EP0265501A1 - Resine de polyamide et procede de preparation de reactifs pour diagnostiques immunitaires - Google Patents

Resine de polyamide et procede de preparation de reactifs pour diagnostiques immunitaires

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Publication number
EP0265501A1
EP0265501A1 EP87903178A EP87903178A EP0265501A1 EP 0265501 A1 EP0265501 A1 EP 0265501A1 EP 87903178 A EP87903178 A EP 87903178A EP 87903178 A EP87903178 A EP 87903178A EP 0265501 A1 EP0265501 A1 EP 0265501A1
Authority
EP
European Patent Office
Prior art keywords
polyamide resin
resin
protide
conjugate
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP87903178A
Other languages
German (de)
English (en)
Other versions
EP0265501A4 (fr
Inventor
Patrick Kanda
Ronald C. Kennedy
James T. Sparrow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baylor College of Medicine
Texas Biomedical Research Institute
Original Assignee
Baylor College of Medicine
Texas Biomedical Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baylor College of Medicine, Texas Biomedical Research Institute filed Critical Baylor College of Medicine
Publication of EP0265501A1 publication Critical patent/EP0265501A1/fr
Publication of EP0265501A4 publication Critical patent/EP0265501A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide

Definitions

  • the present invention relates to the synthesis and use of synthetic peptides and proteins to induce an immune response in experimental animals. More particularly, the present invention relates to a polyamide resin, a method of making that polyamide resin, a method of inducing an immune response in an experimental animal using a conjugate of a peptide or protein synthesized on that resin, and the use of the resin for immunodiagnostic purposes.
  • Solid phase peptide synthesis is a valuable tool for investigating the structure and mechanism of action of proteins.
  • Most such synthetic methods involve the use of a cross-linked polystyrene based resin as the solid phase to which the peptide is anchored during assembly, usually through a linker molecule. Assembly is accomplished by a repetitive cycle of adding a protected amino acid to the solid phase, selectively removing (deprotecting) a protective group on that amino acid, and adding additional suitably protected amino acids (for a review, see Merrifield, R.B., "Solid-phase Peptide Synthesis", 32 Adv. Enzymology 221 (1969)).
  • polystyrene based resins are most commonly used as supports in solid phase peptide synthesis, their relatively hydrophobia character in comparison to the polar organic media required to solubillze reaetants can be problematic in peptide chain assembly. Such media may freely solvate the growing peptide, yet incompletely swell the polystyrene matrix. Within the polymer lattice, impaired diffusion of reagents and steric hindrance can contribute to lowered efficiency during coupling cycles, which, on a repeated basis, lowers final yields appreciably. During the early stages of assembly, when the resin to peptide mass ratio is high and the physical properties of the support dominate, this lowered efficiency is particularly acute.
  • the polyamide resin as the amino methyl derivative, can accommodate synthetic schemes incorporating alternate protection strategies through selection of the appropriate linker molecule, which links the C-terminal residue to the support.
  • the peptide or protein thus synthesized which will be referred to throughout the present disclosure as a "protide”, can be used in a number of investigative applications.
  • protide As an immunogen. It has previously been demonstrated that synthetic peptides analogous to sequences contained in viral encoded proteins have proven useful for identification of native antigen determinants associated with such proteins. Several laboratories have reported studies on the antigenic activity of various HBsAg synthetic peptides. Dreesman, G.R., et al., 295 Nature 158 (1982); Lerner, R.A., et al. 78 Proc. Natl. Acad. Sci. USA 3403 (1981); Prince, A.M., et al., 79 Proc. Natl. Acad. Sci. USA 579 (1982).
  • an object of the present invention to provide a method of preparing a polyamide resin upon which a protide can be synthesized using solid phase synthetic methods which can be injected into an experimental animal to induce an immunogenic response without separation of the protide from the resin.
  • Another object of the present invention is to provide a method of preparing a polyamide resin for solid phase protide synthesis comprising cross-linking a dimethylacrylamide monomer with a molecule containing a functional group by free radical co-polymerization in an aqueous solution which has been emulsified by a detergent when added to an organic solvent medium.
  • Another object of the present invention is to provide a method of inducing an immunogenic response in an experimental animal with a synthetic peptide or protein comprising preparing a polyamide resin, synthesizing a peptide or protein on that polyamide resin, and immunizing an experimental animal with the polyamide resin-synthetic peptide or synthetic protein conjugate.
