Rabbit Antibody

Mary Johnson

Rabbit Antibody

Mary Johnson (han at labome dot com)

Synatom Research, Princeton, New Jersey, United States

DOI

//dx.doi.org/10.13070/mm.en.3.153

Date

last modified : 2023-07-08; original version : 2013-02-16

Cite as

MATER METHODS 2013;3:153

Abstract

This article addresses many questions related to rabbit monoclonal and polyclonal antibodies, unique rabbit antibody isotypes, antibody molecular weight, and antirabbit secondary antibodies.

Rabbit Has Only One IgG Isotype

Humans and mice have five antibody isotypes (IgA, IgD, IgE, IgG, and IgM). So far, four isotypes have been identified (IgA, IgE, IgG, and IgM) in rabbits. Interestingly, while humans have two IgA subclasses, mice have one IgA subclass, rabbits have a total of 13 IgA subclasses [1, 2]. On the other hand, rabbits have two κ light chain types K1 and K2 [3]. For a more in-depth discussion on isotypes, subclasses, heavy and light chains, different isotype compositions in various species, and constant and variable regions, please see Labome article on mouse antibody.

Most of rabbit research antibodies are of IgG isotype. Rabbit has only one IgG subclass, while mouse has IgG1, IgG2a, IgG2b, IgG2c, IgG3 subclasses, and the rat has IgG1, IgG2a, IgG2b, IgG2c subclasses. IgG-κ(K1) accounts for 90% of IgG in New Zealand White strain [3]. Table 1 lists rabbit antibody classes, subclasses, gene names, and links to NCBI and IMGT gene databases.

Isotypes	Subclasses	NCBI accession number	IMGT gene
IgA	IgA1, IgA2, IgA3, ...	Only IgA1 is listed here as an example. X51647	IGHA
IgE		AY386696	IGHE
IgG		L29172	IGHG
IgM		J00666	IGHM

Table 1. Rabbit isotypes/classes, subclasses, and their genes.

Rabbit Antibody Molecular Weight

Different isotypes and subclasses may have different molecular weights due to 1) the presence or absence of interchain disulfide bonds, 2) the oligomerization of immunoglobulin molecules through intermolecular disulfide bonds, and 3) the number of immunoglobulin domains in heavy chains. Labome article about mouse antibody discusses antibody molecular weights in detail.

The common rabbit IgG isotype has a molecular weight of 150 KD, with two heavy chains (about 50 KD each) and two light chains (about 25 KD each) under the non-reducing condition. Under the reducing condition, the molecular weights are 50 and 25 kD for heavy and light chain respectively.

Rabbit Antibody Production

Rabbit polyclonal antibody production

Rabbit is the predominant animal host for polyclonal antibody production, due to its small size, a rapid reproduction cycle, a considerable phylogenetic distance from most research models, and well-developed antibody production protocols. As of April 14, 2021, Labome antibody database lists 414178 rabbit polyclonal antibodies against 18749 human genes, 13330 mouse genes, 11020 rat genes, and a total of 52288 genes from 157 species. The practice of re-branding (that is, different suppliers use different catalog numbers for the same antibody reagent) is prevalent among antibody suppliers. Labome is the only antibody platform that takes active measures to minimize the number of re-branded antibody reagents. Thus the total number of antibodies reported here might be lower than those numbers reported by other articles/websites.

