ANTIBODIES

Abstract

A method for analyzing protein(s) in a sample using an immunoassay kit includes creating protein-reducing and/or protein-denaturing conditions by contacting the sample with a reducing and/or denaturing agent provided in the immunoassay kit, to provide a partially or fully denatured protein population. One or both of a presence and an amount of one or more protein-associated analytes are determined under the created protein-reducing and/or protein-denaturing conditions by contacting the partially or fully denatured protein population with one or more specific antibodies or binding fragments thereof provided in the immunoassay kit. The one or more specific antibodies or binding fragments thereof include one or more chemically-introduced non-disulfide cross-links between at least one heavy chain or binding fragment thereof and at least one light chain or binding fragment thereof.

Claims

1. A method for analyzing protein(s) in a sample using an immunoassay kit, comprising: creating protein-reducing and/or protein-denaturing conditions by contacting the sample with a reducing and/or denaturing agent provided in the immunoassay kit, to provide a partially or fully denatured protein population; and determining one or both of a presence and an amount of one or more protein-associated analytes under the created protein-reducing and/or protein-denaturing conditions by contacting the partially or fully denatured protein population with one or more specific antibodies or binding fragments thereof provided in the immunoassay kit; wherein the one or more specific antibodies or binding fragments thereof comprise one or more chemically-introduced non-disulfide cross-links between at least one heavy chain or binding fragment thereof and at least one light chain or binding fragment thereof.

2. The method of claim 1, wherein the one or more chemically-introduced non-disulfide cross-links define a bond selected from the group consisting of a bismaleimide bond and a thioether bond.

3. The method of claim 2, wherein the one or more specific antibodies or binding fragments thereof are cross-linked at a cross-linking efficiency selected from the group consisting of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%.

4. The method of claim 1, wherein the one or more protein-associated analytes are epitopes hidden in an interior of a native, non-reduced and/or non-denatured folded structure of the protein(s).

5. The method of claim 1, including providing the one or more specific antibodies or binding fragments attached to a support.

6. The method of claim 1, wherein the one or more specific antibodies or binding fragments thereof are selected from the group consisting of monoclonal antibodies or binding fragments thereof or polyclonal antibodies or binding fragments thereof.

7. The method of claim 6, wherein the one or more binding fragments thereof are F(ab′)2 fragments of the one or more specific antibodies.

8. The method of claim 1, including providing the immunoassay kit comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or binding fragments thereof or anti-free light class specific antibodies or binding fragments thereof.

9. The method of claim 1, including providing the immunoassay kit comprising reagents for performing an immunoassay selected from the group consisting of a radioimmune assay comprising one or more radioisotopes, a lateral flow assay comprising a test strip or dipstick, an ELISA-type assay comprising an enzyme adapted to convert a substrate into a detectable label, a nephelometric assay, a turbidimetric assay, a flow cytometry assay comprising one or more detectable particles, a fluorescent assay comprising one or more fluorescent labels, a chemiluminescent assay comprising one or more chemiluminescent labels, and a bead-type assay comprising detectably-labeled beads.

10. The method of claim 1, including selecting the sample from the group consisting of serum, whole blood, plasma, urine, and tissue.

11. A method for analyzing protein(s) in a sample using an immunoassay kit, comprising: creating protein-reducing and/or protein-denaturing conditions by contacting the sample with a reducing and/or denaturing agent provided in the immunoassay kit, to provide a partially or fully denatured protein population; and determining one or both of a presence and an amount of one or more protein-associated analytes under the created protein-reducing and/or protein-denaturing conditions by contacting the partially or fully denatured protein population with one or more specific antibodies or binding fragments thereof provided in the immunoassay kit; wherein the one or more specific antibodies or binding fragments thereof comprise one or more chemically-introduced non-disulfide cross-links between at least one heavy chain or binding fragment thereof and at least one light chain or binding fragment thereof; further wherein the one or more protein-associated analytes are hidden in an interior of a native, non-reduced and/or non-denatured folded structure of the protein(s).

12. The method of claim 11, wherein the one or more chemically-introduced non-disulfide cross-links define a bond selected from the group consisting of a bismaleimide bond and a thioether bond.

13. The method of claim 12, wherein the one or more specific antibodies or binding fragments thereof are cross-linked at a cross-linking efficiency selected from the group consisting of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%.

14. The method of claim 11, including providing the one or more specific antibodies or binding fragments attached to a support.

15. The method of claim 11, wherein the one or more specific antibodies or binding fragments thereof are selected from the group consisting of monoclonal antibodies or binding fragments thereof or polyclonal antibodies or binding fragments thereof.

16. The method of claim 15, wherein the one or more binding fragments thereof are F(ab′)2 fragments of the one or more specific antibodies.

