Claims for Patent: 3,992,516

Summary for Patent: 3,992,516

Title:	Direct fluorescent antibody composition and method for P. Pneumocystis carinii
Abstract:	A Pneumocystis carinii antiserum conjugated with fluorescein isothiocyanate composition, its use being the detection of Pneumocystis carinii organisms and the method of making this composition using an enzyme purifying technique.
Inventor(s):	Lim; Sook Kyung (Ann Arbor, MI)
Assignee:
Application Number:	05/519,217
Patent Claims:	1. The process of producing pneumocystis carinii antiserum conjugated with fluorescein isothiocyanate comprising a. digesting out tissue proteins from a homogenized and clarified pneumocystis carinii organism suspension containing tissue obtained from an infected host, purifying the obtained suspension by density gradient centrifugation to secure pneumocystis carinii organisms which are substantially free of other antigens; b. immunizing a host with the purified pneumocystis carinii organism containing suspension and isolating the antiserum globulin fraction containing the specific antibody for pneumocystis carinii antigens from the hyperimmune serum produced; and c. conjugating said antiserum globulin with a fluorescent dye. 2. The process of claim 1, in which said fluorescent dye is fluorescein isothiocyanate. 3. The process of claim 1, in which there is employed in said digesting step a protolytic enzyme selected from the group consisting of trypsin, chymotrypsin, pepsin, collagenase, and pronase. 4. The process of claim 1, in which there is employed in said digesting step a said protolytic enzyme which is trypsin. 5. The process of claim 1, in which said digested pneumocystis carinii containing material is purified using a sucrose gradient. 6. The process of claim 1, in which said pneumocystis carinii tissue is lung tissue obtained from rats and humans. 7. The process of claim 1, in which said immunized host is selected from the group consisting of rabbits, horses goats and monkeys. 8. The process of claim 1, in which said immunized host is a rabbit. 9. The process of claim 1, in which said process of producing said purified P. carinii suspension comprises homogenizing the infected lung tissue in Eagle minimal essential medium with Hank solution containing penicillin and streptomycin, clarifying the solution obtained and discarding the sediment, digesting this mixture with trypsin, separating and discarding the sediment, centrifuging the supernatant fluid and reconstituting the sediment to make a 10-20% suspension of the antigen organisms, fractionating the suspension by sucrose density centrifugation and collecting the fractions. 10. The process of claim 9, in which said process of producing said antiserum comprises immunizing rabbits with the purified P. carinii suspension, then separating the P. carinii antibodies from other antibodies by absorption of the other antibodies with mouse tissue powder. 11. The process of claim 10 in which said process of conjugating said antiserum globulin with fluorescein isothiocyanate comprises combining an equal volume of antiserum and saturated ammonium sulfate solution having a pH of 7 and incubating the mixture at 4.degree. C, centrifuging the mixture and collecting the precipitate, dialyzing the globulin containing fraction in a saline solution to eliminate ammonium ions, reacting the globulin with the fluorescein isothiocyanate, removing the unbound dyes and fractionating the conjugate in a diethylamino-ethyl-cellulose anion exchange column to obtain the fraction of optimum F/P ratio of antibodies. 12. The process of claim 11, in which said F/P ratio is 1 to 3. 13. The process of claim 1, in which the dye to protein ratio is 1 to 3. 14. The process of detecting pneumocystis carinii organisms comprising staining said organisms in sputum, trachael aspirate or materials which may be infected with said organisms with pneumocystis carinii antiserum conjugated with fluorescent dye and observing fluorescence when the organisms are present. 15. Pneumocystis carinii antiserum globulin conjugated with fluorescein isothiocyanate. 16. The fluorescent dye conjugated pneumocystis carinii antiserum globulin produced by the process of claim 1. 17. The process of producing a Pneumocystis carinii antigen which comprises digesting out serum and tissue proteins from a homogenized and clarified Pneumocystis carinii organism suspension containing tissue obtained from an infected host, purifying the obtained suspension by density gradient centrifugation, to obtain Pneumocystis carinii organisms substantially free of other antigens. 18. The process of producing an antiserum globulin fraction containing the specific antibody for Pneumocystis carinii which comprises: a. digesting out serum proteins from homogenized and clarified Pneumocystis carinii organism suspension containing tissue obtained from an infected host, purifying the obtained suspension by density gradient centrifugation to obtain Pneunocystic carinii organisms which are substantially free of other antigens; b. immunizing a host with the purified Pneumocystis carinii organism containing suspension and isolating the antiserum globulin fraction containing the specific antibody for Pneumocystis carinii antigens from the hyperimmune serum produced. 19. The process of detecting Pneumocystis carinii organisms which comprises making smears of the purified organism or trypsinized impression smears of infected tissue, fixing the smears with acetone, and staining the fixed smears with the labeled antiserum prepared by the method of claim 1. 20. The process of claim 19 which further comprises counterstaining the smear.

Details for Patent 3,992,516

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-03-29