BrdU (bromodeoxyuridine, 5-bromo-2'-deoxyuridine, BUdR, BrdUrd, broxuridine), an analog of deoxythymidine (Fig 1), can be incorporated into DNA by DNA polymerase during the S phase of the cell cycle, and has been used to indicate cell proliferations. BrdU within DNA molecules is recognized by several clones of BrdU antibodies. Table 1 lists these clones, their applications and recent references among the publications Labome has surveyed. These antibodies have been used in a variety of immuno methods - immunohistochemistry, immunocytochemistry, flow cytometry, immunoprecipitation, and Western blotting. BrdU assays are routinely used to measure cell proliferation. Detection of BrdU by BrdU antibodies usually require either acid or enzymatic or copper ion treatment of chromatins to expose BrdU. Commercial kits, such as APC BrdU Flow Kit from BD, are commonly used [1].

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Figure 1. Skeletal formulae of bromodeoxyuridine, deoxythymidine, and uridine (from left to right). From Wikipedia.

BU1/75(ICR1) clone is a rat monoclonal antibody, of IgG2a subcless, and specific against bromouridine and chlorouridine, but not against iodouridine and thymidine [2]. It is the widely used BrdU antibody in the literature Labome has surveyed. Yu K et al injected BrdU solution intraperitoneally into mice and stained the frozen sections from the mice to assay in vivo cell proliferation [3]. Sarek G et al, for example, stained mouse ear fibroblasts pulse-labelled with CldU with this rat anti-BrdU antibody from AbD Serotec (now Bio-Rad) [4]. M Coolen et al quantitated proliferating BrdU+ cells in killifish pallium with this clone [5].

Gonchoroff NJ et al injected Balb/c mice with 5-iodouridine covalently coupled to ovalbumin, fused the spleen cells with the P3/NSl/l-Ag4-1 mouse myeloma, and selected clones reactive with bromouridine and iodouridine but nonreactive with ovalbumin [6]. The resultant clone BU-1, of IgG2a subclass, has a dissociation constant of 80 nM for free bromouridine. It is reactive against bromouridine, chlorouridine, iodouridine, but not fluorouridine and thymidine. Unlike clone B44, BU-1 antibody can detect BrdU in DNA without denaturation [6].

Gratzner HG immunized BALB/c mice with a conjugate of iodouridine and ovalbumin, fused the resultant spleen cells the plasmacytoma line SP2/0Ag14, and selected a clone (B44) highly specific for bromodeoxyuridine and iododeoxyuridine with no cross-reaction with thymidine [6]. This clone can detect BrdU in denatured DNA [6]. Sarek G et al, for example, stained mouse ear fibroblasts pulse-labelled with IdU with this mouse anti-BrdU antibody from BD [4].

G3G4 is a mouse IgG1 clone, deposited by Dr. SJ Kaufman to DSHB in 1993.

Liboska R et al examined the affinities of BMC9318 from Roche, B44 from Becton Dickinson, Bu20a from BioLegend, BU-33 from Sigma-Aldrich, BU6-4 from Genetex, BU5.1 from Millipore, MoBu-1 from Exbio and BU1/75 from Abcam against 2'-deoxy-5-ethynyluridine (EdU)and found that with the exception of MoBu-1 clone, all reacted with Edu [109]. Ligasová A et al evaluated the affinities of anti-bromodeoxyuridine monoclonal antibodies: BMC9318, Bu-33, B44, Bu6-4, Bu20a and Bu5.1, against oligonucleotides with BrdU at 5' or 3' end or in the middle, and found that clones Bu-33 and Bu5.1 had very low affinity with the tested oligonucleotides and all of the tested clones exhibited the highest affinity to the oligonucleotide with BrdU at the 5' end; clones Bu20a, BMC9318 and Bu5.1 clones exhibited the lowest affinity to the oligonucleotide with BrdU at the 3' end; clones Bu-33, B44, and Bu6-4 to the oligonucleotide with BrdU in the central part of the chain [110]. In addition, clones B44 and BMC9318 were found to work with all four protocols: hydrochloric acid (2N and 4N), DNase I and copper ion treatment, for the detection of BrdU in HeLa and HCT116 cells [110]. The authors in a follow-up article attributed the differing affinities of the above clones to their varying speed of dissociation and optimized an enzymatic protocol to achieve higher signal/background ratio, even for those clones with lower affinities [80].

