1. A biosensor for detecting the presence or absence of mycoplasma contamination comprising a support and a detectably labeled substrate for an hydrolytic enzyme produced andor secreted by a mycoplasma, wherein the substrate is attached to a support.
2. The biosensor of claim 1 wherein the enzyme is selected from the group consisting of proteases, reductases and nucleases.
3. The biosensor of claim 1 wherein the enzyme is selected from the group consisting of the gene product of pepA1, MCAP\u20140195, MCAP\u20140267, MCAP\u20140341, MCAP\u20140509, MCAP\u20140675 and mixtures thereof.
4. The biosensor of claim 1 wherein the enzyme is a reductase and the substrate is reactive black 5,5,5\u2032-dithio-bis-(2-nitrobenzoic acid) (DTNB), BODIPY\xaeFL L-cystine, or 2\u2032,7\u2032-difluoro-4\u2032-(2-(5-((dimethyl amino phenyl)azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (DFDMAP-fluorescein).
5. The biosensor of claim 4 wherein the reductase is selected from the group consisting of the gene product of nrdE (such as MCAP\u20140101), MCAP\u20140427, trxB (thioredoxin reductase, such as MCAP\u20140779 or MHP7448\u20140098), MCAP\u20140858 and mixtures thereof.
6. The biosensor of claim 1 wherein the mycoplasma-specific enzyme is a nuclease and the detectable label is an acetoxymethyl ester or maleimide derivative of blue dye number 1 coupled to an aminollyl-dNTP labeled nucleic acid substrate of the mycoplasma-specific nuclease.
7. The biosensor of claim 6 wherein the nuclease is selected from the group consisting of the gene product of the 5\u2032-3\u2032 exonuclease encoded by MCAP\u20140047 or MHP7448\u20140581, the gene product of nfo (such as MCAP\u20140060 or MHP7448\u20140062), vacB (such as MCAP\u20140097 or MHP7448\u20140037), uvrC (such as MCAP\u20140252 or MHP7448\u20140066), mc (ribonuclease III, such as MCAP\u20140492 or MHP7448\u20140398), MCAP\u20140768, uvrB (such as MCAP\u20140773 or MHP7448\u20140648), uvrA (such as MCAP\u20140774 or MHP7448\u20140091) and mixtures thereof.
8. The biosensor of claim 1 wherein the support is a reagent container, a bead, a polymer film, a culture medium container or a cell culture container.
9. A method of detecting mycoplasma contamination of a cell culture comprising the steps of providing a cell-permeable detectable label coupled to a cell-impermeant carrier in the culture medium wherein cleavage of the detectable label by a mycoplasma-specific enzyme is followed by uptake of the detectable label into cells; and detecting labeled cells, thereby detecting mycoplasma contamination of the cell culture.
10. The method of claim 9 wherein the mycoplasma-specific enzyme is a protease and the detectable label is leucine-(7-methoxycoumarin-4-yl)acetyl (leu-MCA), arginine-(7-methoxycoumarin-4-yl)acetyl (arg-MCA), methionine-(7-methoxycoumarin-4-yl)acetyl (met-MCA), or an acetoxymethyl ester.
11. The method of claim 10 wherein the protease is selected from the group consisting of pepA1 (MCAP\u20140157), pepA2 (MCAP\u20140195), pepA (leucyl aminopeptidase, such as MHP7448\u20140464), MCAP\u20140267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP\u20140341 or MHP7448\u20140649), MCAP\u20140509, mapP (methionine amino peptidase, MCAP\u20140675 or MHP7448\u20140173), and mixtures thereof.
12. The method of claim 9 wherein the mycoplasma-specific enzyme is a nuclease and the detectable label is an acetoxymethyl ester of derivative of blue dye number 1 coupled to a nucleic acid substrate of the mycoplasma-specific nuclease
13. The method of claim 12 wherein the nuclease is selected from the group consisting of the 5\u2032-3\u2032 exonuclease encoded by MCAP\u20140047 or MHP7448\u20140581, the gene product of nfo (such as MCAP\u20140060 or MHP7448\u20140062), vacB (such as MCAP\u20140097 or MHP7448\u20140037), uvrC (such as MCAP\u20140252 or MHP7448\u20140066), mc (ribonuclease III, such as MCAP\u20140492 or MHP7448\u20140398), MCAP\u20140768, uvrB (such as MCAP\u20140773 or MHP7448\u20140648), uvrA (such as MCAP\u20140774 or MHP7448\u20140091) and mixtures thereof.
14. A method of determining the presence or absence of mycoplasma in a sample, comprising the steps of:
contacting the sample with a detectably labeled substrate for an enzyme produced andor secreted by a mycoplasma under conditions that result in the modification of the substrate by the enzyme; and
detecting the modification or the absence of the modification of the substrate wherein modification of the substrate indicates the presence of mycoplasma in the sample, and wherein the absence of modification of the substrate indicates the absence of mycoplasma in the sample.
15. The method of claim 14 wherein the level of the detectable label is quantitatively related to the presence or amount of mycoplasma in the sample.
