Immunocytochemical specificity for peptides with special reference to arginine-vasopressin and oxytocin.

Although specificity is generally given as the main characteristic for immunocytolchemical staining (ICC), it becomes more and more clear that nonspecific staining is a major problem in these techniques. The most used criterion for specificity is the absorption test in which the antmserum, witholut any further characterization, is absorbed to) the homololgous antigen I e.g. , ref. 8 1. The limitation (If the ahsolrptmon test can he illustrated very well on the hypothalamoneurohypolphyseal system (HNS) of the rat, in which argmnmnevasopressin )AVP) and oxytolcin (OXT) are synthesized, the more since a mutant is available, the Brattlehoro rat, which is deficient in AVP I 1 1 1. Although anti-AVP plasma could he absorbed totally with AVP, this antibody stained the HNS of the Brattlehoro rat. This staining appeared to) he caused by cross reaction with the structurally closely relatedl OXT. Thus, it appeared that the absorption test can only he used for the elemolnstratmon (If method .opeciJthti (viz. the absence (If staining disturbances caused by mechanisms other than the interaction between antibodies and the antigen to) he localized I. Serum eii.hcit (viz. primary antiboldies must react only with that compound to) which the were raised and not with other compoundls can therefore miolt be proven in this way. The absorption test does not eliminate the’ possibility that antibodies may he able to) react with structurally related compolunds. It even does not exclude binding to) apparently unrelated compounds (i.e., according to) their primary amino) acid sequence (5, 16)1 (for details on method and serum specificity see ref. I “ I I Figure 1 1. Therefore it is necessary to) quantify aspects (If the antigenantmholely reaction. The amount (If cross reaction in ICC can he quantmheel using solid phase model systems I I 1 1. If cross reactmoln with a peptmele occurs, the same solid phase-peptide complex can he used to purify the antmboldv. The basic problem with this procedure is, that it is impossible to) know a prmorm which compounds, present in the tissue, might bind with the antiholelies. Therehlre, it is necessary to characterize the different antigens in the tissue in an alternate way, e.g., by the conventmolnal protein characterization methods (see below) and to) study (e.g., in model systems) the affinity (If the antibody population for these antigens. A number of other criteria should also) he fulfilled (cf. ref. 9); i.e., 1) identical incubation procedure in the model system and the tissue section, 21 the condition of the test antigen in the model system and tissue antigen should he the same. At this time, these requirements cannot all be met in one specificity test. For large antigens (i.e., contractile proteins ) S DS-polyacrylamiele gel electropholresis was successfully applied, followed by longitudinal sectioning of the gel, treatment with fixative, and immunocytochemically staining I 181. For neuropeptides (e.g., AVP, OXT, arginine-vasotocin (AVT)), this kind of separation was not suitable because (If minimal differences in molecular size. Isoelectric focusing seems a promising method to characterize the peptides 1 1). However, since AVP and AVT appeared to have the same isoelectric point, this technique is not yet the solution to the problem (Van der Sluis, unpublished). When the immunocytochemically reacting compounds in the tissue are known, the unwanted antibodies can be removed. If additioln of the antigen does not result in precipitation, solid phase absorption should he used. The result of the purification can be tested again in the above-mentioned model system and on tissue (see below). Remolval (If cross-reacting antibodies is not always possible. The three different antibodies raised against AVT, in our institute, appeared to) show a colnsiderable cross reaction with the closely related helrmone AVP (Swaah, unpublished). Solid phase absorption to) AVP resulted, however, in removal of all the antibodies against AVT. Thus, no specific ICC localization of AVT appeared to) he possible in the presence (If AVP. The final step in proving serum specificity is the applicatioln of purified antisera to the tissue. According to Pool et al. (9), the antiserum must, ideally, give negative staining in the same tissue lacking the antigen. In this respect the homozygous Brattleboro rat, is an ideal mutant to) tC5t the serum specificity of anti-AVP. The parvocellular suprachiasmatic nucleus, which is known to produce only AVP (e.g., refs. 1 1 and I”), might he used to control the cross reactivity (If anti-OXT with AVP. The use (If serial sections, alter-