This invention relates to a method of producing stable immunoassay reagents. More particularly, it relates to a test strip upon which is deposited stabilized enzyme antibody conjugates. Enzyme-based diagnostic assays are used to test for the presence of antigens or antibodies in human body fluids. Many methods are used including the use of liquid reagents, radioimmunoassay methods, the use of dry test strips, and the use of coated test slides. U.S. Pat. No. 3,876,504 to Koffler teaches the use of a glass or plastic slide coated with a gelatin and a porous surface forming material to which is bonded either component of the antibody-antigen reaction of interest in an insolubilized form. The slide is air dried and desiccated. The other component, either antigen or antibody, is conjugated with an enzyme, and this conjugate is mixed with the body fluid to be applied to the slide. A coloring agent activatible by the enzyme is finally applied to the test slide in order to provide an indication of antibody-antigen reaction.
The reagents and indicators for immunoassay are fragile and often deteriorate in normal storage operation conditions. Several methods have been attempted to achieve a more stabilized reagent. U.S. Pat. No. 3,860,484 to O'Malley discloses a process of stabilization of unconjugated enzymes by contacting them with synthetic polymers and copolymers. The resulting solution is then freeze dried to form a dry product.
U.S. Pat. No. 4,806,343 to Carpenter et al discloses a method for preserving the activity of proteins after freezing by exposing the protein to a carbohydrate and a transition metal ion and then freezing the protein. Useful metal ions discussed include divalent ions of Zn, Cu, Cd, Ni, and Co.
U.S. Pat. No. 4,563,425 to Yoshioka et al discloses a method to inhibit enzyme deactivation when the enzyme is contacted with a glucose based substrate solution. This method involves the addition of carrier bound metalions to the substrate solution before termination of the enzyme substrate reaction. The metal ion may be one or more of Ti, V, Cr, Mn, Fe, Co, Cu, Sb, Ce, or Ag. The metal salt may be an acid salt or a complex salt, such as a halogen salt. The carrier may be, for example, a polysaccharide, polyamide, glass, or ion exchanger.
U.S. Pat. No. 4,024,000 to Shibata et al discloses a method for the stabilization of an aqueous solution of unconjugated beta-amylase enzyme during purification by adding a divalent or trivalent metal ion to the solution. The resulting solution is then purified by membrane separation at 45.degree.-55.degree. C. After separation, the concentrated enzyme solution may be salted out by addition of sodium chloride, in order to precipitate a beta-amylase fraction, which may then be dried.
U.S. Pat. Nos. 4,331,761; 4,169,012; and 4,228,240 to Dawson et al disclose a method for stabilizing aqueous peroxidase containing compositions by the addition of polyvalent metal ions to the compositions. Such ions may include Mg, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, and Al. The metal salts may be sulfates, phosphates, halides, or nitrates. The products of the various Dawson et al patents may be either dried or in aqueous solutions. The dried products are disclosed to be dried from aqueous compositions which then may or may not be lyophilized. However, the compositions are usually first lyophilized.
U.S. Pat. No. 4,757,016 to Klenner et al discloses a process for stabilization of a peroxidase enzyme which may be conjugated to an antibody. This is done by incubation of the enzyme with aminopyrine for one hour at 20.degree.-25.degree. C.
U.S. Pat. No. 4,233,405 to Neubeck discloses a spray drying process for preparation of unconjugated enzyme products. Neubeck discloses the concentration of a liquid enzyme solution by ultrafiltration and the addition of water-insoluble salts to the concentrate prior to the spray drying.
Finally, U.S. Pat. No. 4,446,232 to Liotta discloses dry, layered test strips suitable for use in enzyme-linked immunoabsorbant assays (ELISA), having a layer of porous material such as nitrocellulose or DBM paper within which soluble enzyme-linked antibodies are dispersed. However, the only method disclosed by Liotta for application of the enzyme-linked antibodies to the test strip is lyophilization.
Therefore, what is needed is a high temperature stable enzyme-antibody conjugate. What is also needed is a continuous method to produce storage stable dry, test strip layers suitable for use in ELISA.
USPTO Backgrounds
USPTO Backgrounds