Method	Supplier	Catalog number	Sample reference
block/activate	Cell Signaling Technology	7321	[4, 5]
ChIP	Cell Signaling Technology	9733	[6, 7]
ChIP	Abcam	ab110641	[8, 9]
ChIP-Seq	Cell Signaling Technology	9733	[10, 11]
ChIP-Seq	Abcam	ab108341	[12, 13]
ELISA	Sino Biological	40150-R007	[14, 15]
ELISA	Invitrogen	700254	[16, 17]
ELISA	Abcam	ab128906	[18, 19]
ELISA	Cell Signaling Technology	9733	[20, 21]
FC	Cell Signaling Technology	4858	[22, 23]
FC	Sino Biological	40592-R001	[24, 25]
FC	BD Biosciences	559565	[26, 27]
FC	Abcam	ab133357	[28, 29]
FC	Invitrogen	MA5-28983	[30, 31]
IC	Cell Signaling Technology	3195	[32, 33]
IC	Abcam	ab16667	[34, 35]
IC	Sino Biological	40143-R001	[36, 37]
IC	Invitrogen	MA5-14520	[38, 39]
IC	BD Biosciences	559565	[40, 41]
IC	Cosmo Bio	REC-RCAB0002PF	[42, 43]
IC	ReproCELL	09-0023	[44, 45]
IC	Pel-Freez	P40101-0	[46, 47]
IC	SWant	PV 25	[48, 49]
IC	LifeSpan Biosciences	LS-B8482	[50, 51]
IHC	Invitrogen	MA5-14520	[52, 53]
IHC	Cell Signaling Technology	9664	[54, 55]
IHC	Abcam	ab16667	[56, 57]
IHC	BD Biosciences	559565	[58, 59]
IHC	SWant	PV 25	[60, 61]
IHC	Sino Biological	10084-R015	[62, 63]
IHC	Pel-Freez	P40101-0	[64, 65]
IHC	Alpha Diagnostics	CX40-A	[66, 67]
IHC	Ventana	790-2223	[68, 69]
IHC	Biocheckinc	BCH-1/37-2	[70, 71]
IHC	Cell Marque	235R-15	[72, 73]
IHC	Dako	M3616	[74, 75]
IHC	GeneTex	GTX16667	[76, 77]
IHC-F	Cell Signaling Technology	9664	[78, 79]
IHC-F	Abcam	ab16667	[34, 80]
IHC-F	Invitrogen	MA5-14520	[81, 82]
IHC-F	BD Biosciences	559565	[83, 84]
IHC-F	SWant	PV 25	[85, 86]
IHC-F	Pel-Freez	P40101-0	[87, 88]
IHC-F	ReproCELL	09-0024	[89, 90]
IHC-F	Alpha Diagnostics	CX40-A	[91, 92]
IHC-Free	Abcam	ab51253	[93, 94]
IHC-Free	Cell Signaling Technology	2250	[95, 96]
IHC-Free	Pel-Freez	P40101-0	[97, 98]
IHC-Free	SWant	PV 25	[49, 99]
IHC-Free	Invitrogen	G10362	[100, 101]
IHC-P	Abcam	ab16667	[102, 103]
IHC-P	Cell Signaling Technology	9664	[39, 104]
IHC-P	Invitrogen	MA5-14520	[39, 105]
IHC-P	Sino Biological	40143-R001	[36, 106]
IHC-P	GeneTex	GTX16667	[107, 108]
IHC-P	Ventana	790-2991	[109, 110]
IHC-P	Dako	M3616	[111, 112]
IHC-P	Cell Marque	104R-15	[113, 114]
IHC-P	SWant	PV 25	[115, 116]
IHC-P	Abnova	MAB1652	[117, 118]
IHC-P	Alpha Diagnostics	AQP01-A	[119, 120]
IHC-P	BD Biosciences	559565	[121, 122]
IP	Cell Signaling Technology	5246	[123, 124]
IP	Abcam	ab110641	[9, 125]
IP	Sino Biological	11215-R107	[126, 127]
IP	Invitrogen	G10362	[128, 129]
PLA	Abcam	ab40800	[130, 131]
PLA	Cell Signaling Technology	2983	[132, 133]
RIP	Abcam	ab186733	[29, 134]
RPPA	Cell Signaling Technology	2535	[135, 136]
RPPA	Abcam	ab32420	[135, 136]
RPPA	Invitrogen	MA1-39540	[135, 136]
WB	Cell Signaling Technology	4060	[137, 138]
WB	Abcam	ab32503	[139, 140]
WB	Sino Biological	40143-R001	[36, 106]
WB	Invitrogen	44-621G	[141, 142]
WB	Alpha Diagnostics	MTP11-A	[143, 144]
WB	ABclonal Biotechnology	AC026	[145, 146]
WB	Abfrontier	LF-PA0018	[147, 148]
WB	BD Biosciences	559565	[149, 150]
WB	GeneTex	GTX62601	[151, 152]
WB	Dako	M3642	[153, 154]
WB	New England Biolabs	e8023s	[155, 156]
WB	Novus Biologicals	NBP2-66953	[157, 158]
WB	Pel-Freez	P40101-0	[46, 87]
WB	SWant	PV 25	[159, 160]
WB	Ventana	790-4905	[161, 162]
WB	Biocheckinc	BCH-1/37-2	[71, 163]
WB	CalBioreagents	M085	[164, 165]

