METHODS FOR DETECTING NEUTRALIZING ANTIBODIES FOR BONE MORPHOGENETIC PROTEINS
The present invention relates to methods of detecting neutralizing antibodies for bone morphogenetic proteins (BMP). More particularly, it relates to a highly specific, robust, rapid and accurate cell-based assay for detecting the presence of anti-BMP neutralizing antibodies.
1 . A method for the detection of neutralizing antibodies to a bone morphogenetic protein (BMP) in a sample comprising the steps of:
(a) contacting said sample with a BMP;
(b) incubating said sample from step (a) with a BMP-responsive cell comprising at least one endogenous gene, the expression of which is capable of being modulated by exposure to said BMP;
(c) isolating mRNA from said cell;
(d) preparing cDNA corresponding to said mRNA by reverse transcription;
(e) amplifying said gene from said cDNA of step (d) by quantitative real-time polymerase chain reaction (QPCR);
(f) determining the amount of said gene amplified in step (e) in said cells;
(g) determining the amount of said gene amplified according to step (e) in control cells contacted with BMP alone; and
(h) detecting the presence of neutralizing antibodies in said sample if the amount determined in step (f) is less than or greater than the amount determined in step (g).
2 . The method of claim 1 , further comprising the steps of:
(i) amplifying a housekeeping gene from said cDNA of step (d) by quantitative real-time polymerase chain reaction (QPCR);
(j) determining the amount of said housekeeping gene amplified in step (i) in said cells; and
(k) normalizing the amount of said gene determined in step (f) with the amount of said housekeeping gene determined in step (j).
3 . The method of claim 1 , wherein said BMP is selected from the group consisting of OP-1 (BMP-7), OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18, DPP, Vg1, Vgr, 60A protein, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, CDMP-1, CDMP-2, CDMP-3, NODAL, UNIVIN, SCREW, ADMP, NEURAL, and fragments thereof.
4 . The method of claim 3 , wherein said BMP is OP-1 (BMP-7).
5 . The method of claim 1 , wherein said sample is serum or human serum.
6 . (canceled)
7 . The method of claim 1 , wherein said BMP-responsive cell is selected from the group consisting of A549, SAOS-2, ROS and CSC12.
8 . (canceled)
9 . The method of claim 1 , wherein said gene is:
a) an early gene;
b) an early gene selected from the group consisting of Id-1, Id-2, Id-3, Id-4, Msx2, Dlx2, Dlx3, Dlx5, Noggin, Smad6, Smad7 and Runx2;
c) a late gene; or
d) a late gene is selected from the group consisting of HEY1, DIO2, ADAMTS9, HAS3, FGFR3, MFI2, CHI3L1, NOG, BAMBI, GREM1, GREM2 and SOST.
10 . (canceled)
11 . (canceled)
12 . (canceled)
13 . (canceled)
14 . The method of claim 1 , wherein said sample is contacted with a BMP in step (a) for at least 30 minutes.
15 . The method of claim 1 , wherein said sample from step (b) is incubated with a BMP-responsive cell for about 3 hours.
16 . The method of claim 1 , wherein the antibody is specific for OP1 (BMP-7).
17 . The method of claim 1 , wherein said sample is subjected to at least one prescreening assay prior to performing steps (a)-(h).
18 . The method of claim 17 , wherein said prescreening assay is any assay that is capable of detecting neutralizing antibodies to a bone morphogenetic protein (BMP).
19 . The method of claim 18 , wherein said prescreening assay is selected from the group consisting of immunoassay and cell-based assay.
20 . The method of claim 19 , wherein said immunoassay is an enzyme-linked-immunosorbent assay (ELISA).
21 . The method of claim 19 , wherein said cell-based assay involves activation of a reporter gene.
22 . The method of claim 21 , wherein said reporter gene is a luciferase gene.
23 . The method of claim 22 , wherein said luciferase gene is linked to a promoter of a gene that is capable of being modulated by exposure to a BMP.
24 . The method of claim 23 , wherein said gene is an early or late gene.
25 . The method of claim 23 , wherein said gene is selected from the group consisting of Id-1, Id-2, Id-3, Id-4, Msx2, Dlx2, Dlx3, Dlx5, Noggin, Smad6, Smad7, Runx2, fibromodulin, Hey 1 and SFRP-2.
26 . The method of claim 19 , wherein said cell-based assay involves monitoring the activity of alkaline phosphatase.
27 . (canceled)