Brief Description of Common Immunoassay Methods
Competitive Enzyme Linked Immunosorbent Assays (ELISA)
The most commonly used immunologic method for quantitation of DNA damage is competitive ELISA. There are numerous variations of the ELISA using fetal calf serum (FCS), BSA, or milk proteins for blocking nonspecific binding, alkaline phosphatase or peroxidase conjugated secondary antisera for primary antibody detection, and colored, fluorescent, or radioactive substrates for endpoint detection. As an illustration our routine color ELISA for PAH-DNA is described (2). Antigen, either in vitro modified DNA or monoadduct coupled to carrier protein is coated onto 96-micro well plates. Generally 5-10 ng of antigen (sonicated, denatured PAH-DNA) is used and coating is accomplished by incubating 100 µl of antigen in PBS on the plate at 37oC overnight. After washing with PBS containing 0.05% Tween 20, plates are incubated with PBS-Tween with 1% FCS or BSA for 1 hr to block nonspecific binding to the plate. While plates can be hand washed, the most reproducible data are generated with automatic 96-well plate washers. A standard curve is generated by serial dilution of either the modified denatured DNA or monoadduct in PBS (50 µl) and mixed with 50 µl diluted antibody (in PBS-Tween-FCS) before addition to the coated well. Unknowns to be tested are similarly mixed with antibody before addition to the plate. Antibody binding to the antigen on the plate is competed by antigen in solution. After a 1½ hr incubation, the plate is washed and bound primary monoclonal antibody quantitated with 100 µl commercial secondary antiserum, goat anti-mouse IgG-alkaline phosphatase, diluted 1:500 to 1:1000 in PBS-Tween-FCS. After a 1½ hr incubation, the plate is again washed with PBS-Tween then 0.01M diethanolamine to remove residual phosphate which will interfere with the assay. The substrate, p-nitrophenylphosphate, is available in tablet form and must not be touched because of alkaline phosphatase activity on the hands. Wells are incubated with 100 µl of a 1 mg/ml solution in 1 M diethanolamine, pH 8.6 until the absorbance at 405nm is between 0.5 and 1. Appropriate antibody dilutions and coating conditions must be determined by trial and error. Generally higher antibody dilutions and lower coating levels lead to more sensitive assays but must be balanced with time to endpoint detection and signal to noise ratio. The standard curve, % inhibition versus amount of antigen per well, is generated and amount of adduct in the unknown determined from its % inhibition. Although the unknown DNA could be directly coated on the plate and detected with the primary antibody, competitive assays are more sensitive due to the larger amount of test DNA which can be assayed in solution. Plates can usually be coated with no more than 200 ng of DNA but 50-100 µg can be used as competitor. With the highest affinity antibodies, 50% inhibitions in the low femto mole range can be obtained. With this level of sensitivity and the ability to assay 50-100 µg of DNA, adduct levels of approximately 1 adduct per 108 nucleotides can be measured. In assays in which monoadduct is isolated before analysis, sensitivity can be increased provided larger amounts of DNA are available.
Immunohistochemistry
Immunohistochemical detection of DNA damage can be carried out on either fixed cells (e.g., lymphocytes or exfoliated oral or bladder cells) or tissue sections (frozen or paraffin). Before use, microscope slides are precoated with silane by dipping into a solution containing 6 ml 3-aminopropyltriethoxysilane in 300 ml acetone for 2 min. Two slides can be held back-to-back in the staining rack. The slides are then dipped sequentially into tanks, the first containing acetone for 2 min. This solution should be changed after ~200 slides. This is followed by a second acetone wash for 2 min followed by two 2 min dippings in distilled water.
For smeared cells, an appropriate dilution of cells is used so that a single layer is present and the slide is allowed to air dry before fixing the cells in methanol for 3 min (-20oC) followed by dipping in acetone (-20oC). Other fixation protocols (e.g., 70% ethanol) are available and may result in differences in staining. Thus, during methods development various methods should be tested to select optimum conditions. Frozen tissues are fixed by similar methods while paraffin sections must be deparaffinized before staining. Slides containing paraffin sections are treated with a microwave oven heating step with an appropriate buffer such as citric acid or Tris which is selected again by testing which conditions give optimum staining. The 0.01 M citric acid (pH 6.0) or 0.1M Tris solution (pH 2.0) is prewarmed in a microwave oven for 15 min then used to boil the slides in the microwave oven for another 15 min. They are then washed with tap water twice for 5 min each changing the solution between washes in a staining jar on a shaker. Similar wash steps are carried out between each step.
