Novel polypeptide-comprising biopolymer systems.

Covalent bioconjugation between anionic polyelecrolytes and polypeptide antigens chemically synthesized by solid-phase chemistry, were studied in hydrated reversed micelle systems. The epitops of Foot-and-Mouse disease virus VP1 protein (40--60 and 135--160 residues) were used as polypeptide antigens. The polypeptide-comprising Biopolymer Systems were obtained by two methods: 1) Inclusion of peptides into electrostatic polyelectrolyte complexes of polycations with proteins, 2) Inclusion of peptides into Cu(+2)-induced polyelectrolyte complexes of polyanions with similarly (anionic) charged proteins. The immunogenetic properties of polymer-peptide conjugates and peptide-comprising Biopolymer Systems were investigated and the specificity of antibodies produced was analyzed. Polymer-Peptide conjugates, as well as peptide-comprising Biopolymer Systems appeared to possess a high peptide specific immunogenecity even without the addition of traditional adjuvants. It was found that PE-BSA.FMD.Ag conjugates conferred effective immunoprotection against Foot-and-Mouth disease virus.

Fluorescence study of Cu(2+)-induced interaction between albumin and anionic polyelectrolytes.

The Cu(2+)-induced complex formation of bovine serum albumin (BSA) with anionic polyelectrolytes (PEs) (polyacrylic acid (PAA), poly(N-isopropylacrylamide) [poly[NIPAAm]], and copolymers of N-isopropylacrylamide (NIPAAm) and acrylic acid) in aqueous solution was studied by a fluorescence technique and high-performance liquid chromatography analysis. The character of the interactions depends on the monomer composition (r = [COOH]/[NIPAAm]), [Cu(2+)]/[PE], and [BSA]/[PE] ratios and solution pH. Two types of ternary polycomplex (polymer + Cu(2+) + BSA) particles are formed depending on the monomer composition r of the copolymer. At r from 1/3 to 1/1, the protein molecules in the structure of ternary polycomplex particles are densely covered by the shell of a polymer coil and practically "fenced off" from the water environment. At r > or = 3/1 ternary polycomplex PAA-Cu(2+)-BSA particles have more friable structures in which protein molecules are practically exposed to the solution. At low polymer concentration, an intrapolymer ternary polycomplex is formed. This complex aggregates to an interpolymer species upon increase in polymer concentration. Fluorescence data indicate that in ternary complex polymer interacts through Cu(2+) ions with BSA preferentially at the site close to the location of "cleft" tryptophan residue. This leads to static quenching of this tryptophan fluorescence. Cu(2+)-induced complex formation is an equilibrium reaction.

Water-soluble covalent conjugates of bovine serum albumin with anionic poly(N-isopropyl-acrylamide) and their immunogenicity.

We have conjugated bovine serum albumin (BSA) to poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAAm-AA)) by using water-soluble carbodiimide, and the effects of the bulk mass ratio of protein to polymer (r) on the formation of polymer-protein conjugates have been studied. HPLC, electrophoresis, viscosimetry and fluorescence spectroscopy suggest that the mode of covalent binding of BSA to poly(NIPAAm-AA) depends upon the weight concentration ratio (r) of BSA to poly(NIPAAm-AA). At r < or = 1, free poly(NIPAAm-AA) molecules coexist with conjugate, and when r reaches 1 the amount of free polymer is too small to be observed. It is shown that depending on the ratio r, two types of conjugate particles were formed: at r < 1, the protein molecules in the structure of conjugate particles are densely covered as a shell by polymer chain and practically "fenced off" from water environment; at r > 1 the conjugate-forming particles possess more friable structures in which protein molecules are practically exposed to the solvent. The complex formation involving electrostatic interactions between BSA and carbodiimide activated polymer are proposed as the driving force for the covalent binding of BSA to polymer macromolecules. The coil-globule transition of macromolecules in low and thermally induced precipitation in more concentrated solutions of bioconjugates was observed. The immunogenic properties of covalent conjugates of CP-BSA were investigated and the temperature-modulated solubility-immunogenicity alterations was analyzed. A single immunization of mice with conjugates at the thermally precipitating concentration without an adjuvant evoked increased specific immune response to BSA, which practically did not depend on the initial conjugation ratio of components. Such a modulated system is attractive for application as a novel immunogenic system in vaccine technology.

Immune response to progesterone immobilized on Cu2+-induced amphifilic polyelectrolyte-protein complex: antigen specificity and affinity of hybridoma clones.

Cu2+-mediated complex formation between copolymers of acrylic acid with N-isopropyl-acyrlamide (CP1) and negatively charged covalent conjugate of bovine serum albumin with progesterone (BSA.P) was studied in neutral water in the presence of Cu2+. It was shown that under conditions where CP and BSA.P are negatively charged and incapable of binding to one another, the divalent Cu2+ act as "fasteners" promoting the formation of relatively stable water-soluble ternary polycomplexes. The immunogenic properties of ternary mixtures BSA.P-Cu2+-CP1 and BSA.P+IFA were investigated and the production of monoclonal antibodies (MAbs) against progesterone hormone was analyzed. Fusion following the two different immunization procedures resulted in the growth of comparable numbers of progesterone-specific MAbs with apparently similar antigen affinities. Thus, immunizations using antigens in BSA.P-Cu2+-CP1 appear to provide an efficient alternative to incomplete Freund's adjuvant.

Immune response to 17beta-estradiol involved in polymer gels: antigen specificity and affinity of hybridoma clones.

The immunogenic properties of 17beta-estradiol, immobilized in negatively charged polymer gels, were investigated, and the specificity of antibodies produced was analyzed. The polymer gels developed were composed of a hydrophobic estradiol core surrounded by hydrophilic polyanions as corona. As an immunogen, it was conceived to function via a dual mode, that is as a hapten-delivery system (prolongation effect) and as a polyelectrolyte adjuvant. Polymer gels containing estradiol appeared to possess a high estradiol-specific immunogenicity even without the addition of traditional adjuvants. A comparative study of estradiol trapped in polymer gels versus estradiol conjugated to bovine serum albumin (BSA.E) + Incomplete Freund's Adjuvant (IFA) mixtures revealed similar immunogenic properties in terms of induction of specific antibodies. Following a short immunization procedure based on the use of 17beta-estradiol immobilized in polymer gels, we developed 10 specific monoclonal antibodies with Kd values ranging between 1.2 X 10(-7) and 8 X 10(-8) M.