  • Another object of the present invention is to provide an assay for detection of proteins such as antigens and antibodies using the polyamide resin-protide conjugate of the present invention.
  • Another object of the present invention is to provide a method of inducing an immunogenic response in an experimental animal using the polyamide resin-protide conjugate of the present invention.
  • Another object of the present invention is to provide a polyamide resin for solid phase protide synthesis which does not require the separation of the protide from the resin and the subsequent purification of the protide before the use of that protide to, for instance, induce an immunogenic response.
  • Another object of the present invention is to provide a polyamide resin for solid phase protide synthesis which is particularly useful in the mapping of the antigenic determinants of a protein as a result of the elimination of the steps of separation of the protide from the resin and the subsequent purification of the protide before the use of the protide in a binding assay.
  • a method of preparing a polyamide resin for solid phase protide synthesis comprising cross-linking a dimethylacrylamide monomer with a molecule containing a functional group in .a free radical reaction with an initiator and a promotor.
  • the beaded polyamide resin is then polymerized in the emulsion formed when an aqueous solution of those reagents is suspended In an aqueous medium.
  • the beads are then isolated and used as the solid phase for synthesis of a protide.
  • Also provided is a method of inducing an immunogenic response in an experimental animal comprising preparing a polyamide resin, synthesizing a protide on the polyamide resin, and immunizing an experimental animal with the polyamide resin-protide conjugate.
  • An in vitro diagnostic assay comprises preparing a polyamide resin, synthesizing a protide on the resin to form a polyamide resin-protide conjugate, and contacting the polyamide resin-protide conjugate with a body fluid suspected of containing antibodies capable of binding specifically to the protide.
  • the bound antibodies are then detected using, for instance, an enzyme linked immunosorbent assay.
  • FIG. 1 is a graph of optical density at 410 nm as a function of the reciprocal dilution of rabbit antisera to the polyamide resin-HTLV-III peptide 503-532 conjugate obtained by enzyme linked immunosorbent assay.
  • the circles represent data free rabbits immunized with that conjugate, the triangles represent data from those same rabbits before immunization.
  • protide refers to both the peptides and proteins which are synthesized according to the method of the present invention.
  • a significant advantage of the method of the present invention is that the protide synthesized on the polyamide resin can be used to induce an immunogenic response in a mammal without being separated from the resin and purified.
  • the usual method of coupling the protide to polystyrene based resins is through a benzyl ester derivative, and separation of the protide from the resin is usually accomplished by either acidic or basic cleavage.
  • Benzyl esters are susceptible to several such methods of cleavage, but are also stable throughout the multiple deprotection, neutralization and coupling reactions which are characteristic of solid phase synthetic methods.
  • Hydrazine has also been used to separate the protide from the resin (Kessler, W. and B. Iselin, 49 Helv. Chim. Acta 1330 (1966)) as have various ammonolytic (Manning, M., 90 J.Am.Chem.Soc. 1348 (1968)) and other methods.
  • the polyamide resin of the present invention is prepared by cross-linking a commercially available dimethylacrylamide monomer
  • the cross-linker is either N,N'-bisacrylyl- 1,3-di.aminopropane or N,N'-bisacrylyl-1,3-diaminobutane prepared according to the method of Halpern and Sparrow (J.A. Halpern and
  • a functional monomer is included in the cross-linked resin of the present invention.
  • the term "functional monomer” refers to those alkenyl amines which are used to anchor the C-terminal amino acid of a synthetic protide to the resin.
  • the functional monomer when protected with the methylsulfonylethyloxycarbonyl
  • MSC alkenyl amine 7 Int. J. Peptide Protein Res. 295 (1975)
  • Those functional monomers are prepared by reaction of the commercially available chloride derivative with the alkenylamine, and the MSC protective group is subsequently removed with base. However, the MSC group is not required.
  • the polyamide resin of the present invention is also prepared by simply adding an excess of the allylamine, followed by filtering or other method to remove the resulting fines. The amount of functional monomer added is selected to yield a resin substitution of between about 0.1 mmol and about
  • the initiator can be any of the initiators known to those skilled in the art such as a persulfate or riboflavin, and is preferably ammonium persulfate.
  • the aqueous phase refers to an organic phase which, when combined with the aqueous phase and stirred, results in a suspension from which the resin of the present invention is obtained.