Table 2. Top rabbit monoclonal antibodies from different suppliers for different immunological methods and their references, based on a survey of formal publications in Labome's Validated Antibody Database, as of July 6, 2023. Publications are limited to those published since 2014. Only one rabbit antibody from each supplier is listed for each method.

Rabbit monoclonal antibody

Efforts have been ongoing for the last few decades to generate rabbit monoclonal antibodies. An antibody produced in rabbits is crucial to those antigens that are not immunogenic in rodents, which are the most commonly used host animals for monoclonal antibody production. Success was achieved through the transgenic rabbit animals with c-myc and v-abl transgenes to produce plasmacytoma cell lines initially, and hence rabbit monoclonal antibodies were produced [166]. Rabbit monoclonal antibodies can also be generated through phage display technologies and alternative methods, especially since rabbit hybridoma technology is patented [3]. Rabbit monoclonals have also been postulated to be candidates for anti-cancer therapeutics [167], and rapid generation of rabbit monoclonal antibodies through an array system has been tried [168].

Immune response mechanisms between mice and rabbits are different. Rabbit immune response has a broader repertoire. Rabbit can elicit a robust immune response against foreign antigens which is useful for antigens with weak immunogenicity or antigens derived from rodents [169].

Many commercial suppliers are now producing and/or distributing rabbit monoclonal antibodies, through mostly proprietary rabbit monoclonal antibody generation technologies. As of June 5, 2022, Labome antibody database lists 39315 rabbit monoclonal antibodies against 5863 human genes, 4414 mouse genes, 3772 rat genes and a total of 696 genes from other species. One of most commonly cited rabbit monoclonal antibody is the SP6 clone against Ki-67, a marker of cell proliferation. The clone is available from multiple suppliers (Thermo Fisher MA5-14520, Abcam ab16667 and ab21700, GeneTex GTX16667, and Bio-Techne NB600-1252, among others). Other rabbit monoclonal antibodies include rabbit recombinant anti-S100 beta antibody as an astrocyte marker [170], anti-αVβ3 integrin antibody MA5-32195 from Thermo Fisher, or anti-HA antibody from CST (3724) [171].

Labome surveys literature to develop Validated Antibody Database. Table 2 lists some of the most commonly cited rabbit monoclonal antibodies among the articles Labome has curated.

Rabbit hybridomas tend to be less stable than rodent hybridomas [172], and valuable rabbit monoclonal antibodies are often sequenced and made into recombinant expression. Rabbit monoclonal antibodies have a higher affinity than mouse monoclonal antibodies, and this mandates harsher buffer conditions for the stripping of rabbit monoclonal antibodies in Western blotting for re-blotting or in sequential immunostaining. An SDS buffer with the reducing agent 2-mercaptoethanol has been proposed to be the method of choice instead of an acidic (pH 2) glycine buffer or a 6 M urea hot buffer [173]. For the transient expression of rabbit monoclonal antibody clone 4G3 against PLXDC2, it was found that hypothermic culture (at 32 C vs. 37 C) and purification by CIMmultus DEAE chromatography (vs. Protein A/G or HiTrap DEAE FF) yielded the best quality, as examined by western blot, possibly due to more homogenous glycosylation and less aggregation [174].

