Aging Aging

Immunochemical Assay for DNA Damage 1653.4. Sandwich ELISA1. Make 100% ssDNA samples from an aliquot of the DNA samples to be assayed:Add, at room temperature, 25 µL of DNA sample to a cluster tube, and add200 µL of a solution of 1.3 M NaCl + 0.02 M NaOH, pH 12.3. Shake for 1 s on areaction tube mixer or sonicate (set at 2.5, duration 1 s), and neutralize with25 µL of 0.25 M NaH 2 PO 4 . In the case of 100% ssDNA samples of sperm, theseare always sonicated before neutralization.2. Add to the first row of a D1B-coated “high-binding” polystyrene plate (with a12-channel pipet) 120 µLPT and in all other rows 70 µL.3. Place the samples in rows of 12, including the 100% ssDNA samples, accordingto the scheme prepared in advance.4. Add of each sample (with a 12-channel pipet) 20 µL in a well of the first row of theplate. In the case of sperm DNA samples add the 20-µL aliquots with a single pipet.5. Make 1:1 serial dilutions until the last row.6. Add to wells 5–8 of the last row of each plate 140 pg of single-stranded DNA(intended to correct for plate-to-plate variations).7. Incubate at room temperature under continuous vibration for at least 5 min (seeNote 7, 2 × 8 plates/vibrator).8. Wash 3× with PT buffer (hand wash apparatus or plate washer).9. Add 100 µL of D1B-AP solution/well (with dispenser, use filter plate up to 200×,or with 12-channel pipet; see Note 5).10. Incubate again at room temperature under continuous vibration for at least 5 min(up to 2 × 8 plates/vibrator).11. Wash 3× with PT buffer (hand wash apparatus or plate washer). The last washshould be extensive and done using the hand wash apparatus (see Notes 8 and 9).12. Add 100 µL of MUP solution/well (with dispenser or with 12-channel pipet).13. Incubate for 1 h at 37°C in a humidified incubator (cover on plate, do not stack plates).14. Read fluorescence in all wells (Cytofluore II, gain 45) and transfer data to a PCwith lotus or excel spreadsheet (see Note 6).3.5. Calculation of ssDNA PercentageProvided limited amounts of DNA are used a linear relationship is obtainedbetween the input amount of DNA and the level of fluorescence, both for completelyand for partially single-stranded DNA. To calculate the fraction ofsingle-strandedness in a particular sample, the ratio of the fluorescence of theDNA dilutions to that of the corresponding completely single-stranded DNAdilutions, both corrected for background fluorescence (fluorescence in wellswithout DNA), is divided by a factor of 10 (because of the predilution of thecompletely single-stranded DNA sample) according to the following formula:(fl sample – fl background )% single-strandedness = —————————————— × 100%10 × (fl 100% ss – fl background )