Antibodies, anti-polynucleotide

With the exception of antibodies reacting only with native DNA all the different kinds of anti-polynucleotide antibodies described above can be induced experimentally. 

The first description of antibodies against polynucleotides was that of the anti-DNA antibody. Such antibodies, in sera of patients, were demonstrated by precipitation, by complement fixation, and by hemagglutination techniques (Seligmann, 1957 Ceppelini et al., 1957 Seligmann and Milgrom, 1957 Robbins et al., 1957). 

We have seen in various sections of this article that anti-RNA antibodies induced by immunization with ribosomes and the antibodies to pol3uibo-nucleotide complexes — MBSA have made possible a more direct approach to the study of the conformation and structure of synthetic polynucleotides and both viral and non-viral natural ribonucleic acids. 

Subtract the mean background values from the mean values for wells conudning polynucleotide-spermidine complex. In the example given, subtract the mean obtained in Step 15 from that in Step 14. The result is the mean optical density owing to the binding of monoclonal anti-Z-DNA antibody to the polynucleotide. 

In order to define the specificity of the antibodies and to attempt to identify the antigenic determinants (or groups of antigenic determinants) Nahon-Merlin et al. (1973 a) studied the cross-reactions of anti-poly I poly C antibodies with different polynucleotides and polynucleotide complexes by direct precipitation and by specific absorption of antibodies. The rabbits respond to immunization with poly I poly C — MBSA, with a production of antibodies which varies according to the rabbit the sera contain from 700 to 1000 [i.g/ml of antibodies. 

Analysis of the specific absorption of anti-poly I poly C antibodies by double-stranded polynucleotide complexes reinforces both the idea of specificity of these antibodies for double-stranded structures and also the immunochemical differences between the three double-helical complexes. Thus poly rA poly rU, poly dG poly dC and poly rG poly rC absorb, respectively, 81.5, 73.3 Sind 57.7% of the antibodies in the homologous system. In order to inhibit 50 % of the reaction with the homologous antigen, 5-7 [xg of poly A poly U, 12.5 xg poly dG poly dC, and 85 [xg of poly rG poly rC... 

The antisera thus precipitate a polynucleotide which is one of the components of the immunogen and also a heterologous polynucleotide. The fact that anti-poly I poly C precipitates poly I in single-stranded form suggests the possibility that the sera contain, in addition to antibodies specific for... 

Cross-reaction of the antibodies with poly G is reminiscent of that previously observed with anti-poly I — MESA antibodies (Lacour et al., I968), and also of the cross-reaction between anti-inosine — ESA antibodies with guanosine reported by Inouye et al., (1971). Whatever the interpretation of these reactions with poly I and poly G, these two polynucleotides, which can both exist in multistranded hydrogen-bonded homopolynucleotide structures, precipitate only a small proportion of the population of antibodies induced... 

The anti-poly I poly C antibodies react not only with RNA of reovirus but also, though to a lesser degree, with RNA extracted from mammalian cells (Fig. 4). Comparison of the efficiency of inhibition of the cross-reaction with mammahan RNA by RNA of reovirus, by double-helical complexes of synthetic polynucleotides and by single-stranded polynucleotides, has shown that the antibodies anti-poly I poly C react especially with double-stranded conformational determinants of the RNA. None of the single-stranded polynucleotides is capable of totally inhibiting the reaction with RNA whatever... 

Differences in the capacity of inhibition by polynucleotides not involved in complementary hydrogen bonds and by double-helical complexes of synthetic polyribonucleotides, or double-stranded viral RNA allow the conclusion that it is above all the regions of associated base pairs which are recognized in the RNA by anti-poly I poly C antibodies. Such complementary double-stranded helical regions have been described especially in tRNA but they have also been shown to exist in ribosomal RNA. These two kinds of RNA were therefore isolated and studied separately. Although both fractions precipitate anti-poly I poly C antibodies, their reactivity is nevertheless very different and rRNA precipitates eight times as much antibody as tRNA. Since tRNA possesses an important tertiary structure, this low reactivity could be explained by the non-accessibility of antigenic sites. 