Rabbit Monoclonal vs. Mouse Monoclonal

Several studies have been conducted to compare rabbit monoclonal antibodies (RabMAb) vs. mouse monoclonal antibodies. In 2005, Sabrina Rossi et al. published a comparative study evaluating RabMAbs against estrogen receptor, progesterone receptor, Ki-67, cyclin D1, CD3, CD5, CD23, and synaptophysin in routine diagnostic immunohistochemistry on a large number of tumor types and normal tissues. In direct comparison with mouse monoclonal antibodies, the authors concluded that RabMAbs had similar specificity as mouse monoclonals, while displaying higher sensitivity, thus permitting higher working dilutions (5 to 10 times on average), and in some cases, the antigen retrieval became unnecessary for optimal staining [175]. Similar observations were published by Rafael Rocha et al. in 2008 [176]. Rafael Rocha found that rabbit monoclonal antibodies against estrogen receptor and progesterone receptor showed a higher intensity of staining than the commonly used mouse monoclonal antibodies and rabbit monoclonal antibody against HER2 was more sensitive than mouse monoclonal antibody in 2009 [177]. However, mouse monoclonal antibody against HER2 is more specific than the rabbit counterpart. On formalin-fixed, paraffin-embedded canine tissues, however, Jose G. Vilches-Moure and Jose A. Ramos-Vara found that rabbit and mouse monoclonal antibodies recognized COX-2, Ki67, synaptophysin, and vimentin; but only rabbit monoclonal antibodies detected CD3, chromogranin A, and progesterone receptor, and just mouse monoclonal antibodies detected CD79a, calcitonin, and estrogen receptor [178]. Table 3 lists the advantages and disadvantages of mouse and rabbit monoclonal antibodies.

host	advantages	disadvantages
mouse	widely exploited and used uniform, pure, and reliable model readily available inbred strains at low cost mice are easy to manipulate mouse mAb responds well to foreign proteins numerous commercially available anti-mouse secondary antibodies and kits humanized antibodies to reduce HAMA-mediated responses	mice are limited by a small spleen, are usually inbred, and offer less diversity of immune responses
rabbit	higher binding affinity with no apparent loss of specificity wider repertoires reduce antigen-retrieval pretreatments a bigger spleen to provide more hybridomas for selecting the desired mAb simpler structure of rabbit immunoglobulin easy to be humanized	commercially available product not suitable for other species hybridomas are less stable than mouse hybridomas; thus often the antibody sequences from the hybridomas are obtained to enable recombinant expression

Table 3. Comparisons between mouse and rabbit monoclonal antibodies.

Anti-Rabbit Secondary Antibodies

Goats, sheep, and donkeys are common hosts for generating anti-rabbit secondary antibodies, according to Labome survey on secondary antibodies. See a list of anti-rabbit secondary antibodies.

Anti-Rabbit Gene Antibodies

Rabbits, while not as popular as rodents, are a valuable research animal. Rabbits serve as a useful research model for tumorigenesis [179], and injury and wound healing [180, 181], among other applications. Rabbit reticulocyte lysate is a commonly used in vitro cell-free translation system [182, 183]. An interesting line of research on rabbits is the co-evolution of European rabbits and myxoma virus, which was introduced to control the rabbit populations in Australia and Europe in the 1950s [184].

As of June 5, 2022, Labome antibody database lists monoclonal antibodies against 204 rabbit genes and polyclonal antibodies against 138 rabbit genes. Antibodies against all these genes can be searched and compared at Labome website using the search box above, for example, antibodies against rabbit LDHA, rabbit ALDOA, rabbit CD4, rabbit NHE-3, and rabbit GAPDH.

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