  • the orgtnic phase comprises a mixture of hexane and carbon tetrachloride.
  • the emulsifier is added during the stirring to allow for the formation of beads of uniform size.
  • the emulsifier can be any detergent known to those skilled in the art, and In a presently preferred embodiment, is either sorbitan sesquioleate, sorbitan monolaurate or sorbitan monodecanoate.
  • the amount of detergent added is adjusted to give a spherical resin of approximately uniform size. A decrease in the amount of detergent results in an emulsion which yields increased amounts of larger, amorphous material, which could contribute to a reduction to the internal growing chains of amino acids. An increase in the amount of detergent increases the amount of fine material, which is difficult to remove without the loss of significant amounts of the resin. Those fines clog the reaction vessels of the peptide synthesizer as well as the associated lines and valves.
  • a promoter is than added to promote the polymerization of the monomers in the suspension, resulting in the formation of beads of the polyamide resin of the present Invention.
  • a number of promoters are known to those skilled in the art, but particular success in preparing the polyamide resin of the present invention has been obtained with N,N,N',N'-tetramethylethylenediamine (TEMED).
  • TEMED N,N,N',N'-tetramethylethylenediamine
  • the resulting beads are then filtered and washed, the MSC group (if present) is removed with base, and the beads are dried.
  • the beads may then be sifted through a mesh sieve to insure relatively uniform size. Overall yields using the method of the present invention ranged from about 87% to about 94% from starting monomers.
  • the aminomethyl, cross-linked polydimethylacrylamide resin of the present invention provides maximum exposure of the protide in an aqueous solution, and the resin-polymer backbone does not restrict the protide conformationally.
  • the exposure of the protide is the result of the ability of the polyamide resin to swell to many times its dry bed volume when highly solvated by water.
  • linker refers to a linking group which links the carboxyl group of the first amino acid of the protide to the polymeric resin.
  • this linker is an oxyalkyl benzole acid (OBA) to which an amino acid residue is coupled to serve as the first amino acid in the protide chain.
  • OBA linker is used to attach the C-texminal amino acid to the polyamide resin of the present invention, anhydrous hydrogen fluoride can be used to remove the side chain protecting groups from the protide without significant loss of the protide from the resin.
  • the amino acid of choice is glycine, which is protected with the t-butyloxyearbonyl (t-Boc) protecting group, but it will be understood by those skilled in the art who have the benefit of this disclosure that the amino acid could be any amino acid, particularly, the amino acid which is the first amino acid in the protide to be synthesized, and that other protecting groups are equally suitable.
  • the glycine residue serves the additional function of a spacer between the protide and the resin-polymer backbone.
  • the Boc-glycyl-4-(oxymethyl) benzole acid which is the presently preferred linker was prepared by a modification of the method described by Mitchell, et al. (Mitchell, A.R., S.B.H. Kent, M. Engelhard and R.B. Merrlfleld, A new synthetic route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl) phenylacetamidomethyl-resin, an improved support of solid-phase peptide synthesis, 43 J. Org. Chem. 2845 (1978), which is incorporated herein in its totality by this specific reference thereto.
  • An important modification of the Mitchell, et al., method is the elimination of the use of dimethylformamide as a solvent.
  • the activator used to couple the linker to the polyamide resin prepared as described above is diisopropyl carbodiimide and 4-dimethylaminopyridine, but it will be understood by those skilled in the art that other activators such as dicyclohexylcarbodiimide and 4-methylpyrrolindinopyridine are equally suitable for such a purpose.
  • the polyamide resin-protide conjugate is used for a number of purposes, including in vitro assays, inducing an immunogenic response in experimental animals, or mapping antigenic determinants.
  • an in vitro assay is conducted by crushing the beaded polyamide resin-protide conjugate with a mortar and a pestle and absorbing the crushed conjugate onto a solid phase such as a microtiter test plate with neutral pH buffer.
  • Serum or other body fluid suspected of containing an antibody capable of specifically binding the protein or peptide on the resin is then incubated with the absorbed conjugate, unbound antibodies are removed by washing, and the bound antibodies are detected by enzyme linked immunosorbent assay, biotin-avidin amplified assay or other detection methods such as are known in the art.