The specificity of antibodies induced in mice by poly I poly C MBS A, or by poly I poly C in emulsion with complete Freund s adjuvant, or in the hamster by poly I poly C — MBS A, has been studied less extensively. While in sera to poly I poly C — MBS A of rabbits the presence of anti carrier protein antibodies has been detected easily, this is not true of the sera of mice immunized by the same immunogen, which react essentially with synthetic polynucleotides (Lacour et al., 1971). 

It is essentially the cross-reactions with another double-helical complex, poly A poly U, which have been studied with immune sera of mice and hamsters. These cross-reactions have been observed very frequently with immune sera of RAP mice and B/W mice and to a lesser degree with the sera of hamsters poly A poly U is the best inhibitor of the reaction between anti-poly I poly C of mice and the homologous antigen (Lacour et al., 1971 Steinberg et al., 1971). The role of the bases in this immunoreaction does not appear to be essential. It is probable that, as in rabbit, these antibodies recognize double-helical structures. While there is similarity in the reactions of the sera of the three species with synthetic polynucleotide double-helical complexes, the cross-reactions of the anti-poly I poly C antibodies with nucleic acids are very different in the rabbit, the mouse, and the hamster (Table 4). 

Cross-reactions with poly I poly C have not always been observed. Thus the antibodies induced by Koffler et al. (1971) with poly A-poly U — MBSA in rabbits of unstated origin, reacted only with the homologous antigen. Other differences of reactivity, in particular with heterologous polynucleotides, existing among anti-poly A poly U antibodies obtained in rabbits by different groups of workers will be mentioned and discussed later. 

Anti-poly A poly U antibodies also react with natural polynucleotides, including reovirus RNA, tRNA and ribosomal RNA from mammalian cells. Inhibition studies have shown that the poly A poly U absorbs all the antibodies reacting with RNA from mouse ascites cells. 

Anti-poly G poly C antibodies have been demonstrated by immunodiffusion and by complement fixation in the sera of rabbits immunized with poly G-poly C — MBSA (Michelson et al., 1971 Nahon-Merlin et al., 1971). The anti-poly G poly C antibodies react not only with poly G poly C but also with a large number of double-helical complexes such as poly A poly U, poly I poly C and poly dG poly dC. It is nevertheless to be noted that the complex poly A poly I in which two purine polynucleotides are involved is not precipitated by these antibodies, which is readily explained by the special stereochemical structure of this complex. The anti-poly G poly C antisera nevertheless precipitate poly iso A poly I, but in this case it can be considered that displacement of the glycosyl-hnkage from N to in polyisoadenylic acid converts this polynucleotide into an analogue of poly C. The complex is thus effectively between a poly purine ribonucleotide and a poly pyrimidine ribonucleotide as in the case of other complexes such as poly A poly U and poly I poly C. Immune sera against poly G poly C also react with the triple-stranded complex 2 poly G poly C. In addition they can precipitate one or the other of the component polynucleotides of the homologous complex. 

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... 

Immune sera against poly A poly U, anti-poly I poly C and anti-poly G were used as controls. Anti-poly G does not precipitate any of the RNAs tested, whereas antibodies to the two complementary complexes precipitate all the RNAs, regardless of origin or fraction (Table 7 to 9). Thus antipoly G poly C antibodies show a specificity for ribosomal RNA from animal cells, and for certain viral RNAs furthermore they distinguish between rRNA and tRNA. In contrast, antibodies obtained by immunization with other polynucleotide complexes such as poly A poly U and poly I poly C show no specificity for any RNA whatever its type or origin, with the exception however of double-stranded viral RNA (Stollar, 1970). 

The antibodies produced against double-helical complexes of polynucleotides have also been used to demonstrate the presence of double-stranded RNA in cells. The presence and the localization of double-stranded reovirus RNA has been revealed in cells infected by this virus using anti-poly I poly C — MBSA antibodies marked with fluorescein isocyanate (Silverstein and ScHUR, 1970). 

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