 5
Alexa Fluor 594 4
Alexa Fluor 647 5
Alexa Fluor 633 2
biotin 8
Cy2 2
Cy3 10
Cy5 4
DyLight 649 1
horseradish peroxydase 2
nanogold 1
PE 1
rhodamine 1
Table 9. Common conjugates of secondary antibodies used in immunocytochemistry or immunohistochemistry.

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, and Sigma 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.

Invitrogen / Molecular Probes / Life Tech 64
Jackson Immuno Research 23
GE Healthcare 11
Jackson Immunoresearch 10
DakoCytomation 9
Santa Cruz Biotechnology 7
Sigma Chemical Co 5
Calbiochem / EMD Millipore 4
Vector Laboratories 4
Li-Cor Bioscience 3
Dianova 3
Cell Signaling 2
MP Biomedicals 2
eBioscience 2
Southern Biotech 2
R & D Systems 1
Perkin Elmer 1
Thermo Fisher Pierce 1
BD 1
Beckman Coulter 1
Leica Microsystems 1
Biorad 1
Rockland 1
Abcam 1
Nanoprobes 1
Miltenyi 1
Table 10. Suppliers and their numbers of instances among the analyzed publications.
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, Sigma, GE Healthcare Bio-Science (including Amersham Biosciences), Bio-Rad, Santa Cruz Biotechnology were the most cited suppliers (Table 10).

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