ANTIBODIES

Abstract

The invention provides an immunoassay kit providing an immunoassay kit comprising one or more analyte specific antibodies or fragments thereof, characterised that the antibody or fragment thereof comprises one or more non-disulphide cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the analyte- specific antibodies or fragments thereof.

Claims

1. An immunoassay kit, comprising: one or more analyte specific antibodies or fragments thereof, wherein the antibody or fragment thereof comprises one or more non-disulphide cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the analyte-specific antibodies or fragments thereof.

2. The immunoassay kit according to claim 1, additionally comprising one or more reducing agents.

3. The immunoassay kit according to claim 1, wherein the immunoassay is a radioimmune assay, a lateral flow assay, an ELISA-type assay, a nephelometric assay a turbidimetric assay, a flow cytometry assay, a fluorescent assay, a chemiluminescent assay, or a bead-type assay.

4. The immunoassay kit according to claim 1, wherein the cross-link comprises a bismaleimide or a thioether bond.

5. The immunoassay kit according to claim 1, wherein the antibody or fragment thereof is attached to a support.

6. The immunoassay kit according to claim 1, wherein the antibody or fragment is a monoclonal antibody or fragment thereof or a polyclonal antibody or fragment thereof.

7. The immunoassay kit according to claim 1, wherein the antibody is a fragment of an antibody and is an F(ab).sub.2 fragment.

8. The immunoassay kit according to claim 1, comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or fragments thereof or anti-free light class specific antibodies or fragments thereof.

9. A method of detecting an analyte in a sample comprising: adding a reducing or denaturing agent to the sample and using an immunoassay kit according to claim 1 to detect the presence of, or an amount of, the analyte in the sample.

10. The immunoassay kit according to claim 2, wherein the cross-link comprises a bismaleimide or a thioether bond.

11. The immunoassay kit according to claim 2, wherein the antibody or fragment thereof is attached to a support.

12. The immunoassay kit according to claim 4, wherein the antibody or fragment thereof is attached to a support.

13. The immunoassay kit according to claim 2, wherein the antibody is a fragment of an antibody and is an F(ab).sub.2 fragment.

14. The immunoassay kit according to claim 4, wherein the antibody is a fragment of an antibody and is an F(ab).sub.2 fragment.

15. The immunoassay kit according to claim 5, wherein the antibody is a fragment of an antibody and is an F(ab).sub.2 fragment.

16. The immunoassay kit according to claim 2, comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or fragments thereof or anti-free light class specific antibodies or fragments thereof.

17. The immunoassay kit according to claim 4, comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or fragments thereof or anti-free light class specific antibodies or fragments thereof.

18. The immunoassay kit according to claim 5, comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or fragments thereof or anti-free light class specific antibodies or fragments thereof.

19. The immunoassay kit according to claim 6, comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or fragments thereof or anti-free light class specific antibodies or fragments thereof.

20. The immunoassay kit according to claim 7, comprising one or more additional anti-immunoglobulin class or immunoglobulin-type specific antibodies or fragments thereof or anti-free light class specific antibodies or fragments thereof.

Description

[0126] 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.

[0127] 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.

[0128] 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

[0129] Bismaleimidoethane (BMOE) crosslinking of anti-lambda total F(ab)2 fragments

[0130] 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).sub.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.

[0131] An ELISA plate was coated with polyclonal IgG Lambda and BMOE-treated or untreated anti-total lambda F(ab).sub.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).

[0132] BMOE Cross-Linked Anti-Free Lambda and Anti-Free Kappa Antisera

[0133] 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.

[0134] 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.

[0135] Stabilisation of Anti-Prealbumin Antibodies by Treatment with BMOE.

[0136] 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.

[0137] The Antigen Binding Activity of BMOE-Stabilised Anti-Prealbumin is Resistant to Reducing and Detergent Conditions.

[0138] 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 g/mL) were coated onto microwell plates and treated with a range of chemical conditions prior to the addition of purified prealbumin protein (0.1 g/mL). Bound prealbumin was detected with biotinylated anti-prealbumin (0.2 g/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.

[0139] Anti-IgG Antibodies are Stabilised by BMOE Cross-Linking

[0140] 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.

[0141] 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.