  • the polyamide resin-protide conjugate can also be used to map antigenic determinants by simply removing a portion of the polyamide resin-protide conjugate at intervals during the synthesis of the protide, deprotecting the protide, and testing each removed portion in serial fashion to determine that point in the synthesis at which the protide binds antibody. This method is made possible by the elimination of the separation and purification steps required in other synthetic methods.
  • the conjugate can also be tested for its ability to bind antibody by crushing and absorbing to a solid support such as a microtiter test plate and assayed as described above. Separation of the protide from the resin and purification of the protide is not required for such an assay.
  • the polyamide resin-protide conjugate is also useful as an immunogen.
  • the conjugate is used directly for Immunization of experimental animals with or without an adjuvant.
  • the experimental animals of primary interest are mammals, but an immunogenic response can be induced in other experimental animals such as birds using the method of the present invention.
  • an immune response specific for hepatitis B was induced by immunization of rabbits using a conjugate comprised of a synthetic peptide with the same sequence as the hepatitis B antigen (HBsAG) peptide 119-159 emulsified in Freund's complete adjuvant.
  • HBsAG hepatitis B antigen
  • N,N'-bisacrylyl-1,3-diaminopropane was prepared according to the method set out in Helpern and Sparrow, supra. Briefly, diaminopropane (Aldrich) was dissolved in acetonitrile and added dropwise to an acrylyl chloride-acetonitrile solution at 4oC, allowed to warm to room temperature and stirred. The diaiminopropane dihydrochloride was removed by filtration, washed with warm acetonitrile, and the combined filtrates were concentrated in vacuo. N,N'-bisacrylyl-1,3-diaminopropane was crystallized at 4oC overnight and the resulting plates filtered and dried in vacuo.
  • the degree of functionalization was checked by coupling Bocalanine to 100 mg of the resin using diisopropylcarbodiimide as activator and 4-dimethylaminopyridine (recrystallized from ethyl acetate) as catalyst.
  • Amino acid analysis showed a substitution of 0.15 to 0.35 mmol/g resin, depending on the lot, and resins were prepared with as little as about 0.1 and as much as about 0.5 mmol/g resin depending upon the amount of allylamine added.
  • the loaded resin gave no detectable staining with picryl-sulfonic acid, indicating the absence of unreached free amine.
  • the beads When swollen in methylene chloride, the beads occupied about 2.5 times their dry bed volume.
  • dimethylfoxmamide or an aqueous solution the beads occupied approximately four and six times their dry bed volume, respectively.
  • the linker Boc-glycyl-4-(oxymethyl) benzoic acid was prepared by modification of the method of Mitchell, et al., supra. Briefly, the 4-(bromomethyl) benzole acid phenylacylester was prepared by dissolving 10.3 ml redistilled diisopropylethylamine and 12.05 g (60.6 mmol) bromoacetophenone in 450 ml ethyl acetate. 4-(bromomethyl) benzole acid (13.89 g, 60.6 mmol) was added in seven equal portions over a three hour period to the stirred solution at 40-50oC. Stirring was continued for two more hours at room temperature.
  • the 4-(bromomethyl) benzole acid phenylacylester was converted to Boc-glycyl-4-(oxymethyl) benzole acid by dissolving Boe-L-glycine (25 mmol, 4.38g) in 15 ml methanol and titrating to neutrality with tetramethylammonium hydroxide (25% in methanol). Solvent was removed azeotropically with chloroform in vacuo, and the salt dissolved in 150 ml acetonitrile. To the stirred solution was added 5.8 g (17.5 mmol) of the 4-(bromomethyl) benzoie acid phenacyl ester prepared as described.
  • Boc-glycyl-4-(oxymethyl) benzole acid prepared as described in Example IV was coupled to the aminomethyl polyamide resin (1.2 g) prepared as described in Example III on a Biosearch Sam II automated peptide synthesizer using diisopropylcarbodiimide and dimethylaminopyridine as activator in a 1:1 methylene chloride:dimethylformamide solution. Both methylene chloride
  • hepatitis B surface antigen (HBsAg) peptide 119-159 was assembled on the aminomethyl, cross-linked polydimethylacrylamide resin prepared as described in Example III, having the Bocglycyl-4-(oxymethyl) benzole acid linker prepared as described in
  • Example IV attached thereto using the method described in Example
  • the peptide included the following substitutions to control the specific formation of disulfide loops: serine for cysteines 121, 138, and 149.
  • the cysteines 139 and 147 sulfhydryls were blocked by the 4-methoxybenzyl group, while the sulfhydryls of cysteines at 124 and 137 were protected as the S-acetamidomethyl derivatives.
  • ⁇ -N-tBoc protected amino acids were purchased from Baches. Additional side chain protecting groups were as follows: formyl group for the indole nitrogen of tryptophan; benzylethers for threonine and serine hydroxyls; acetamidomethyl or
  • DIEA Diisopropylethylamine
  • Halocarbon Trifluoroacetic acid
  • Human serum can be assayed for the presence of antibody specific for the HBsAg peptide 119-159 by the following in vitro assay.