17. The method of claim 11, including providing the immunoassay kit comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or binding fragments thereof or anti-free light class specific antibodies or binding fragments thereof.

18. The method of claim 11, including providing the immunoassay kit comprising reagents for performing an immunoassay selected from the group consisting of a radioimmune assay comprising one or more radioisotopes, a lateral flow assay comprising a test strip or dipstick, an ELISA-type assay comprising an enzyme adapted to convert a substrate into a detectable label, a nephelometric assay, a turbidimetric assay, a flow cytometry assay comprising one or more detectable particles, a fluorescent assay comprising one or more fluorescent labels, a chemiluminescent assay comprising one or more chemiluminescent labels, and a bead-type assay comprising detectably-labeled beads.

19. The method of claim 11, including selecting the sample from the group consisting of serum, whole blood, plasma, urine, and tissue.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The invention will now be described by way of example only with respect to the following figures:

[0108] FIG. 1 shows Coomassie Blue stained gels illustrating the formation of thioether bonds in anti-free kappa F(ab′)2 and anti-free lambda F(ab′)2 antibody fragments. The formation of cross-linked light and heavy chains of the F(ab′)2 molecule are indicated in the SDS-PAGE gel image (arrow at approximately 50 kDa). The degree of cross-linking is higher at 7 days.

[0109] FIG. 2 shows Coomassie Blue stained gels of treated (to induce thioether bond formation) and non-treated anti-free kappa F(ab′)2 fragments. (NR) non-reduced samples (R) reduced samples. Interpretation of the structures in each band is also shown.

[0110] FIG. 3 similarly shows anti-free lambda F(ab′)2 antibody fragments.

[0111] FIG. 4 shows the binding activity of thioether cross-linked and non-cross-linked anti-free kappa and anti-free lambda F(ab′)2 antibodies to free light chains (FLC) and normal human serum (NHS).

[0112] FIG. 5 shows the cross-linking of anti-lambda total F(ab′)2 antibodies by bismaleimidoethane (BMOE), as measured by reducing Coomassie Blue stained SDS-PAGE.

[0113] FIG. 6 shows the binding of anti-lambda total F(ab′)2 antibodies to IgG lambda before and after bismaleimidoethane (BMOE) cross-linking.

[0114] FIG. 7 shows anti-free lambda F(ab′) 2 (10 μM) cross-linked with 2 mM bismaleimidoethane (BMOE) and anti-free kappa whole molecule (101.1M) cross-linked with 4 mM BMOE, as measured by reducing Coomassie Blue stained SDS-PAGE. Scanning densitometry of the reduced SDS-PAGE lanes indicated a cross-linking efficiency of >70% for anti-free lambda and >90% for anti-free kappa.

[0115] FIG. 8 shows untreated and bismaleimidoethane (BMOE) cross-linked anti-free kappa whole molecule binding to kappa free light chains. Cross-reactivity to IgG, IgA and IgM is also shown.

[0116] FIG. 9 shows untreated and bismaleimidoethane (BMOE) cross-linked anti-free lambda F(ab′)2 binding to lambda free light chains. Cross-reactivity to IgG, IgA and IgM is also shown.

[0117] FIG. 10 shows the cross-linking and stabilisation of anti-prealbumin antibodies by bismaleimidoethane (BMOE) treatment, as illustrated by Coomassie Blue stained SDS-PAGE analysis. Without prior cross-linking (left panel), the antibodies spontaneously disassociated into their constituent heavy (H.sub.1) and light (L.sub.1) chain fragments in the presence of reducing agent (50 mM Dithiothreitol; +DTT). Following treatment with BMOE (right panel) the antibodies became resistant to reducing conditions and migrated at apparent molecular weights consistent with the formation of stable heavy-light chain pairs (H.sub.1L.sub.1 and H.sub.2L.sub.2). Interpretation of the structures in each band is shown.

[0118] FIG. 11 shows the binding activity of BMOE-stabilised anti-prealbumin antibodies (Xa.PA) by ELISA analysis. Anti-prealbumin antibodies were treated with a range of chemical conditions prior to the addition of purified prealbumin protein and bound prealbumin was detected with biotinylated anti-prealbumin using Streptavidin-HRP. Binding activity is shown relative to unstabilised anti-prealbumin (a.PA). (PBS), Phosphate Buffered Saline; (DTT), 50 mM Dithiotheitol; (Tween), 0.2% Tween-20; (SDS), 0.1% Sodium Dodecyl Sulfate.

