Selection of monoclonal antibodies against 6-oxo-M(1)dG and their use in an LC-MS/MS assay for the presence of 6-oxo-M(1)dG in vivo

Abstract

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.

Scheme 1. Conjugation Reaction between 6-oxo-M₁Guo and the Carrier Protein (BSA or mcKLH)

Figure 1

6-oxo-M₁dG, M₁dG, and structural analogues that were used as competitive antigens during ELISA analysis of murine sera, hybridomal supernatant, and purified antibodies.

Figure 2

Schematic of monoclonal antibody generation and isolation procedure. Eight mice were inoculated with adduct/protein conjugate, followed by tail bleeding and testing for the presence of 6-oxo-M₁dG antibodies using direct ELISA analysis (step 1). Splenocytes from a single mouse, Balb/CJ R, were fused with myeloma cells, plated on twenty-four 96-well plates, and screened using direct and competitive ELISA analyses (step 2). From this screen, 180 hybridoma cell lines showed anti-6-oxo-M₁dG mAb production. These were replated and screened (step 3). Five parental cell lines were chosen for subcloning and further screening (step 4). On the basis of these final screenings, the cell line 6C9BA4C12 was chosen for scale-up and antibody purification (step 5).

Figure 3

6-oxo-M₁dG antibodies are present in the serum of mouse BALB/cJ R. The serum of mouse BALB/cJ R was subjected to competitive ELISA analysis with 6-oxo-M₁dG used as the competitor. The displayed results represent the average of spectroscopic readings at 15 and 30 min postaddition of ABTS substrate.

Figure 4

ELISA analysis of specificity of various subclonal antibodies. The specificity of antibodies produced by two hybridoma cell lines (B and E2) subcloned from the parental hybridoma cell line 6C9 were assessed using competitive ELISA analysis. Xanthine and 6-oxo-M₁dG were used as competing antigens. The displayed results represent the average of spectroscopic readings of subclones E2 and B at 15 and 30 min postaddition of ABTS substrate.

Figure 5

Specificity determination of antibodies produced from subclone 6C9BA4C12. Purified antibodies from the hybridomal subclone (6C9BA4C12) were screened for specificity for 6-oxo-M₁dG in the presence of structural analogues using competitive ELISA analyses.

Figure 6

6-oxo-M₁dG is present in rat feces. A representative LC-MS/MS chromatogram of 6-oxo-M₁dG and the internal standard [¹⁵N₅]-6-oxo-M₁dG isolated from rat feces is displayed. The inset represents a chromatogram of internal standard alone, purified from PBS.