Gender differentiation of bovine sperm cells

• US 20020115055A1
• Filed: 09/27/2001
• Published: 08/22/2002
• Est. Priority Date: 09/28/2000
• Status: Abandoned Application

• Current Assignee: Marcos Fernando De Resende Matta
• Original Assignee: Marcos Fernando De Resende Matta
• Inventors: Matta, Marcos Fernando de Resende
• Application Number:
  US09/965,017
• Publication Number:
  US 20020115055A1
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: G01N 2333/4716 Complement proteins, e.g. anaphylatoxin, C3a, C5a
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism

Abstract

A method to make an immuno-differentiation of bovine sperm cells using monoclonal antibodies against male specific antigen, 17.18 kDa protein, associated to proteins of the classic pathway complement system is described. Monoclonal antibodies directed towards a gender-specific protein associated to the complement, provoke cellular rupture. For this methodology to be useful, an elimination of the alternative pathway of the complement is necessary. The sperm cells are then put in contact with a monoclonal antibody specifically for the gender-marking protein. Thus, the classic pathway complement system causes selected rupture of the marked sperm cells. The sperm cells of the treated semen are then ready to be used in artificial insemination, cryo-preservation or “in vitro” fertilization.

Claims

1. A method for gender differentiation of bovine spermatozoa wherein monoclonal antibodies against male specific antigen, 17.18 kDa protein, associated with the classical pathway of the complement system are used after the inactivation of the alternative complement pathway, comprising:

a. incubating heated guinea pig serum with sperm cells and monoclonal antibodies, said monoclonal antibodies being specific enough to recognize only male specific antigens, and

b. selectively rupturing the male sperm cells, leaving a majority of the live sperm cells as female.

2. The method of claim 1 wherein the serum is selected from the group including cow serum, goat serum, sheep serum, pig serum and guinea pig serum.

3. The method of claim 1 wherein the serum is guinea pig serum.

4. The method of claim 1 wherein the serum and ascitic fluid are heated at 52.2°

C.+0.3 for 30 minutes.

5. The method of claim 1 wherein the sperm cells is incubated with monoclonal antibodies for 45 minutes at 37°

C.

6. The method of claim 1 further comprising following step b. washing the sperm cells to remove excess of antibodies, and then adding the serum and seminal plasma to the sperm and then incubating for 1 hour at 37°

C.

7. A method for gender differentiation of bovine spermatozoa wherein monoclonal antibodies against male specific antigen, 17.18 kDa protein, associated with the classical pathway of the complement system are used after the inactivation of the alternative complement pathway, using the following steps:

a. Inactivating the alternative pathway of the complement system by heating guinea pig serum and ascitic fluid at 52.2°

C.+0.3 for 30 minutes or purified monoclonal antibodies; and

b. Incubating the sperm cells with monoclonal antibodies, originated from one or more different clones (hybridomas), for 45 minutes at 37°

C.; and

c. Washing the sperm cells to remove excess of antibodies, and then adding the inactivated guinea pig serum and seminal plasma to the sperm cell and then incubating for 1 hour at 37°

C.

8. The method of claim 7, wherein the B protein in a guinea pig serum inactivates the alternative pathway of the complement.

9. The method of claim 7 comprising eliminating the B protein at temperatures varying from 50°

to 53°

C.

10. The method of claim 7 comprising eliminating the alternative pathway of the complement by methods selected from column affinity, genetic modification of animals, and utilization of protein H is foreseen in this patent.

11. The method of claim 7, wherein monoclonal antibodies are incubated with the sperm cells for the time necessary for there to be an antigen-antibody binding.

12. The method of claim 7, wherein the serum is incubated for a time sufficient for the reaction of the classic pathway of the complement system to proceed.

13. The method of claim 7 wherein the antibody and complement are incubated simultaneously with the sperm cells.

14. The method of claim 7 wherein the monoclonal antibodies are reacted with different epitopes for better differentiation.