[0119] FIG. 12 shows the cross-linking of anti-IgG antibodies by BMOE (A) and the binding activity of BMOE-stabilised anti-IgG antibodies following chemical treatment (B), as illustrated by Coomassie Blue stained SDS-PAGE analysis. Panel (A) shows that BMOE-treated anti-IgG antibodies are resistant to 50 mM DTT. In panel (B), stabilised antibodies were immobilised on a solid matrix and treated with Phosphate Buffered Saline (CTRL) or a mixture of Glycine (0.1M, pH 3), tris(2-carboxyethyl)phosphine (TCEP, 2 mM) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 0.3%) containing varying amounts of Urea. Following the addition of purified human IgG, specifically bound proteins were eluted with 0.1M Glycine pH 2.5 and analysed by SDS-PAGE.

DETAILED DESCRIPTION

[0120] The conversion of antibody disulphide bonds to thioether bonds may be induced in alkaline environments at raised temperature. Formation of such bonds is generally known in the art, such as in Zhang et al IgG1 Thioether Bond formation in vivo. JBC, 288:16371-16382, 2013. Zhang and Flynn. Cysteine racemization IgG heavy and light chains, JBC, 288:34325-34335, 2013.

[0121] Anti-kappa free light chain and anti-lambda free light chain F(ab).sub.2 antibodies were investigated to see whether thioether bonds could be introduced into the fragments. The Applicant manufactures anti-kappa and anti-lambda antisera and sells immunoassays that utilise anti-kappa and anti-lambda antisera under the trade mark Freelite™. These antibodies bind either free lambda or free kappa light chains. The data shown in FIGS. 1-4 shows that it is possible to introduce thioether cross-links into F(ab′)2 fragments by treatment with 50 mM Glycine-NaOH, pH 9 at 50° C. Approximately 20% cross-linking efficiency is observed compared to untreated F(ab′)2 after 7 days (FIGS. 1-3). Whilst there is some reduction in ELISA activity, as shown in FIG. 4, activity still remains in the treated antibodies.

[0122] The binding activity of anti-free kappa and anti-free lambda antibodies was assessed by ELISA after 0 and 7 days alkaline treatment (FIG. 4). Antibodies were coated onto ELISA plates and presented with purified kappa light chains, purified lambda light chains or Normal Human Serum. Binding activity was detected by measuring absorbance at 450 nm using Tetramethylbenzidine (TMB) chromogenic substrate and anti-light chain antibodies conjugated to Horse Radish Peroxidase (HRP). Whilst there is some reduction in ELISA activity, as shown in FIG. 4, activity still remains in the treated antibodies.

[0123] Bismaleimidoethane (BMOE) Crosslinking of Anti-Lambda Total F(Ab)2 Fragments

[0124] Anti-lambda F(ab′)2 antibodies were investigated to see if antibody chains could be cross-linked by BMOE. Anti-lambda F(ab′)2 fragments were reduced with 1 mM (TCEP). The TCEP was removed using Hi-Trap Desalting columns and the reduced anti-lambda total F(ab′) 2 was cross-linked at 100-500× fold molar excess of BMOE and then analysed by Coomassie Blue stained SDS-PAGE run under reducing conditions. FIG. 5 shows that BMOE can cross-link F(ab′)2 fragments with an efficiency of over 50%. Moreover, the resulting antibody chain cross-links are resistant to reducing conditions.

[0125] An ELISA plate was coated with polyclonal IgG Lambda and BMOE-treated or untreated anti-total lambda F(ab′) 2 was bound to the plate. Binding activity by anti-total lambda was measured by light absorbance at 450 nm using anti-sheep-HRP and TMB substrate. Under conditions that produce >50% BMOE cross-linking (FIG. 5), anti-total lambda antibody retains over 70% IgG Lambda binding activity (FIG. 6).

[0126] BMOE Cross-Linked Anti Free Lambda and Anti Free Kappa Antisera

[0127] Commercially available Freelite™ antibodies were investigated to see whether antibody chains could be cross-linked by BMOE treatment. Whole molecule anti-free kappa and F(ab′).sub.2 anti-free lambda were reduced with 1.5 mM and 1.0 mM TCEP, respectively. The TCEP was removed using Hi-Trap Desalting columns and the anti-free kappa and anti-free lambda antibodies were cross-linked at 400-fold and 200-fold molar excess of BMOE, respectively. Samples were analysed by Coomassie Blue stained SDS-PAGE run under reducing conditions. As shown in FIG. 7, the BMOE cross-linking efficiency is over 50% for antibodies in either F(ab′)2 or whole molecule formats.

[0128] Activity ELISA assays were performed whereby BMOE-treated and untreated antibodies were coated onto ELISA plates and presented with purified immunoglobulins (IgG, IgA, IgM, free kappa, free lambda). Binding activity was detected by measuring absorbance at 450 nm using anti-light chain-HRP and TMB substrate. The results in FIGS. 8-9 show that cross-linked antisera still maintain specific antigen binding activity. Stabilisation of anti prealbumin antibodies by treatment with BMOE.

