COLLAGEN IV BINDING ASSAY FOR THE DETECTION OF COLLAGEN VII

1 . A method of detecting and/or quantifying collagen VII or fragment thereof in a sample, the method comprising:

contacting the sample with collagen IV or a fragment thereof which binds to collagen VII; and

detecting binding of collagen VII or a fragment thereof to collagen IV or the fragment thereof, to thereby detect and/or quantifying the amount of collagen VII or the fragment thereof in the sample.

2 . The method of claim 1 , wherein the collagen VII or fragment thereof comprises, consists essentially of, or consists of: an amino acid sequence which is at least 70, 80, 90, 95, 99 or 100% percent identical to the amino acid sequence of SEQ ID NO: 1; an amino acid sequence of at least 2000, 2500, or 2900 contiguous amino acids of SEQ ID NO: 1; or an amino acid sequence of SEQ ID NO: 1.

3 . The method of claim 1 , wherein full length collagen VII is detected or quantified.

4 . The method of claim 1 , wherein collagen VII or a fragment thereof comprising both the NC1 and NC2 domains is detected or quantified.

5 . The method of claim 1 , further comprising contacting the sample with an anti-collagen VII antibody, which antibody binds collagen VII or a fragment thereof when complexed to collagen IV or a fragment thereof, e.g., it binds collagen VII in a collagen VII-collagen IV complex.

6 . The method of claim 1 , further comprising contacting the sample with an anti-collagen VII antibody, which binds to an epitope of collagen VII or a fragment thereof which is accessible after the binding of collagen VII or a fragment thereof to collagen IV or the fragment thereof.

7 . The method of claim 6 , wherein the anti-collagen VII antibody binds to the NC2 domain of collagen VII.

8 . The method of claim 6 , wherein the anti-collagen VII antibody binds to the NC1 domain of collagen VII.

9 . The method of claim 1 , further comprising contacting the sample with a second antibody, which binds to the anti-collagen VII antibody, e.g., a second antibody that has a detectable label.

10 . The method of claim 9 , comprising detecting the presence of the anti-collagen VII antibody, e.g., detecting anti-collagen VII antibody bound to collagen VII or a fragment thereof.

11 . The method of claim 9 , comprising quantifying the amount of anti-collagen VII antibody, e.g., quantifying anti-collagen VII antibody bound to collagen VII or a fragment thereof.

12 . The method of claim 1 , further comprising measuring the level of collagen VII bound to collagen IV. The method of claim 1 wherein, the collagen IV or the fragment thereof is disposed on a support, e.g., an insoluble or solid support, e.g., it is support, e.g., an insoluble or solid support prior to contacting with collagen VII or a fragment thereof.

13 . The method of claim 1 wherein, the sample is biological sample, e.g., a tissue biopsy, skin biopsy, a biological fluid, blood, or culture medium.

14 . The method of claim 1 wherein, the sample is a drug product preparation or a drug substance preparation.

15 . A method of detecting and/or quantifying collagen VII or a fragment thereof in a sample, wherein the collagen VII or fragment thereof detected or quantified comprises, consists essentially of, or consists of an amino acid sequence which is at least 70, 80, 90, 95, 99 or 100% percent identical to the amino acid sequence of SEQ ID NO: 1;

an amino acid sequence of at least 2000, 2500, or 2900 contiguous amino acids of SEQ ID NO: 1; or

an amino acid sequence of SEQ ID NO: 1;

the method comprising:

contacting the sample with collagen IV or a fragment thereof which binds to collagen VII; contacting the sample with an anti-collagen VII antibody which binds to the NC2 domain of collagen VII;

detecting the binding of the anti-collagen VII antibody;

to thereby detect and/or quantify collagen VII or fragment thereof in the sample.

16 . The method of claim 15 , wherein full length collagen VII is detected or quantified.

17 . The method of claim 16 , wherein collagen VII or a fragment thereof comprising both the NC1 and NC2 domains is detected or quantified. The method of claim 1 , further comprising contacting the sample with an anti-collagen VII antibody, which antibody binds collagen VII or a fragment thereof when complexed to collagen IV or the fragment thereof, e.g., it binds collagen VII in a collagen VII-collagen IV complex.

18 . The method of claim 16 , further comprising contacting the sample with a second antibody, which binds to the anti-collagen VII antibody, e.g., a second antibody that has a detectable label.

19 . The method of claim 18 , comprising detecting the presence of the anti-collagen VII antibody, e.g., detecting anti-collagen VII antibody bound to collagen VII or a fragment thereof.

20 . The method of claim 19 , comprising quantifying the amount of anti-collagen VII antibody, e.g., quantifying anti-collagen VII antibody bound to collagen VII or a fragment thereof.

21 . The method of claim 16 , further comprising measuring the level of collagen VII bound to collagen IV.

22 . The method of claim 16 wherein, the collagen IV or the fragment thereof is disposed on a support, e.g., an insoluble or solid support, e.g., it is support, e.g., an insoluble or solid support prior to contacting with collagen VII or fragment thereof.

23 . The method of claim 16 wherein, the sample is biological sample. In certain embodiments the sample is a tissue biopsy, skin biopsy, a biological fluid, blood, or culture medium.

24 . The method of claim 16 wherein, the sample is a drug product preparation or a drug substance preparation.

25 . A method of analyzing or processing a collagen VII preparation, e.g., to determine whether to accept or reject a batch of a collagen VII, or to guide or control of a step in the production of collagen VII, e.g., by a method described herein.

26 . A method of evaluating or processing a collagen VII preparation, e.g., a collagen VII drug substance preparation or a collagen VII drug product preparation, e.g., by a method described herein.

27 . The method of claim 26 , wherein the method comprises:

acquiring an evaluation of the presence or amount of collagen VII, e.g., full length collagen VII, in a sample;

providing a determination of whether the value (e.g., a value correlated with absence, presence or amount) determined meets a preselected criteria, e.g., is full length collagen VII present and/or present in a sufficient amount in the sample;

thereby evaluating or processing the collagen VII preparation.

28 . The method of claim 27 , wherein the presence or amount of collagen VII is determined by a method described herein.

29 . The method of claim 28 , wherein the method further comprises providing a comparison of the value determined with a reference value to thereby evaluate the sample.

30 . The method of claim 29 , wherein the comparison includes determining if the test value has a preselected relationship with the reference value, e.g., determining if it meets the reference value.

31 . The method of claim 28 , wherein the method further comprises determining if a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).

32 . The method of claim 31 , wherein the test value, or an indication of whether the preselected relationship is met, can be memorialized, e.g., in a computer readable record.

33 . The method of claim 28 , wherein the sample is further processed depending on whether a preselected relationship is met, e.g., a decision or step is taken, e.g., the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, labeled, packaged, released into commerce, or sold or offered for sale, depending on whether the preselected relationship is met.

34 . The method of claim 33 , wherein the preselected relationship is met, and the sample is processed into drug product or formulated.

35 . The method of claim 33 , wherein the preselected relationship is met, and the sample is subjected to purification.

36 . The method of claim 28 , further comprising a determination of whether a test batch of a collagen VII is to be expected to have one or more of the properties of the collagen VII.

37 . The method of claim 28 , further comprising a determination of the stability and/or shelf life of a collagen VII drug product preparation.