  • a quantity ot the HBsAg peptide 119-159-polyamide resin prepared as described in Example VI is crushed with a mortar and pestle. A microscope may be used to verify that the polyamide resin-peptide conjugate has been crushed.
  • Approximately 100 ⁇ l of a solution containing between about 200 nanograms and about 10 micrograms of the crushed polyamide resin-peptide conjugate in a neutral pH buffer such as phosphate buffered saline (PBS) is absorbed to a solid phase such as Dynatech Immunolon II Mierofilter test plate. Nonspecific binding sites are blocked with 10% normal goat serum (NGtS) and the plate is washed with Tween 20 PBS (T-PBS) to remove unbound antibodies.
  • NtS normal goat serum
  • T-PBS Tween 20 PBS
  • Human sera suspected of containing antibodies specific for HBsAg peptide 119-159 and rabbit antisera produced by immunizing rabbits with the polyamide resin-HBsAg peptide 119-159 conjugate diluted in 10% NGtS is then added to the polyamide resin-peptide-coated plate and incubated for one hour at 37oC, followed by washing with T-PBS.
  • Biotln goat anti-human IgG or biotin goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) is then incubated with the bound human and rabbit sera, respectively, for one hour at 37oC.
  • Av-HRP horseradish peroxidase
  • T-PBS horseradish peroxidase
  • peroxidase activity is determined using a 1 mM solution of 1,2'-azino-di(3-ethyl-benzthiazoline sulfonie acid) (Sigma Chemical Co.) and 0.03% H 2 O 2 as substrate.
  • the reaction is stopped with 5% (w/v) sodium dodecyl sulfate in water prior to quantitatlng spectrophotometrically at 410 nm using a Dynatech plate reader.
  • Optimal dilutions of each reagent are selected by titration. All reagents for determining specific binding except the substrate are diluted in 10% NGtS. EXAMPLE VIII IN VITRO ASSAY FOR PRESENCE OF HBsAg ANTIBODY
  • Human serum was assayed for the presence of antibody to hepatitis B surface antigen by the following in vitro assay.
  • the polyamide resin-peptide conjugate prepared as described in Example VI was used to induce an immunogenic response in rabbits as follows. New Zealand white female rabbits were immunized with three monthly intramuscular injections of either 200 ⁇ g HBsAg peptide 119-159 (as the peptide-resin conjugate) or only glycyl-resin emulsified in Freund's complete adjuvant (range of immunogen, 50 ⁇ g to 1 mg for rabbits). Serum was collected after bi-weekly bleeding and checked for anti-HBsAg activity using a commercially available radioimmunoassay (RIA) kit (AUSAB, Abbott Laboratories). The recognition of the native HBsAg surface antigen by the anti-peptide 119-159 antibody response induced in the rabbits is demonstrated by the following data developed by that RIA.
  • RIA radioimmunoassay
  • the HTLV-III peptide gp 120 503-532 was assembled on the cross-linked polydimethylacrylamide resin prepared as described in Example III, having the Boc-glycyl-4-(oxymethyl) benzole acid linker prepared as described in Example IV attached thereto using the method as described in Example V, in the same method as described for the synthesis of the HBsAG peptide 119-159 in Example VI, the only difference being the order in which the protected amino acids were added.
  • 503-532 conjugate produced a specific anti-peptide response as determined by an enzyme linked immunosorbent assay conducted according to the method of Example VII (with the use of antisera produced by immunizing the rabbits with the polyamide resin-HTLV-III peptide 503-532 conjugate rather than the conjugate which included the HBsAg peptide 119-159 as used in that Example).
  • the results of that immunoassay are presented graphically in Fig. 1.
  • the data represented by the circles is data from rabbits immunized with that conjugate, the data represented by triangles is from those same rabbits before immunization (the solid circles and triangles are both from one rabbit; the open circles and triangles are both from one rabbit; the open circles and triangles are both from a second rabbit).
  • the ability of the rabbit antisera generated to peptide 503-532 to neutralize HTLV-III infectivity was assessed on the basis of a reduction of reverse transcriptase activity using a tenfold dilution of the HTLV-III stock with a constant amount of antisera. Barre-Sinoussi, F., et al., 220 Science 868 (1983).
  • a single rabbit anti-peptide 503-532 antiserum efficiently reduced HTLV-III replication at day 10 compared to pooled human sera from AIDS patients at tenfold dilutions of virus.
  • a second rabbit antiserum to that peptide failed to reduce HTLV-III replication and so was used as a control throughout the RT assay. No anti-HTLV-III activity was detected in this particular antiserum based on radioimmunoprecipitation even though the rabbit received a similar immunogen and produced a detectable anti-peptide response.
  • the antiserum that neutralizes HTLV-III detected both gp 120 and gp 160 envelope glycoproteins. This rabbit antiserum was found to be less efficient in neutralizing HTLV-III compared to human AIDS serum on day 12 and 15 following HTLV-III infection.