[0129] Anti-human prealbumin antibodies were investigated to see if antibody chains could be cross-linked by BMOE. Anti-prealbumin fragments were reduced with 250-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP). The TCEP was removed using Hi-Trap Desalting columns and the reduced anti-prealbumin was cross-linked at 400-fold molar excess of BMOE and then analysed by Coomassie Blue stained SDS-PAGE run in the presence or absence of reducing agent (50 mM DTT). FIG. 10 shows that without prior cross-linking, the antibodies spontaneously break down into their constituent heavy and light chains upon DTT treatment. In contrast, greater than 80% of the BMOE-treated antibodies remained associated as heavy-light chain pairs (in H.sub.2L.sub.2 or H.sub.1L.sub.1 forms) irrespective of the presence of DTT, thus indicating a high degree of structural stability.

[0130] The antigen binding activity of BMOE-stabilised anti prealbumin is resistant to reducing and detergent conditions.

[0131] Conventional mammalian antibodies (e.g. IgG) express an antigen binding site (also known as the Complementarity Determining Region or Paratope) on each F(ab) fragment. These sites are formed by the associated variable domains from paired heavy and light chains, which both contribute to antigen recognition and binding activity. Therefore, under conditions that disrupt heavy-light chain pairing, the binding activity of antibodies for their target antigen would be compromised. As BMOE-treatment was shown in FIG. 10 to stabilise heavy-light chain pairing, the binding activity of anti-prealbumin was investigated to determine whether BMOE-stabilisation protects the antibodies from reducing conditions and protein denaturants. In FIG. 11, anti-prealbumin antibodies (5 μg/mL) were coated onto microwell plates and treated with a range of chemical conditions prior to the addition of purified prealbumin protein (0.1 μg/mL). Bound prealbumin was detected with biotinylated anti-prealbumin (0.2 μg/mL) using Streptavidin-HRP and 3,3′,5,5′-Tetramethylbenzidine chromogenic substrate (TMB). The data in FIG. 11 show that BMOE cross-linking of anti-prealbumin (Xa.PA, PBS) only had a marginal effect on prealbumin binding activity when compared to unstabilised anti-prealbumin (a.PA, PBS). Furthermore, treatment of BMOE-stabilised anti-prealbumin with 50 mM DTT had no significant effect on activity, even in the presence of non-ionic detergent (Tween 20, 0.2%). It was also observed that BMOE-stabilised anti-prealbumin retained antigen binding activity after exposure to Sodium Dodecyl Sulfate (SDS, 0.1%), an anionic detergent generally known in the art to act as a protein denaturant. Therefore, the data in FIGS. 10 and 11 illustrate that cross-linking of anti-prealbumin antibodies with BMOE confers a high degree of structural stability, minimally affects antigen binding activity under physiological conditions and protects the antigen binding activity from reducing agents and protein denaturants.

[0132] Anti-IgG Antibodies are Stabilised by BMOE Cross-Linking

[0133] Anti-human IgG antibodies were investigated to see if antibody chains could be cross-linked by BMOE. Anti-IgG fragments were reduced with 250-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP). The TCEP was removed using Hi-Trap Desalting columns and the reduced anti-prealbumin was cross-linked at 400-fold molar excess of BMOE and then analysed by Coomassie Blue stained SDS-PAGE run in the presence or absence of reducing agent (50 mM DTT). The results in FIG. 12A show that greater than 80% of the BMOE-treated antibodies remained associated as heavy-light chain pairs irrespective of the presence of DTT, thus indicating a high degree of structural stability.

[0134] The BMOE-stabilised anti-IgG antibodies were also investigated to determine what effect exposure to a combination of low pH, reducing agent, detergent and chaotropic agent would have on the antigen binding activity. Antibodies were cross-linked with BMOE and covalently attached to a solid matrix by conventional amine-linkage chemistry. The antibodies were subsequently treated with Phosphate Buffered Saline as a control (CTRL) or Glycine (pH 3 buffer), TCEP (reducing agent) and CHAPS (detergent) containing varying concentrations of Urea (Chaotrope). Purified human IgG was applied and the specifically captured proteins were eluted using Glycine pH 2.5 buffer and analysed by SDS-PAGE. As shown in FIG. 12, the BMOE-stabilised anti-IgG antibodies retained antigen binding activity under all of the conditions tested. This illustrates that BMOE-stabilisation renders anti-IgG antibodies resistant to a range of chemical conditions.

[0135] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. Obvious modifications and variations are possible in light of the above teachings. All such modifications and variations are within the scope of the appended claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.