Method for x and y spermatozoa separation

• US 4,999,283 A
• Filed: 08/18/1989
• Issued: 03/12/1991
• Est. Priority Date: 01/10/1986
• Status: Expired due to Term

• Current Assignee: University Of Kentucky Research Foundation (University of Kentucky)
• Original Assignee: University Of Kentucky Research Foundation (University of Kentucky)
• Inventors: Zavos, Panayiotis M., Dawson, Karl A.
• Primary Examiner(s): Teskin, Robin
• Application Number:
  US07/396,738
• Time in Patent Office: 571 Days
• Field of Search: 435/7, 435/2, 435/240.1, 435/240.2, 436/503, 436/501, 436/510, 436/519, 436/517, 436/518, 436/811, 436/813, 436/824, 436/906, 530/387, 530/806, 530/809, 530/413
• US Class Current: 435/2
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: A01N 1/0231 Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
• CPC Class Codes: A61K 35/52 Sperm; Prostate; Seminal fluid; Leydig cells of testes
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function
• CPC Class Codes: Y10S 436/813 Cancer
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 436/906 Fertility tests

Abstract

A method for separating male and female determining spermatozoa includes the initial step of exposing freshly ejaculated spermatozoa in a substantially protein free diluent to an excess concentration of a monoclonal antibody directed against H-Y antigen that binds substantially exclusively with male determining spermatozoa. The method continues with the suspending of the exposed spermatozoa together with a conjugate of (1) an immunoglobulin G antibody that binds substantially exclusively to the monoclonal antibody and (2) an immunoabsorbant substrate in a substantially protein free diluent. This forms a conjugate/spermatozoa preparation. The method concludes with the recovering of the separated male and female determining spermatozoa.

Claims

1. The method for separating male and female determining spermatozoa for the purpose of controlling the sex of offspring prior to conception, comprising the steps of:

exposing the spermatozoa in a protein free diluent to an excess concentration of a first antibody that binds male determining spermatozoa;

suspending said exposed spermatozoa together with a conjugate of a second antibody that binds exclusively to said first antibody and an immunoabsorbent substrate in a protein free diluent to form a conjugate/spermatozoa preparation; and

recovering the female determining spermatozoa from the conjugate/spermatozoa preparation while said male determining spermatozoa are bound to said immunoabsorbent substrate.

2. The method for separating male and female determining spermatozoa recited in claim 1, including the additional step of recovering said male determining spermatozoa from said immunoabsorbent substrate.

3. The method for separating male and female determining spermatozoa recited in claim 1, wherein said exposing step includes the additional step of diluting said spermatozoa to a concentration of between 10×

10⁶ and 100×

10⁶ active spermatozoa per milliliter of protein free diluent.

4. The method for separating male and female determining spermatozoa recited in claim 1, wherein said first antibody is monoclonal antibody directed against H-Y antigen of said male determining spermatozoa.

5. The method for separating male and female determining spermatozoa recited in claim 1, including the steps of recovering said exposed spermatozoa from said excess concentration of said first antibody and washing said exposed spermatozoa to remove unbound first antibody.

6. The method for separating male and female determining spermatozoa recited in claim 5, wherein said recovering is by centrifugation.

7. The method for separating male and female determining spermatozoa recited in claim 5, including the step of resuspending said washed, exposed spermatozoa in a protein free diluent.

8. The method for separating male and female determining spermatozoa recited in claim 1, including the additional step of incubating said spermatozoa at approximately 37°

C. during said exposing and suspending steps.

9. The method for separating male and female determining spermatozoa recited in claim 1, wherein said immunoabsorbent substrate is agarose beads.

10. The method for separating male and female determining spermatozoa recited in claim 1, wherein said second antibody is anti-immunoglobulin antibody capable of binding said first antibody.

11. The method for separating male and female determining spermatozoa recited in claim 1, wherein said suspending step includes the additional step of agitating said conjugate/spermatozoa preparation to prevent the immunoabsorbent substrate from settling.

12. The method for separating male and female determining spermatozoa recited in claim 1, wherein said female determining spermatozoa recovering step includes the steps of draining said protein free diluent and female determining spermatozoa from said immunoabsorbent substrate and then centrifuging said protein free diluent and female determining spermatozoa to separate said female determining spermatozoa from said diluent.

13. The method for separating male and female determining spermatozoa recited in claim 1, including the additional step of filtering the separated female determining spermatozoa so as to substantially remove dead spermatozoa and increase overall efficacy.

14. The method for separating male and female determining spermatozoa recited in claim 13, wherein said filtering step includes the step of passing the separated female determining spermatozoa through an unbound sephadex bead column.

15. The method for separating male and female determining spermatozoa recited in claim 2, including the additional step of filtering the separated male determining spermatozoa so as to substantially remove dead spermatozoa and increase overall efficacy.

16. The method for separating male and female spermatozoa recited in claim 2, including the additional steps of separately filtering the separated male and female determining spermatozoa so as to remove dead spermatozoa and increase overall efficacy and separately collecting said separated male and female determining spermatozoa in a protein containing media following filtering, said protein containing media serving to support the separated spermatozoa until subsequent use in artificial insemination.

17. The method for separating male and female determining spermatozoa recited in claim 1, wherein said protein free diluent is a sodium citrate buffer.