DNA—MBSA

Three or four days before carrying out a spleen cell fusion give the chosen animal an iv injection (0.5 mL) of a suspension of DNA/mBSA complex prepared as in Step 2. 

Immunization with denatured DNA-MBSA gives rise to antibodies, often mainly of the IgM class, that react with single-stranded DNA but not with native DNA. The largest antigenic determinant for such antibodies is about the size of a pentanucleotide. When DNA with an unusual base, such as the glucosylated hydroxymethylcytosine of T-even phage, is used, the specificity is directed largely to the modified base and much more IgG may be produced.Similarly, when UV-irradiated DNA or photooxidized DNA is used, the modified bases of the lesions provide the major specificity determinants. - ... 

In order to elucidate the role of conformation of the immunogen, animals were immunized with complexes of polyribonucleotides (double- or triple-stranded) associated with MBS A. The antibodies obtained reacted specifically with double- or triple-stranded complexes and it was concluded that the specificity was determined by the macromolecular conformation of the immunogen (Nahon et al., 1967 Lacour et al., 1968 Michelson et al., 1971). Immunization of rabbits with DNA-MBSA or with double-stranded polynucleotide complexes adsorbed to MBS A ehcits antibodies belonging to the macroglobulin class (Stollar and Sandberg, 1966 Nahon-Merlin et al., 1973). 

For every 1 mL of this solution, add 15 pL of mBSA solution, keeping the soludon agitated during the addidon. The soludon immediately becomes cloudy as the result of the formadon of an electrostadc complex between the mBSA and DNA see Note 8). 

Peyer s Patch Immunization The DNA solution should be at a concentration of about 1 mg/mL see Note 11). Allow about 80 pL of DNA solution per rat per immunization. Add mBSA solution, such that the weights of mBSA and DNA are equal. Then add 1.1 vol of adjuvant and emulsify as described in Step 3 of the previous section, taking care not to make the emulsion too viscous, as it must be injected via a very fine needle. 

Immunization with homopolynucleotide-MBSA complexes gives rise to antibodies that are specific for an oligonucleotide segment of the polymer. They probably recognize a number of bases in sequence, perhaps in a stacked array. Poly(I), poly(C), and poly(A) each induce specific antibodies that show little cross-reaction with the other homopolynucleotides, and slight cross-reaction with denatured DNA. ... 

In contrast to single-stranded DNA, native DNA adsorbs poorly to polystyrene (Engvall, 1976). This problem can be overcome by treating the polystyrene with a 1% solution of aqueous protamine sulfate for 90 min (Klotz, 1982), followed by three washes with distilled water and drying. Other polycationic substances, such as MBSA (Section 5.2.3.3, Ferrua et al., 1983) or poly-L-lysine (type VII B Sigma), may also be used (Leipold et al., 1983). Poly-L-lysine is suspended in water at a concentration of 1 mg/ml (which may... 

No specificity for a particular DNA is obtained when dinucleotides or ohgonucleotides are conjugated with a carrier protein, when DNA complexed to MBSA, or whole bacteria, are used as immunogens the antibodies thus induced react with a variety of denatured DNAs (Beiser and Erlanger, 1966 Plescia et al., 1964 Halloran and Parker, 1966 Tan and Natali, 197O Forsen et al., 1970 Christian, 1965 Boros and Olitzki, 1967 Favors KAYA, 1970). 

Table 4. Reactions of anti-poly I poly C-MBSA sera with RNA and DNA...

Poly G — MBSA, like poly A, poly I or poly C, induces antibodies in which the specificity is associated with the nature of the base. Nevertheless these anti-poly G antibodies do not react with nucleic acids whereas the other antibodies to single-stranded polynucleotides react with denatured DNA (Seaman et al., 1965). This could be due to partial renaturation especially... 

With the objective of obtaining antibodies reacting with native DNA, Stollar (1970) immunized rabbits with complexes of polydeoxyribonucleo-tides such as poly dG poly dC — MBSA, poly dAT — MBSA and the hybrid... 

Abbreviations DNA deoxyribonucleic acid RNA ribonucleic acid rRNA ribosomal RNA tRNA transfer RNA. A adenine U uracil G guanine C cytosine, poly A polyadenylic acid poly U polyuridylic acid poly C polycytidylic acid poly I poly-inosinic acid poly G polyguanylic acid. The letters r and d refer to the ribose or deoxy-ribose series. Complexes between the polynucleotides are written as follows polyG poly C double-stranded helical complex of poly G and poly C 2 poly G poly C, triple-stranded complex. BSA bovine serum albumin MBSA methylated bovine serum albumin. N nitrogen. AMV Avian myeloblastosis virus NDV Newcastle disease virus. SLE systemic lupus erythematosus. 

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