COMPOSITIONS FOR SEPARATION OF A PLURALITY OF DISTINCT TARGETS FROM A SAMPLE

Abstract

The present invention generally relates to compositions that may be used to separate targets from non-targets. More specifically, the present invention relates to the separation of at least two distinct targets from a sample containing a mixture of targets and non-targets by contacting the sample with a composition comprising a plurality of heterogeneous epitope binding agents having affinity for the distinct targets.

Description

BRIEF DESCRIPTION OF THE FIGURES

This application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a graph and concomitant table depicting colorimetric spectroscopic absorbance data obtained following the gel filtration of S-acetylthioacetic acid succinimide (SATA)-activated antibodies. The absorbance at 280 nm (A₂₈₀) value of sequentially collected two ml fractions was measured. Fractions 5-6 were pooled. Fraction 7 was concentrated and added to the pool. (See Example 1).

FIG. 2 is a graphical representation depicting the percent separation/depletion for sixteen high abundance plasma proteins as a function of resin volume, following application of a fixed volume of plasma (25 μl) to various volumes of antibody-conjugated resin (200, 350, 500 and 750 μl), and subsequent ELISA analyses of the separated/unbound/depleted fraction. (See Example 1).

FIG. 3 is a graphical representation depicting the percent separation/depletion for sixteen high abundance plasma proteins as a function of resin volume, following application of a fixed volume of plasma (25 μl) to various volumes of antibody-conjugated resin (200, 350, and 500 μl), and subsequent ELISA analyses of the separated/unbound/depleted fraction. (See Example 2).

FIG. 4 is a graphical representation depicting the percent separation/depletion for eighteen high abundance plasma proteins as a function of resin volume, following application of a fixed volume of plasma (25 μl) to various volumes of antibody-conjugated resin (200, 350, and 500 μl), and subsequent ELI SA analyses of the separated/unbound/depleted fraction. (See Example 3).

FIG. 5 is a photograph of blank resins exposed to various conjugation conditions and then incubated overnight at 37° C. Condition 1 resin is bromoacetamide-activated using 3 mM bromoacetic acid-NHS ester. Condition 2 resin is maleimide-activated using 1 mM maleimide-NHS ester. Condition 3 resin is maleimide-activated using 3 mM maleimide-NHS ester. Condition 4 resin is maleimide-activated using 3 mM maleimide-NHS ester, conjugated to 20 antibodies. (See Example 4).

FIG. 6 is a graphical representation depicting the percent separation/depletion for twenty high abundance plasma proteins as a function of resin volume, following application of a fixed volume of plasma (25 μl) to various volumes of antibody-conjugated resin (250 and 500 μl), and subsequent ELISA analyses of the separated/unbound/depleted fraction. (See Example 4).

FIG. 7 is a bar graph representation of the percent separation/depletion of twenty high abundance plasma proteins following application of 25 μl plasma to 500 μl of resin (maleimide-activated). (See Example 4).

FIG. 8 is a bar graph representation of the percent separation/depletion of twenty high abundance plasma proteins following application of 25 μl plasma to 500 μl of resin (bromoacetamide-activated). (See Example 4).

FIG. 9 is a bar graph representation of the percent separation/depletion of twenty high abundance plasma proteins following application of 100, 200 or 300 μl of plasma to 3.75 ml of bromoacetamide-activated resin for one or two sequential cycles. The second depletion (p) represents a reapplication of the depleted plasma from the first cycle. (See Example 5).

FIG. 10 is a plot of the absorbance at 280 nm versus time during immunodepletion column chromatography. Human citrated plasma (100 μl) was injected into a 10 ml column of immunoaffinity resin in the presence of 1× PBS. Fractions (1 ml) of the flow-through (depleted plasma) were collected. The 20 high abundance plasma proteins bound to the column resin were eluted with 0.1 M glycine-HCl, pH 2.5, and 0.1% (w/v) octyl β-D-glucopyranoside.

FIG. 11 presents photograph images of SDS-PAGE of the depleted plasma fractions. An aliquot of the fractions collected during chromatography to remove the 20 high abundance protein from plasma were resolved by SDS-PAGE, as were an aliquot of the pooled fractions and whole plasma. Panel A shows a gel stained with Coomassie blue. Panel B shows a silver-stained gel.

FIG. 12 is a graphical representation depicting the percent depletion of the 20 high abundance proteins from 100 μl of plasma using a 10 ml immunoaffinity column. Protein levels were determined using an ELISA assay.

Claims

1. A method for separating at least two distinct targets from a sample and simultaneously fractionating at least two distinct non-targets from the sampler the method comprising: a. contacting the sample with a composition comprising a plurality of heterogeneous antibodies stochastically conjugated en mass to a solid support, the antibodies having affinity for distinct targets such that when the antibodies contact the targets they bind to the targets thereby separating the targets from the sample; andb. collecting at least two fractions of the target-depleted sample, such that each fraction comprises at least one distinct non-target.

2. The method of claim 1, wherein the sample is liquid and is contacted with the composition by perfusion over the solid support.

3. The method of claim 2, wherein the solid support is selected from the group consisting of resins, beads, emulsions, glass supports, silica supports, polymer supports, copolymer supports, magnetic supports, powders, nano-capillaries, and other nano-materials.

4. The method of claim 3, wherein the resins, beads, or emulsions are contained in a column.

5. The method of claim 4, wherein the beads are selected from the group consisting of agarose beads, polyacrylamide beads, polyacrylic beads, copolymer beads, silica beads, and magnetic beads.

6. The method of claim 5, wherein the fractions are collected during a procedure selected from the group consisting of spin-column chromatography, fast protein liquid chromatography (FPLC), low pressure liquid chromatography, and high performance liquid chromatography (HPLC).

7. The method of claim 1, wherein the sample is derived from a species selected from the group consisting of mouse, rat, cow, dog, cat, chicken, rhesus monkey, chimpanzee, zebrafish, Drosophila, Dictyostelium, yeast, Arabidopsis, and rice.

8. The method of claim 1, wherein the sample is derived from a human.

9. The method of claim 1, wherein the sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, tears, urine, feces, saliva, vaginal fluid, nipple aspirate/lactation fluid, semen, perspiration, peritoneal fluid, ravages, cell lysates, cell culture supernatants, and cell culture lysates.

10. The method of claim 1, wherein the antibodies are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single chain antibodies, peptide epitopes, and antibody fragments.

11. The method of claim 1, wherein the antibodies are a mixture of IgG's, IgY's, and single chain antibodies.

12. The method of claim 1, wherein the antibodies bind from about 5 to about 100 distinct targets.

13. The method of claim 1, wherein the target and the non-target are selected from the group consisting of proteins, peptides, protein-containing complexes, nucleic acids, and small metabolites.

14. The method of claim 1, wherein the sample is human plasma, the targets and the non-targets are proteins, the solid support comprises size exclusion agarose beads, such that the non-targets are separated on the basis of size, and the fractions of the target-depleted sample are collected during liquid chromatography.