While some studies directly incubate fixed material with primary antibody, procedures to enhance antibody binding are usually carried out to increase assay sensitivity. These procedures normally include treatment with proteases to remove histone and nonhistone proteins from the DNA and with RNase to eliminate potential cross-reactivity with RNA adducts. This can be followed by acid or base treatment to denature the DNA and further increase antibody accessibility to the adduct. Clearly the adduct of interest must be stable to the denaturing treatment chosen. For PAH-DNA adduct detection (53,54)(3), we routinely incubate slides for 1 hr at 37oC in 100 µg/ml RNase in Tris buffer (10 mM Trizma base, 1 mM EDTA, 0.4 M NaCl adjusted pH to 7.5 with HCl) followed by washing with PBS 5 min, changing the buffer twice. Proteinase K treatment is then carried out (10 µg/ml in Tris buffer, pH 7.5) for 7 min at room temperature. After this step slides should be checked under the microscope to ensure cells are intact. Slides are again washed with PBS for 5 min, twice. The DNA is then denatured by treatment with 4 N HCl for 7 min at room temperature but the treatment time may vary according to the cell line and tissue type. Slides are neutralized with 50 mM Tris base treatment for 5 min at room temperature then washed with PBS for 5 min, three times.
From this step on there are two commonly used detection systems, immunofluorescence or immunoperoxidase. For the immunofluorescence method, slides are next incubated with 10% normal goat serum for 37oC for 45 min to block nonspecific binding. This is followed by incubation with an appropriate dilution of adduct specific antibody in blocking solution for 45 min at 37oC or at 4oC overnight. Washing is followed by goat anti-mouse IgG conjugated to the fluorescent tag, usually fluorescein isothiocyanate (FITC) for 45 min at 37oC. More recently, we have used Alexa conjugates (Molecular Probes, Eugene Oregon) because of the greater stability of this fluorescent tag in the stained slides. The antibody dilution is selected by staining cells with or without the adduct of interest with serial dilutions of antibody so that conditions giving the lowest background binding to negative control cells but clear positive staining with positive control cells can be determined. Cells can be counterstained with propidium iodide (1 µg/ml at room temperature for 45 sec) to allow visualization of nuclei in adduct negative preparations. Dehydration is carried out by serial dipping in 95% ethanol for 3 min each in two staining tanks followed by 100% ethanol for two 3 min and one 5 min dipping. Slides are then cleaned with xylene, two 3 min dipping and one 5 min. Cover slips are then mounted.
For the peroxidase method, nonspecific binding is blocked by incubation with 1.5-10% normal horse serum or 2% BSA for 1 hr at 37oC. Incubation with the primary antibody (in blocking solution ) is carried out overnight at 4oC. Slides are washed with PBS three times for 5 min. Endogenous peroxidase activity is then quenched by incubation with 0.3% H2O2 in methanol for 15 min at room temperature. After washing with PBS, slides are incubated with reagents contained in Vector ABC kits (Vector Laboratories, Burlinghame, CA) as detailed by the vendor. After washing slides with PBS for 5 min, three times they are rinsed in 1% Triton X-100 (in PBS) for 30 sec, then again with PBS. This step is followed by incubation with diaminobenzidine (DAB, 7 min, room temperature), also available in kit form from Vector, in the presence of H2O2, as directed by the vendor. Polymerization of the benzidine results in a permanent brown precipitate proportional to the amount of antigen present. Sensitivity is increased due to amplification of the signal by the enzyme. However, care must be taken since benzidine is a carcinogen. The slides are then washed in tap water for 5-10 min with 2-3 changes of solvent. Slides can be counterstained with ethyl green (10 mg/ml 0.1M sodium acetate pH 5.2 for 8-11 min) to visualize adduct negative nuclei. Dehydration and mounting is then carried out.
Samples can be evaluated in a qualitative or quantitative manor. Qualitative assessment uses subjective estimation of staining intensity and/or number of positively stained cells with an arbitrary scale. Quantitative measurements for both fluorescence and peroxidase staining directly measures intensity of either fluorescence or absorption of the nucleus, respectively. Sophisticated and expensive video cameras and software are required. For samples with no or low adducts and thus minimal staining, counter staining for DNA with propidium iodide or ethyl green, for fluorescence or peroxidase staining, respectively, is necessary for the software to identify the nuclei automatically.
For immunohistochemical assays, it is important to run control experiments to demonstrate the specificity of cell staining. These controls can include cells treated in culture or tissues from animals treated in vivo with or without the chemical of interest. Additional controls routinely include samples containing adducts but treated with DNase (500 µg/ml for 1 hr at 37oC) before incubation with primary adduct specific antibody or with primary antibody which has been preabsorbed with the DNA adduct of interest. Nonspecific antibodies are also frequently used to confirm specificity of staining.
The major advantages of the immunohistochemical method are its ability to detect adducts in specific cell types within a tissue and its applicability to small amounts of sample. Less than one ml of blood is required for analysis of lymphocytes compare to the 30 ml required for isolation of sufficient amounts of DNA for ELISA. It is also applicable to stored paraffin sections widely expanding the types of studies that can be carried out. The disadvantages of the immunohistochemical method are similar to those of the ELISA. Cross-reacting antibodies will result in errors in quantitation and, in general, the method is not as sensitive as the competitive ELISA.
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