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Abstract

Résine de polyamide servant à la synthèse de peptides et de protéines et procédé de préparation et d'utilisation de ladite résine. On prépare la résine de polyamide en réticulant un monomère de diméthylacrylamide par copolymérisation avec un monomère fonctionnel dans une solution aqueuse en émulsifiant la solution aqueuse dans un solvant organique et en isolant les perles de résine de polyamide formées par addition d'un initiateur et d'un promoteur. Les perles sont utilisées en tant que phase solide pour la synthèse de peptides et de protéines selon les procédés déjà connus. Le conjugat de la résine de polyamide et du peptide ou de la protéine synthétisés est utilisé directement pour des immuno-analyses ou pour des procédés d'immunisation, sans qu'il soit nécessaire de séparer le peptide ou la protéine de la résine et sans qu'il soit nécessaire de recourir ultérieurement à un procédé de purification.
EP19870903178 1986-04-30 1987-04-29 Resine de polyamide et procede de preparation de reactifs pour diagnostiques immunitaires. Withdrawn EP0265501A4 (fr)

Applications Claiming Priority (2)

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US85821686A 1986-04-30 1986-04-30
US858216 1986-04-30

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EP0265501A1 true EP0265501A1 (fr) 1988-05-04
EP0265501A4 EP0265501A4 (fr) 1988-11-09

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EP19870903178 Withdrawn EP0265501A4 (fr) 1986-04-30 1987-04-29 Resine de polyamide et procede de preparation de reactifs pour diagnostiques immunitaires.

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EP (1) EP0265501A4 (fr)
JP (1) JPS63503150A (fr)
AU (1) AU7309487A (fr)
CA (1) CA1339670C (fr)
WO (1) WO1987006594A1 (fr)

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US5126399A (en) * 1986-04-30 1992-06-30 Board Of Regents, The University Of Texas System Methods and compositions for the preparation and use of site-directed immunologic reagents
JP2811336B2 (ja) * 1989-11-06 1998-10-15 株式会社エヌエムビー 新規なセメント分散剤
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AU7309487A (en) 1987-11-24
CA1339670C (fr) 1998-02-10
EP0265501A4 (fr) 1988-11-09
JPS63503150A (ja) 1988-11-17
WO1987006594A1 (fr) 1987-11-05

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