We claim:
1. A high quality reagent for testing male infertility, comprising a properly glycosylated sperm binding glycopeptide complexed to a detection agent.
2. A reagent as described in claim 1, wherein the glycopeptide is selected from the group consisting of: properly glycosylated rhZP3; properly glycosylated peptide comprising amino acid portion 310 to 345 of rhZP3; properly glycosylated peptide comprising amino acid portion 200 to 351 of rhZP3; properly glycosylated peptide comprising amino acid portion 14 to 351 of rhZP3; properly glycosylated peptide comprising at least one binding site for human sperm; properly glycosylated peptide comprising at least two binding sites for human sperm, properly glycosylated peptide comprising at least three binding sites for human sperm; rhZP3; rhZP3ZP2 complex; rhZP3ZP2 fusion protein prepared as a single gene product; and rhZP3ZP2ZP1.
3. A reagent as described in claim 1, wherein the detection agent is selected from the group consisting of: GFP; a fluor; an enzyme; beta galactosidase; alkaline phosphatase; horseradish peroxidase; an antigenic peptide; Alexa488; fluorophore-GFP conjugate, an antigenic peptide, a peptide that comprises HA, a peptide that comprises V5, a peptide that comprises Myc, and biotin.
4. A reagent as described in claim 1, wherein the properly glycosylated sperm binding glycopeptide is prepared by culture of a uterogenital origin cell line or a mammary cell line.
5. A reagent as described in claim 4, where in the cell line is PA-1 or 293.
6. A reagent comprising recombinantly produced ZP2 capable of forming a ZP2ZP3 complex upon incubation with ZP3.
7. A reagent comprising recombinant ZP2ZP3 complex capable of binding specifically to human sperm.
8. A reagent as described in claim 7, wherein the molar ratio of ZP3 to ZP2 is equal to or greater than 1.
9. A reagent as described in any of claim 1, wherein the ZP2 is human ZP2.
10. A composition comprising purified human ZP2ZP3 complex.
11. A kit for testing male infertility, comprising a reagent as described in any of claim 1, and at least a buffer salt or salt solution.
12. A method for detecting infertility of a male, comprising the step of contacting a reagent as described in any of claim 1 with a sperm sample of the male.
13. A method for detecting infertility of a male, comprising:
a) providing a high quality reagent as described in claim 1;
b) contacting a sperm sample of the male with the reagent for a period of time sufficient to allow binding between the glycopeptide and the sperm (note we define glycopeptide broadly to include proteins such as ZP3); and
c) detecting the detection agent bound to sperm.
14. A method as described in claim 13, further comprising the step of washing the contacted sperm sample of step b) before step c).
15. A method as described in claim 13, wherein the detection agent is an antigen, and detection step c) comprises incubating with a fluorescence labeled antibody to generate a fluorescence signal associated with bound detection agent.
16. A method as described in claim 15, wherein the fluorescence labeled antibody is Alexa 488 as well as other coupling dye coupled to an antibody that recognizes an antigenic site on the reagent.
17. A method as described in claim 12, wherein the reagent is ZP3GFP.
18. A method for determining infertility of a male, comprising detecting one or more substances released from a sperm sample upon induction of the acrosome reaction in sperm of the sample.
19. The method of claim 18, wherein the one or more substances are selected from the group consisting of an enzyme, acid phosphatase, protein, membrane fragment, phospholipid, cholesterol, peptide, lipoprotein, nucleotide, nucleoside and glycoprotein.
20. The method of claim 18, wherein the substance is phospholipid.
21. The method of claim 18, wherein the substance is acrosin and wherein the method further comprises incubating the sperm with an acrosin inhibitor.
22. The method of claim 21, wherein the acrosin inhibitor is selected from the group consisting of a low pH solution, HCl, acetic acid, acidified amino acid and acidified glycine.
23. An acrosome scoring method for determining infertility of a male, comprising:
a) inducing acrosome reaction in a sample of sperm from the male;
b) detecting at least two different morphological types of acrosome reacted sperm; and
c) calculating a score based on differential weighting of the detected at least two different morphological types of acrosome reacted sperm.
24. An acrosome scoring method as described in claim 21, wherein at least three different sperm morphologies are detected and given differential weighting.
25. An artificial zona for detecting sperm binding, comprising a solid phase and at least one sperm binding agent comprising a minimum portion of rhZP3 that contains sperm binding site that binds sperm.
26. The artificial zona of claim 25, wherein the solid phase is a particle.
27. The artificial zona of claim 26, wherein the solid phase comprises a substance selected from the group consisting of agarose, Sephadex, acrylamide, latex, polystyrene, glass, gold and insoluble protein.
28. The artificial zona of claim 26, wherein the solid phase is between 25 and 250 microns in diameter.
29. The artificial zona of claim 25, wherein the sperm binding agent is selected from the group consisting of rhZP3, ZP2, ZP2-ZP3 complex, ZP1, ZP1-ZP3 complex, ZP1-ZP2-ZP3 complex, GFP-ZP3 and GFP-ZP3-ZP2.
30. The artificial zona of claim 29, wherein the sperm binding agent is attached to the solid phase in a manner that exposes a sperm binding portion of the sperm binding agent to the outside of the particle surface.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A particle inspection apparatus comprising:
an irradiation unit configured to apply a light beam onto each of front and back surfaces of an object to be inspected, the object including a transparent member;
a first detection unit configured to detect scattering light from the front surface;
a second detection unit configured to detect scattering light from the back surface;
a calculation unit configured to conduct a particle inspection on the front and back surfaces on the basis of outputs from the first detection unit and the second detection unit; and
a control unit configured to control the irradiation unit, the first detection unit, the second detection unit, and the calculation unit,
wherein the irradiation unit includes a light-shielding selection unit configured to selectively block the light beam applied to the front surface or the light beam applied to the back surface, and
wherein the control unit causes the light-shielding selection unit to simultaneously apply the light beam onto the front and back surfaces, determines whether the light beam should be selectively applied onto the front surface or the back surface based on outputs made by the first detection unit and the second detection unit corresponding to simultaneous application of the light beam, causes the light-shielding selection unit to selectively apply the light beam onto the front surface or the back surface when it is determined that the light beam should be selectively applied, and causes the calculation unit to conduct the particle inspection based on outputs made by the detection unit corresponding to selective application of the light beam.
2. An exposure apparatus that exposes a substrate via an original, the apparatus comprising:
the particle inspection apparatus according to claim 1, the transparent member is the original.
3. The exposure apparatus according to claim 2, wherein the object includes a pellicle.
4. A device manufacturing method comprising the steps of:
exposing a substrate with the exposure apparatus according to claim 2; and
developing the exposed substrate.