Luteinizing Hormone-Releasing Hormone: Sequential Versus Conformational Specificity of Antiluteinizing Hormone-Releasing Hormone Sera*

Abstract

The structural requirements for binding of LHRH to 11 antisera were analyzed using 21 analogs and 20 fragments of LHRH and monoiodo-[¹²⁵I]LHRH. Analysis of the binding characteristics of these anti-LHRH sera permitted them to be classified as either conformational or sequential. Conformational anti-LHRH sera (group I) are highly specific antisera which did not recognize any of the numerous fragments of LHRH tested, thus requiring the entire decapeptide for binding in RIA. In contrast, sequential antisera (group II) recognized a well definable portion of the sequence of the LHRH molecule; RRi and IJ₂₉ sera bind the 6 –9 and 7 –10 sequences of LHRH, respectively. In the case of conformational antisera, selective amino acid monosubstitutions of the LHRH molecule resulted in variable loss of binding. Furthermore, the distribution of amino acids essential to the binding site(s) was quite variable from one antiserum to another. These antisera appear to recognize a three-dimensional configuration of the LHRH molecule. For sequential antisera, binding to the LHRH analogs is abolished only in cases of a nonconservative substitution in the specific sequence recognized by the antiserum. Used in RIA for the detection of LHRH in biological fluids, sequential antisera would detect fragments carrying the binding sequence defined in each case. A subgroup of sequential anti-LHRH sera (group III) represents sera recognizing several fragments of LHRH without precise location of the binding site; they are most likely composed of two or more populations of antibodies of the sequential type. These antisera, like those of group II, could detect LHRH fragments other than native LHRH as well as native LHRH fragments in biological fluids. In summary, these results reemphasize the necessity of characterizing antiserum specificity before use in RIA. They also provide some empiric evidence for the existence of a preferred molecular conformation of LHRH.