16. The method of claim 14 wherein the enzyme is a hydrolytic enzyme selected from a protease, a nuclease or a reductase.
17. The method of claim 14 wherein the enzyme is selected from the group consisting of the gene product of pep A1 (MCAP\u20140157), pepA2 (MCAP\u20140195), pepA (leucyl aminopeptidase, such as MHP7448\u20140464), MCAP\u20140267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP\u20140341 or MHP7448\u20140649), MCAP\u20140509, mapP (methionine amino peptidase, MCAP\u20140675 or MHP7448\u20140173), and mixtures thereof.
18. The method of claim 14 wherein the enzyme is selected from the group consisting of the 5\u2032-3\u2032 exonuclease encoded by MCAP\u20140047 or MHP7448\u20140581, the gene product of nfo (such as MCAP\u20140060 or MHP7448\u20140062), vacB (such as MCAP\u20140097 or MHP7448\u20140037), uvrC (such as MCAP\u20140252 or MHP7448\u20140066), mc (ribonuclease III, such as MCAP\u20140492 or MHP7448\u20140398), MCAP\u20140768, uvrB (such as MCAP\u20140773 or MHP7448\u20140648), uvrA (such as MCAP\u20140774 or MHP7448\u20140091) and mixtures thereof.
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 fastener comprising:
a grommet including a leg portion; and
a pin including a head portion, a tail portion and a body portion, the body portion being positioned between the head and tail portions and for contacting the leg portion at first and second operating positions, the head and tail portions each having an outer circumference greater than a maximum outer circumference of the body portion.
2. The fastener of claim 1, wherein the body portion includes first and second shoulders, the first shoulder being positioned between the head portion and the second shoulder.
3. The fastener of claim 2, wherein the leg portion of the grommet ends with a toe portion having a convex surface.
4. The fastener of claim 3, wherein the tail portion includes a tail surface, the convex surface of the leg portion contacts the tail surface of the tail portion at a third operating position.
5. The fastener of claim 4, wherein the convex surface of the leg portion includes a surface point contacted by the tail surface, where the surface point is most distal of all points on the pin away from the grommet in the longitudinal direction.
6. The fastener of claim 4, wherein the pin is positioned for axial rotation in any of the first, second and third positions.
7. The fastener of claim 2, wherein the first shoulder includes a first beveled surface and a second beveled surface positioned between the first beveled surface, the first beveled surface defining a surface angle B2 and the second beveled surface defining a surface angle A1 relative to the central longitudinal axis, respectively.
8. The fastener of claim 7, wherein the second shoulder includes a third beveled surface and a fourth beveled surface positioned between the third beveled surface and the tail portion, the third beveled surface defining a surface angle B1 and the fourth beveled surface defining a surface angle A2 relative to the central longitudinal axis, respectively.
9. The fastener of claim 8, wherein the first and third beveled surfaces are of different angles relative to the common longitudinal axis.
10. The fastener of claim 8, wherein the second and fourth beveled surfaces are of different angles relative to the common longitudinal axis.
11. The fastener of claim 7, wherein the first shoulder further includes a first end surface defined between the first and second beveled surfaces.
12. The fastener of claim 8, wherein the second shoulder further includes a second end surface defined between the third and fourth beveled surfaces.
13. The fastener of claim 8 wherein the second and third beveled surfaces together define a first neck recess to receive a part of the leg portion at an operating position.
14. The fastener of claim 8, wherein the fourth beveled surface and the tail portion together define a second neck recess to receive a part of the leg portion at an operating position.
15. The fastener of claim 4, wherein the leg portion has a toe portion most distal away from the grommet, the toe portion being positioned between the tail portion and head portion at any of the first, second and third operating positions.
16. A fastener with a central longitudinal axis, comprising:
a grommet including a leg portion which ends with a toe portion; and
a pin including a head portion, a tail portion and a body portion, the body portion including a first shoulder defined by first and second beveled surfaces and a second shoulder defined by third and fourth beveled surfaces, the second and fourth beveled surfaces are of different angles relative to the central longitudinal axis.
17. The fastener of claim 16, wherein the first and third beveled surfaces are of different angles relative to the central longitudinal axis.
18. The fastener of claim 16, wherein the first and second shoulders further include a first end surface defined between the first and second beveled surfaces and second end surface defined between the third and fourth beveled surfaces, respectively.
19. The fastener of claim 16, wherein the second and third beveled surfaces further define there between a first neck recess, and the fourth beveled surface and the tail portion further define there between a second neck recess.
20. A fastener with a central longitudinal axis, comprising:
a grommet including a leg portion which ends with a toe portion; and
a pin including a head portion, a tail portion and a body portion, the head and tail portions each having an outer circumference greater than a maximum outer circumference of the body portion, the body portion including a first shoulder defined by first and second beveled surfaces and a first end surface positioned there between, and a second shoulder defined by third and fourth beveled surfaces and a second end surface positioned there between, the first and third beveled surfaces are of different angles relative to the central longitudinal axis, the second and fourth beveled surfaces are of different angles relative to the central longitudinal axis.