ANTIBODIES FOR THE DETECTION OF INTEGRIN COMPLEXES IN FFPE MATERIAL
First Claim
1. Monoclonal rabbit antibody, or a fragment thereof, against integrin with insect-derived glycosylation pattern and integrin with another eukaryotic glycosylation pattern, each comprising at least a light chain variable region (VL) and a heavy chain variable region (VH), wherein the antibody has antigen binding specificity for a non-occluded epitope of an extracellular integrin domain or extracellular integrin chain domain, and wherein the antibody is able to bind to intact heterodimers of integrin in formalin fixed paraffin embedded (FFPE) material and on viable cells with the substantially same specificity.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention relates to antibodies that are capable to bind the extracellular domain of integrin. Another object of the invention concerns the use of said antibodies for detecting integrins in archival formalin fixed paraffin embedded (FFPE) tissue. The invention also relates to methods for preparing monoclonal rabbit antibodies, wherein the immunogen is an insect expression culture-derived recombinant extracellular integrin domain, and another method for screening anti-integrin antibodies that discriminate between closest integrin homologues and that are especially suited for immunohistochemistry in FFPE material.
-
Citations
15 Claims
- 1. Monoclonal rabbit antibody, or a fragment thereof, against integrin with insect-derived glycosylation pattern and integrin with another eukaryotic glycosylation pattern, each comprising at least a light chain variable region (VL) and a heavy chain variable region (VH), wherein the antibody has antigen binding specificity for a non-occluded epitope of an extracellular integrin domain or extracellular integrin chain domain, and wherein the antibody is able to bind to intact heterodimers of integrin in formalin fixed paraffin embedded (FFPE) material and on viable cells with the substantially same specificity.
- 8. Recombinant immunogen consisting of an extracellular integrin domain with insect-derived glycosylation pattern, optionally coupled as delta-trans membrane form.
-
12. Method for preparing monoclonal rabbit antibodies comprising the steps of:
-
(a) recombinantly expressing an extracellular integrin domain in insect cells, (b) purifying the expressed extracellular integrin domain, (c) immunizing a rabbit with the purified extracellular integrin domain, (d) taking polyclonal antiserum comprising polyclonal antibodies from the rabbit, and (e) preparing the monoclonal antibodies.
-
-
13. Method for manufacturing a recombinant monoclonal antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH) with the steps of:
-
(a) introducing at least one vector comprising antibody-encoding nucleic acid sequences of (i) SEQ ID NO;
115 (VL-α
vβ
3) and SEQ ID NO;
116 (VH-α
vβ
3),(ii) SEQ ID NO;
35 (VL-α
vβ
5) and SEQ ID NO;
36 (VH-α
vβ
5),(iii) SEQ ID NO;
155 (VL-α
vβ
6) and SEQ ID NO;
156 (VH-α
vβ
6),(iv) SEQ ID NO;
195 (VL-α
vβ
8) and SEQ ID NO;
196 (VH-α
vβ
8), or(v) SEQ ID NO;
235 (VL-α
v) and SEQ ID NO;
236 (VH-α
v) into a host cell,(b) cultivating the host cell in a culture medium, thereby expressing the encoded antibody, and (c) purifying the expressed antibody.
-
-
15. Method for screening anti-integrin antibodies, which are capable of discriminating between the respective closest homologues of integrin α
- -subunit and/or β
-subunit and suitable for immunohistochemistry in formalin fixed paraffin embedded (FFPE) material, comprising the steps of;(a) providing a sample of antibodies being capable of binding a selected integrin, (b) aligning integrin sequences to identify the closest homologue of the α
-subunit and/or β
-subunit of the selected integrin,(c) performing a differential ELISA on native forms of the selected integrin and the closest homologue(s) thereof with the antibody sample, thereby accumulating antibodies against the selected integrin (primary screen), (d) performing another differential ELISA on native forms of the selected integrin and another integrin with the accumulated antibodies of step (c), thereby further accumulating antibodies against the selected integrin (secondary screen), (e) performing immunohistochemistry of FFPE cell lines with the accumulated antibodies of step (d), wherein at least one cell line is capable of expressing the selected integrin and optionally another cell line is not capable of expressing the selected integrin, thereby further accumulating antibodies against the selected integrin (tertiary screen), (f) performing immunohistochemistry of FFPE cell lines of step (e) with the accumulated antibodies of step (e), wherein the cell line is grown as xenograft tumor in a mammal, thereby further accumulating antibodies against the selected integrin (quaternary screen), and (g) performing immunohistochemistry of archival FFPE tumors with the accumulated antibodies of step (f), thereby further accumulating antibodies against the selected integrin (quintenary screen).
- -subunit and/or β
Specification