Preparation of a semisynthetic antibody

Abstract

The antibody combining site of the mouse myeloma protein 3 15 is contained in the Fv fragment, composed of the variable region of light chain (VL) and heavy chain (VH) [Hochman, J., et al. ( 1 973) Biochemistry 12, 11 301. It was also shown that the binding site for the dinitrophenyl (Dnp) ring is located in VL Biochemistry 16, 3 1541. We now describe the solid phase synthesis of a linear polypeptide of 1 15 amino acid residues with the sequence of VL of protein 31 5. The synthetic product was removed from the resin and the single disulfide bond was formed after reduction in 8 M urea. The synthetic material showed common antigenic determinants with native VL and 40% of it was adsorbed on anti-VL-Sepharose. Fractionation on Dnp-lysylSepharose showed that 24% of the synthetic VL can be adsorbed specifically on this column. Equilibrium dialysis with [3H] Dnp-lysine demonstrated that the affinity-purified syn-A n t i b o d i e s are a family of closely related proteins with diverse specificity. The sequence variability of antibodies of the same class is confined to the N-terminal 110-120 residues (VL or VH)] of the light and heavy chains. The principal variability is localized in three hypervariable regions in each V domain (Wu & Kabat, 1970) which are clustered together to form the antibody combining site (Amzel et al., 1974). The rest of the sequence in the V domains may be regarded as constructing a rigid framework whose three-dimensional structure is very similar in different antibodies, and to which are attached the hypervariable loops forming the specific site (Padlan et al., 1977). It is not yet possible to correlate precisely sequence and specificity, or to predict which combining site will be generated by a particular sequence. Also, it is not always clear what the relationship is between replacements in hypervariable regions and in framework residues. It is conceivable that synthetic homologues of antibodies will enable a systematic step-by-step replacement of amino acids in the V region and will help to elucidate the contribution of each segment in the V domain to the overall structure and to the specificity of the combining site. It is hoped that, if the synthetic methods will be found suitable for the synthesis of V domains, they can also be utilized to prepare synthetic antibodies with known specificity.The experience with the present methods of solid phase synthesis of proteins was predominantly with polypeptides of sizes no longer than 120-1 50 residues, such as RNase (Gutte & Merrifield, 1971), lysozyme (Sharp et al., 1973), or growth hormone (Li & Yamashiro, 1970). Previously we reported the From the Departments of Chemical Immunology (M.G., R.Z., and D.G.) and Biophysics (M.W.), The Weizmann Institute of Science, Rehovot, Israel. Receiced August 2, 1977. Supported in part by a grant from the Israel Commission for basic science. I Abbreviations used: V L and VH, variable portion of light and heavy chain, respectively; V,(s), synthetic VL: Fv, variable fragment of antibody composed of V L and VH; ...

Applications of nonequilibrium Kubo formula to the detection of quantum noise

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Protection of rat liver against hepatic ischemia-reperfusion injury by a novel selenocysteine-containing 7-mer peptide

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“…GPX activity was determined using the method of Wilson ( 14 ) using the supernatant of tissue luminal content. The enzymatic reaction contained the following components: NADPH, reduced GSH and GSH reductase, and was initiated by addition of H 2 O 2 .…”

Stiffnessometer, a Magnetic-Field-Free Superconducting Stiffness Meter and Its Application

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