Abstract: The present invention makes possible the catalytic production of sequence specific oligonucleotides through the use of a specially designed template sequence. The reaction can be made to proceed isothermally in the presence of an excess of nucleoside triphosphates, an agent for polymerization, and a cutting agent. Because the process is catalytic with respect to the template sequence, an unlimited amount of oligonucleotide product can theoretically be generated from a single molecule of template. Where the process is initiated by the presence of a nucleic acid target sequence, the method of the present invention can be used for diagnostic purposes as an amplification method to improve sensitivity. Diagnostic sensitivity can be further enhanced by employing a cascade of these template sequences.

Abstract: A method for refolding recombinant platelet-derived growth factor from a high expression host cell system, such as E. coli, is provided in accordance with the present invention. The recombinant platelet-derived growth factor is isolated from inclusion body proteins, after which the free sulfydryl groups of the reduced monomeric recombinant protein are blocked. The blocked recombinant protein can then be brought into a biologically active conformation, without interference from the formation of incorrect disulfide bonds. Once in a biologically active conformation, the recombinant platelet-derived growth factor may be unblocked to allow the desired disulfide bonds to form, thus locking the recombinant protein into a biologically active conformation. The present invention also provides a novel mixed disulfide intermediate wherein the free sulfhydryl groups of reduced recombinant platelet-derived growth factor are derivatized to a disulfide blocking agent.

Abstract: The present invention provides an efficient and economical method for reducing carryover contamination in an amplification procedure. The method of the present invention enables background caused by contaminant amplification product to be reduced or eliminated through the incorporation of at least one modification into the amplification product. The modified amplification product is readily distinguishable from the target sequence in a test sample. Prior to amplifying the target in a new test sample, the sample may be treated to selectively cleave the contaminant amplification product so that it cannot be amplified in the new sample.

Abstract: Novel platelet-derived growth factor (PDGF) analogs are provided in accordance with the present invention. Also provided is a method for the production of homogeneous quantities of these novel analogs. The novel analogs of the present invention, when refolded, have substantially the same biological activity as naturally occurring PDGF B.sub.109. The method of the present invention employs the use of a stop codon on the c-sis gene, or other coding sequence for a precursor protein of PDGF B.sub.109, or analogs thereof, at a position corresponding to a location from about amino acid 111 to about amino acid 160. The method of the present invention results in the production of relatively large homogeneous quantities of recombinant PDGF B analogs from high expression host cells, such as E. coli.

Abstract: Monoclonal antibodies specific for epitopes found on the B chain of PDGF (including v-sis, c-sis and platelet-derived forms) may be bound to columns and used for purification of rPDGF B. A solution containing a polypeptide possessing at least part of the structural conformation of rPDGF B is passed over such a column and the rPDGF B is bound to the antibody. The rPDGF B may then be eluted from the column to yield rPDGF B of greater than 95% purity as determined by SDS-PAGE.

Abstract: A novel method for refolding reduced IGF-I is provided in accordance with the present invention. The method is carried out by providing additional positive charge at the amino terminal end of the IGF-I molecule by the presence of a short leader sequence. The additional positive charge on the met-end of the IGF-I molecule has been found to enable recombinant IGF-I to refold simply by stirring solubilized inclusion body protein for between 2-16 hours, or overnight. The present invention also provides a novel fusion protein intermediate of IGF-I attached to the positively charged leader sequence.

Abstract: Novel platelet-derived growth factor (PDGF) analogs are provided in accordance with the present invention. Also provided is a method for the production of homogeneous quantities of these novel analogs. The novel analogs of the present invention, when refolded, have substantially the same biological activity as naturally occurring PDGF B.sub.109. The method of the present invention employs the use of a stop codon on the c-sis gene, or other coding sequence for a precursor protein of PDGF B.sub.109, or analogs thereof, at a position corresponding to a location from about amino acid 111 to about amino acid 160. The method of the present invention results in the production of relatively large homogeneous quantities of recombinant PDGF B analogs from high expression host cells, such as E. coli.

Abstract: A novel agarose gel is provided for use in the high resolution electrophoretic (HRE) separation of serum proteins. The agarose gel employs a novel gel buffer which contains either hippurate, glycine, or a mixture thereof, in combination with barbital and Tris. The agarose gel of the present invention yields improved electrophoretic separation of the serum proteins in a sample whether the same gel buffer is also used as the running buffer or whether a standard barbital running buffer is used.

Abstract: A novel competitive assay for theophylline wherein caffeine-like (7-substituted) labeled conjugates are used to detect the presence and/or amount of theophylline present in a test sample. The use of such conjugates in a competitive assay for theophylline results in improved sensitivity of the assay method. Where the assay method is a nephelometric or turbidimetric inhibition immunoassay procedure, the assay was found to be less temperature dependent than prior art immunoassays.

Abstract: A method for obtaining an actual linear standard curve in a sandwich type of immunoassay where a first antibody bound to an insoluble support and a second unbound labelled antibody complex with the antigen contained in a test sample to form an insoluble antibody:antigen:labelled antibody complex which is then detected. Unbound unlabelled first antibody and/or unbound unlabelled second antibody are added to the reaction mixture to divert excess antigen away from the desired end-product complex, thus rendering the antigen of interest the rate-limiting factor in the overall immunoreaction. This results in a pseudo first-order reaction which produces an actual linear standard curve.

Abstract: An electrophoretic gel which, when used in an electrophoretic process for the separating chylomicrons, beta lipoproteins, pre-beta lipoproteins, and alpha lipoproteins, enables the separation of the alpha lipoprotein band from the faster moving free fatty acid albumin band. The electrophoretic gel comprises a matrix selected from the group consisting of polysaccharides and derivatives thereof, a buffer having a buffering capacity in an alkaline pH range, and an effective amount of a poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymer.An improved electrophoretic technique characterized in that the above electrophoretic gel is employed therein.

Abstract: A method comprising (a) applying a sample to at least two application areas on an electrophoretic gel; (b) electrophoresing the gel; (c) aligning a template onto the electrophoresed gel, the template having a template slot corresponding to each electrophoresed area; (d) applying a composition capable of fixing proteins in situ to at least one template slot and applying an antisera capable of reacting with one protein to at least one of the remaining template slots; (e) incubating the resultant product of step (d); (f) removing the template from the incubated, electrophoresed gel; (g) washing the incubated electrophoresed gel of step (f); (h) drying the washed gel of step (g); (i) staining the dryed gel of step (h); (j) destaining the stained gel of step (i); (k) drying the destained gel of step (j); and (l) analyzing the dryed gel of step (k).

Abstract: An improved composition of the type comprising a known concentration of at least one enzyme. The composition is characterized in that substantially all of at least one of the enzymes is combined with a stabilizing amount of a reversible inhibitor thereof to form an inhibitor-enzyme complex, thereby stabilizing the enzyme moiety of the complex.A method of stabilizing a composition comprising a known concentration of at least one enzyme. The method comprising combining substantially all of at least one of the enzymes with a stabilizing amount of a reversible inhibitor thereof to form an inhibitor-enzyme complex, thereby stabilizing the enzyme moiety of the complex.