Compared to BrdU antibodies, researchers cite anti-DNA antibodies much less frequently. Table 2 lists the cited DNA antibody clones, their strand specificity, and other information. One active research area is auto-antibodies in diseases such as systemic lupus erythematosus and isolated cases of viral infections. DNA auto-antibodies are found in 100% cases of systemic lupus erythematosus, and also in inbred mouse strains with an auto immune disorder similar to systemic lupus erythematosus. Most of these auto-antibodies react more strongly with denatured than with native DNA [111]. Some discriminate among helical DNAs of differing base sequence [111]. Quite often, these auto-antibodies with high affinity contain arginine, asparagine, lysine, and tyrosine in the CDRs, enabling the formation of electrostatic interactions between positively charged CDRs and negatively charged phosphodiester-deoxyribose backbone and hydrogen bonds between these amino acids and DNA. While review articles such as the one by Hu Z et al [2], list many more clones, most of them are either not available commercially or through non-profit organizations, or not cited in recent literature.

DNA antibodies have been used in a variety of applications. For example, Amini P et al detected NET formation in primary mature mouse neutrophils using monoclonal mouse anti-dsDNA antibody ( MAB1293) from MilliporeSigma [112].

Scheer U et al immunized BALB/c mouse with cytoskeletal preparations, fused the spleen cells with mouse myeloma line Ag 8.653, and screened for clones with IHC on frozen rat and mouse liver sections [128]. The resultant clone, with IgM subclass, may have resulted from the DNA present in the immunogen or from an autoantibody producing spleen cell [128]. AC-30-10 binds to both double and single-stranded DNA and has no cross-reactivity with deoxyribonucleotides and RNA [128].

Frankfurt OS obtained monoclonal antibody F7-26 from a mouse immunized with DNA treated by nitrogen mustard (HN2) [129]. The clone, of IgM subclass, binds to ssDNA, but not to dsDNA or RNA [2].

Sarek G et al stained mouse ear fibroblasts with the anti-ssDNA antibody (MilliporeSigma, MAB3034) in order to ascertain no broken DNA tracks in DNA combing [4].

8-OHdG (8-hydroxy-2'-deoxyguanosine) results from DNA oxidative damage by hydroxy radical, singlet oxygen and direct photodynamic action. 8-OHdG antibody clone N45.1 from Genox / Nikken SEIL detects 8-OHdG in tissues, sera, urine and other biomaterials with IHC and ELISA. It does not cross react with RNA oxidation products such as 8-hydroxy-guanine and 8-hydroxy-guanosine. Aoki A et al used 8-OHdG staining by clone N45.1 as an oxidative stress marker in mouse placentas [123].

One of the commonly used RNA antibody clones is Y10b. Lerner EA et al fused spleen cells from an unimmunized female MRL/1 mouse with MRL/l mice were bred and maintained myeloma SP 2/0 and screened to obtain the clone Y10b, of the IgG2a class, which is specific to rRNA [130, 131].

Boguslawski SJ et al immunized BALB/c mice with a DNA:RNA hybrid preparation combined with methylated thyroglobulin and fused mouse spleen celles with myeloma cells (Sp2/0-Ag14). The selected clone, S9.6, is of IgG2a subclass with kappa light chains, and has a high affinity for DNA:RNA duplex (Kd = 1.18 x 10^-11 M), and 1/1000 or less affinity to single- or double-stranded DNA or RNA [141]. S9.6 antibody has been used by, for example, Lambo S et al [137] and NK Karanam et al [138] to study R-loops, and Jiang YF et al to indicate compromised mtDNA replication in flies [140].