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Last Updated: March 28, 2024

Claims for Patent: 4,351,824


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Summary for Patent: 4,351,824
Title: Polystyrene latex reagents, methods of preparation, and use in immunological procedures
Abstract:Methods of making and using a stable diagnostic reagent for use in serological testing procedures, and in particular, non-auto-agglutinating polystyrene latex particles with surfaces containing a protein, substantially methylated serum albumin, and a bound, negatively-charged polymeric compound. The particles may be sensitized with a number of negatively-charged compounds, including proteins, polysaccharides and various cell nuclear components. When the coated latex particles are adsorbed with native deoxyribonucleic acid, the resultant immunological reagent can be used to detect certain autoimmune diseases, particularly serum lupus erythematosus.
Inventor(s): Lehrer; Harris I. (Long Beach, CA)
Assignee: ICL Scientific (Fountain Valley, CA)
Application Number:06/231,683
Patent Claims:1. A method for producing stable sensitized polystyrene latex particles useful as a diagnostic reagent in immunological procedures, the method comprising the steps of:

(i) coating a polystyrene latex particle with a water-soluble protein;

(ii) adsorbing a substantially methylated serum albumin onto the particle to form a matrix on the surface of the particle; and

(iii) non-covalently binding a substantially negatively-charged, polymeric compound, having antigenic sites, to the matrix whereby a sensitized particle is made;

wherein a mixture of sensitized particles in solution agglutinates in the presence of antibodies capable of binding antigenic sites of the polymeric compound, but does not exhibit significant auto-agglutination tendencies.

2. The method of claim 1 wherein the coating occurs by adsorption.

3. The method of claim 1 wherein the coating and adsorbing steps are coincident.

4. The method of claim 1 wherein the water-soluble protein is an albumin.

5. The method of claim 4 wherein the water soluble albumin is bovine serum albumin or human serum albumin.

6. The method of claim 1 wherein the negatively-charged polymeric compound is selected from the group consisting of nucleic acids, negatively charged polysaccharides, negatively charged proteins, and mixtures thereof.

7. The method of claim 6 wherein the nucleic acid is deoxyribonucleic acid.

8. The method of claim 1 wherein the water-soluble protein is bovine serum albumin, the methylated albumin is methylated bovine serum albumin, and the polymeric compound is deoxyribonucleic acid.

9. A method of making an immunological reagent for the in vitro diagnosis of rheumatic, autoimmune diseases, the method comprises adsorbing a water-soluble albumin and a substantially methylated bovine serum albumin onto surfaces of polystyrene latex beads, non-covalently binding a nuclear component to the methylated bovine serum albumin on the beads' surfaces, whereby the beads are sensitized with the nuclear component, and placing the beads in a preservative suspension, wherein the sensitized beads do not agglutinate in the presence of normal serum, but agglutinate in the presence of serum containing significant amounts of autoimmune antibodies.

10. The method of claim 9 wherein the water soluble albumin is adsorbed onto the beads' surfaces before the methylated bovine serum albumin.

11. The method of claim 9 wherein the water soluble albumin is human serum albumin or bovine serum albumin.

12. The method of claim 9 wherein the nuclear component is selected from the group consisting of chromatin, ribonuclear protein, deoxyribonuclear protein, and mixtures thereof.

13. The method of claim 9 wherein the nuclear component is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid and mixtures thereof.

14. The method of claim 13 wherein the deoxyribonucleic acid in a buffer exhibits a relative viscosity greater than about 1.2 and less than about 4.0.

15. The method of claim 14 wherein the deoxyribonucleic acid exhibits a relative viscosity from about 2.5 to about 3.2.

16. The method of claim 13 wherein the deoxyribonucleic acid is isolated from chicken blood, calf thymus, herring sperm or salmon testes.

17. The method of claim 13 wherein prior to the binding of the deoxyribonucleic acid the relative viscosity of the deoxyribonucleic acid is adjusted by ultrasonication, whereby breakage and separation of strands in the deoxyribonucleic acid is minimized.

18. A method for producing a reagent capable of agglutinating an aliquot of serum from a patient suffering from serum lupus erythematosus, the method comprising the steps of:

(i) combining 1 volume of a concentrated suspension of polystyrene latex beads, with 0.2 volumes of from about 10 micrograms/ml to 100 mg/ml bovine serum albumin in solution, and stirring;

(ii) adding 0.5 volumes of from about 10 micrograms/ml to 50 mg/ml methylated bovine serum albumin in solution, and stirring;

(iii) mixing in about 10 volumes of from about 50 micrograms/ml to about 5000 micrograms/ml, native deoxyribonucleic acid in solution, the deoxyribonucleic acid (DNA) preferably having a relative viscosity of about 1.2 to about 4.0, and stirring;

(iv) washing the beads by centrifugation to separate the beads from unbound albumins or deoxyribonucleic acid; and

(v) storing the washed beads as a suspension, containing preservatives.

19. The method according to claim 18 wherein the suspension of polystyrene latex beads has a concentration of about 10 grams/100 ml, the solution of bovine serum albumin has a concentration of about 33.3 mgs/ml, the solution of methylated bovine serum albumin has a concentration of about 33.3 mgs/ml, and the solution of DNA has a concentration of about 200 micrograms/ml.

20. The method according to claim 18 wherein the relative viscosity of the deoxyribonucleic acid is from about 2.5 to about 3.2.

21. The method according to claim 20 wherein the relative viscosity of the deoxyribonucleic acid is about 2.8.

22. The method according to claim 18 wherein the preservatives are a detergent and a bacteriostatic agent.

23. A latex bead made according to the method of claims 1, 8, 9 or 18.

24. A reagent for determining the presence of autoimmune antibodies in human serum comprising latex particles, having an average diameter from about 0.05 microns to about 1.1 microns, coated with a mixture of water-soluble albumin and substantially methylated bovine serum albumin, to which is non-covalently bound native deoxyribonucleic acid, the deoxyribonucleic acid exhibiting a relative viscosity between about 1.2 and 4.0 in a pH 6.8, 0.002 molar sodium phosphate buffer containing 0.2 grams sodium chloride and 0.01 grams thimerosal per 100 mls of the buffer.

25. The reagent of claim 24 wherein the water-soluble albumin is human serum albumin or bovine serum albumin.

26. The reagent of claim 24 wherein the diameter of the particles is about 0.6 microns.

27. The reagent of claim 24 wherein the relative viscosity of the deoxyribonucleic acid is from about 2.5 to about 3.2.

28. The reagent of claim 27 wherein the relative viscosity of the deoxyribonucleic acid is about 2.8.

29. The reagent of claim 28 wherein the deoxyribonucleic acid is isolated from chicken blood, salmon testes, herring sperm or calf thymus.

30. A method of testing in vitro for the presence of anti-native deoxyribonucleic acid antibodies in human fluids which comprises placing about one drop of human serum on a slide, adding one drop of a 1 gram per 100 mls suspension of the reagent of claim 24, tilting the slide for a time sufficient to allow agglutination, and observing the mixture for the presence of agglutination.

Details for Patent 4,351,824

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Grifols Therapeutics Llc ALBUKED, PLASBUMIN-20, PLASBUMIN-25, PLASBUMIN-5 albumin (human) For Injection 101138 10/21/1942 ⤷  Try a Trial 2040-01-28
Baxalta Us Inc. BUMINATE, FLEXBUMIN albumin (human) Injection 101452 03/03/1954 ⤷  Try a Trial 2040-01-28
Csl Behring Ag ALBURX albumin (human) Injection 102366 07/23/1976 ⤷  Try a Trial 2040-01-28
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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