1. A high-performance liquid chromatograph apparatus comprising:
liquid feed means (4) for feeing an eluent by varying a mixing ratio of two or more types of eluents;
sample injecting means (8) for injecting a sample into the eluent fed from the liquid feed means (4);
a separation column (10) configured to be supplied with the eluent into which the sample is injected by the sample injecting means (8) and to thereafter separate a target component contained in the sample;
a detector (11) for analyzing the target component separated by the separation column (10); and
control means (13) for controlling operation of the liquid feed means (4), the sample injecting means (8), the separation column (10), and the detector (11), the control means (13) including:
a constant velocity gradient program storage unit (13c) for storing a constant velocity gradient program that feeds the eluent at a constant velocity while varying the mixing ratio of two or more types of eluents;
a constant pressure gradient program converting unit (13b) that converts the constant velocity gradient program to a constant pressure gradient program that feeds the eluent at a constant pressure while varying the mixing ratio of two or more types of eluents; and
a flow velocity setting unit (13d) for controlling a flow velocity of the eluent fed by the liquid feed means (4) according to the constant pressure gradient program converted by the constant pressure gradient program converting unit (13b).
2. The high-performance liquid chromatograph apparatus according to claim 1, further comprising:
a pressure sensor (7) for measuring pressure of the eluent fed from the liquid feed means (4), wherein:
the control means (13) further includes a pressure value trace storage unit (13a) for storing a pressure value measured by the pressure sensor (7); and
the constant pressure gradient program converting unit (13b) of the control means (13) converts the constant velocity gradient program to the constant pressure gradient program based on the pressure value stored in the pressure value trace storage unit (13a).
3. The high-performance liquid chromatograph apparatus according to claim 1, further comprising:
a pressure sensor (7) for measuring pressure of the eluent fed from the liquid feed means (4), wherein
the control means (13) converts the constant velocity gradient program to the constant pressure gradient program based on a current pressure value measured by the pressure sensor (7) and an amount of eluent fed by the liquid feed means (4).
4. The high-performance liquid chromatograph apparatus according to claim 2, wherein:
the constant velocity gradient program storage unit (13c) stores a plurality of constant velocity gradient programs;
the control means (13) includes a setting unit (13d) in which one of the constant velocity gradient programs and a flow velocity are set; and
the constant pressure gradient program converting unit (13b) converts the constant velocity gradient program to the constant pressure gradient program based on the constant velocity gradient program and the flow velocity that are set in the setting unit (13d).
5. The high-performance liquid chromatograph apparatus according to claim 4, wherein:
in the setting unit (13d), time at which the constant pressure gradient program is to be applied is set; and
the control means (13) controls the flow velocity of the eluent fed by the liquid feed means (4) according to the converted constant pressure gradient program effective after the set time.
6. The high-performance liquid chromatograph apparatus according to claim 2, wherein
the control means (13) includes a display and causes the display to display data showing a relationship among elapsed time according to the converted constant pressure gradient program, the eluent mixing ratio, and the flow velocity.
7. The high-performance liquid chromatograph apparatus according to claim 3, wherein
the control means (13) includes a display and causes the display to display a current pressure value measured by the pressure sensor (7).
8. The high-performance liquid chromatograph apparatus according to claim 1, wherein
the separation column (10) uses a packing material comprising a plurality of particles, each particle having an average particle size of about 2.5 micrometers or less.
9. A method for feeding a liquid to a high-performance liquid chromatograph apparatus that feeds an eluent by varying a mixing ratio of two or more types of eluents, injects a sample into the eluent fed, supplies the eluent into which the sample has been injected to a separation column (10), separates a target component contained in the sample, and analyzes using a detector (11) the target component separated, the method comprising the steps of:
storing a constant velocity gradient program that feeds the eluent at a constant velocity while varying the mixing ratio of two or more types of eluents;
converting the constant velocity gradient program to a constant pressure gradient program that feeds the eluent at a constant pressure while varying the mixing ratio of two or more types of eluents; and
controlling according to the converted constant pressure gradient program a flow velocity of the eluent to be fed.
10. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 9, further comprising the steps of:
measuring pressure of the eluent fed; and
storing a pressure value measured, wherein
the constant velocity gradient program is converted to the constant pressure gradient program based on the pressure value stored.
11. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 9, further comprising the step of:
measuring pressure of the eluent fed, wherein
the constant velocity gradient program is converted to the constant pressure gradient program based on a current pressure value measured and an amount of eluent fed.
12. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 10, further comprising the step of:
storing a plurality of constant velocity gradient programs, wherein:
one of the constant velocity gradient programs and a flow velocity are set; and
the constant velocity gradient program is converted to the constant pressure gradient program based on the set constant velocity gradient program and the set flow velocity.
13. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 12, wherein:
time at which the constant pressure gradient program is to be applied is set; and
the flow velocity of the eluent fed by liquid feed means (4) is controlled according to the converted constant pressure gradient program effective after the set time.
14. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 10, wherein
data showing a relationship among elapsed time according to the converted constant pressure gradient program, the eluent mixing ratio, and the flow velocity is displayed on a display.
15. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 11, wherein
the measured current pressure value is displayed on the display.
16. The method for feeding a liquid to a high-performance liquid chromatograph apparatus according to claim 9, wherein
the separation column (10) uses a packing material comprising a plurality of particles, each particle having an average particle size of about 2.5 micrometers or less.
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 method for producing an antibody specific for a polypeptide conformer, the method comprising:
isolating from a polyclonal antibody, which is produced in an animal and raised against a polypeptide preparation enriched for a polypeptide conformer of interest, an antibody specific for the polypeptide conformer of interest;
determining the polypeptide sequence of the antibody specific for the polypeptide conformer of interest;
using the polypeptide sequence to design one or more degenerate oligonucleotides comprising nucleotide sequences corresponding to polynucleotides encoding the antibody specific for the polypeptide conformer of interest; and
using the one or more degenerate oligonucleotides to identify polynucleotides encoding the antibody specific for the polypeptide conformer of interest.
2. The method of claim 1, further comprising, depleting the polyclonal antibody of antibodies that are not specific for the particular polypeptide conformer.
3. The method of claim 1, wherein isolating the polypeptide conformer of interest is performed by immunoprecipitating the antibody specific for the polypeptide conformer of interest with the conformer of interest.
4. The method of claim 1, wherein the a polypeptide composition enriched for a polypeptide conformer of interest is produced using a polynucleotide encoding a signal peptide selected from the group consisting of a heterologous signal peptide, a modified signal peptide, a chimeric signal peptide, and combinations, thereof.
5. The method of claim 1, wherein the polyclonal antibody is recovered by bleeding or exsanguination.
6. The method of claim 1, wherein the one or more degenerate oligonucleotides comprising nucleotide sequences corresponding to polynucleotides encoding the antibody specific for the polypeptide conformer of interest additionally comprise nucleotide sequences useful for inserting the oligonucleotides into a heterologous DNA sequence.
7. The method of claim 1, wherein the one or more degenerate oligonucleotides are used to identify the coding sequence of the antibody specific for the polypeptide conformer of interest from a library.
8. The method of claim 1, wherein the polynucleotide sequences encoding the antibody specific for a polypeptide conformer of interest are inserted into exogenous polynucleotides for expressing said antibody.
9. The method of claim 1, wherein the polynucleotides encoding the antibody specific for the polypeptide conformer of interest are further modified to express a modified version of said antibody selected from the group consisting of a single-chain antibody, a humanized antibody, a chimeric antibody, and an antibody fragment.
10. An antibody specific for a polypeptide conformer of interest produced by the method of claim 1.
11. The antibody of claim 10, wherein the antibody is selected from the group consisting of a single-chain antibody, a humanized antibody, a chimeric antibody, and an antibody fragment.
12. A method of using the antibody of claim 11, to identify a polypeptide conformer of interest in a sample comprising a plurality of conformers of a polypeptide.
13. The method of claim 12, wherein the sample is provided under non-denaturing conditions.
14. A method for conferring passive immunity to a patient suffering from a disease mediated by a polypeptide conformer, the method comprising administering to the patient an antibody of claim 12 along with a suitable pharmaceutical carrier.
15. A method for immunizing a patient against a disease mediated by a polypeptide conformer of interest, the method comprising administering to the patient an immunogenic amount of a polypeptide composition enriched for a polypeptide conformer of interest.
16. A kit of parts comprising an antibody of claim 12, instructions for use of the antibody in an assay to detect the presence of a polypeptide conformer of interest in a sample, and instructions for interpreting results from said assay.
17. An isolated conformer-specific antibody obtained from a polyclonal antibody raised against a polypeptide preparation comprising a conformer of interest, comprising: identifying amino acid sequences of conformer-specific antibodies present in the polyclonal antibody, using the amino acid sequences to produce polynucleotides encoding the conformer-specific antibody, expressing the conformer-specific antibody encoded by the polynucleotides in an organism or a cell free translation system, and isolating the conformer-specific antibody so expressed.
18. The isolated conformer-specific antibody obtained as in claim 17, additionally comprising depleting the polyclonal antibody of antibodies not specific for conformer of interest, then incubating the depleted polyclonal antibody in the presence of a polypeptide preparation enriched for the conformer of interest and isolating complexes comprising the conformer specific antibody and conformer of interest.
19. A conformer-specific antibody isolated from a polyclonal antibody raised against a conformer of interest by incubating the polyclonal antibody in the presence of a conformer of interest, isolating antibody-conformer of interest complexes, determining the amino acid sequence of antibodies present in the complexes, and designing polynucleotides for expression of such conformer-specific antibodies.