Antibodies and immunoassays for soluble cytokeratins (including CYFRA21-1)

Monoclonal antibodies against soluble cytokeratins
Monoclonal antibodies were obtained against preparations of cell culture lysate derived fraction corresponding to soluble cytokeratin mixture containing cytokeratins 8,11 and 19. The preparations (R236) are available from XEMA, see our Reagent catalog.

Matching of XEMA anti-cytokeratin mAbs and reference antibodies
Matching of mAbs was performed to obtain an immunometric pairs measuring soluble cytokeratins in tumor cell lysates and human sera (see results in the table below)

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KS19-1 SA*

KS19-2 SA*

23*

29*

30*

35*

42*

46*

34b-SA*

37b-SA*

45b-SA*

46b-SA*

47b-SA*

KS19.1

N/A

+++CYFRA21-1ref

+++

N/A

KS19.2

+++

N/A

XC23

+++

XC29

hi zero

XC30

XC34

+++

XC35

XC42

+++
K236 v1

+++

XC43

+++

XC45

+++

XC46

+++

XC41

+++

XC47

XC49

XC51

Antibodies coated directly with HRP (*) or biotin and visualized by streptavidin-HRP (SA*)
Reference antibody pair KS19.1-KS19.2 (forming CYFRA21-1 assay) was purchased from Progen GmbH.
Some matching was not done yet (ND), some are not applicable (N/A) due to their apparent negativity based on cross-inhibition data (not shown). The degree of signal in + is based on titration of cell lysate derived cytokeratin preparation AND sera of cancer patients. In some pairs (labeled as “?”), these results are conflicting.
Monoclonal antibody XC42 was shown to recognize an epitope close to or identical to that of reference mAb KS19.1 and therefore chosen as capture antibody against KS19.2 (Progen GmbH) in our CYFRA21-1 assay (K236) version 1.
Other mAb combination detect different variants of soluble cytokeratin antigens and are under research for their possible applications in experimental and clinical oncology.

Applications of XEMA anti-cytokeratin mAbs: immunoprecipitation
The identity of epitope target of XC42 mAb to reference KS19.1 was confirmed by immunoprecipitation with consequent MALDI-TOF analysis of precipitated antigen (data not shown)

Applications of XEMA anti-cytokeratin mAbs: immunofluorescence
Our mAbs can be used to stain the cells and reveal different patterns of cytokeratin staining in tumor cell lines. In the attached IF report mammary carcinoma cell line MCF7 was used as a model object.

Applications of XEMA anti-cytokeratin mAbs: Western immunoblotting
Cell lysis and sample preparation
To prepare total lysates, the cells were washed twice with ice-cold PBS and lyzed on ice in RIPA lysis buffer (20 mM Tris-HCl (pH 7,5); 150 mM NaCl; 1% Triton X-100; 0,5% Na deoxycholate; 0,1% SDS; 1 mM NaF; 1 mM Na2EDTA; 1 mM EGTA; 1 mM Na3VO4; 1 mM PMSF and protease inhibitors cocktail (Sigma, P8340, dilution 1:300) for 10 min. The lysate was clarified by centrifugation at 15000 x g for 15 min. Protein content was determined by the method of Bradford. The cell lysates were mixed with 5 x Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1% ?-mercaptoethanol, 0,02% bromophenol blue final concentrations) and heated at 99?C for 5 min. Samples containing equal amounts of protein (50 µg) were analyzed by SDS– PAGE (10% gel) and transferred to nitrocellulose membrane (Bio-Rad) according to standard manufacture protocol (Bio-Rad Laboratories).

Western immunoblotting procedure
Membrane was washed with water and transferred proteins were visualized with Ponseau S (Sigma). All antibody incubation was carried out at room temperature with gentle agitation. Membrane was washed with TTBS buffer (20 mM Tris-HCl (pH 7,4), 150 mM NaCl, 0,1% Tween-20) and then blocked for 1 hour in TTBS with 5% w/v nonfat dry milk. Then membrane was washed three times for 5 minutes with TTBS and incubated for 1 hour in primary antibody, diluted at the appropriate concentration in TTBS with 5% w/v BSA. After washing (three times for 5 minutes) membrane was incubated for 1 hour in appropriate secondary HRP-conjugated antibody (Sigma) (diluted according with manufacture recommendation in TTBS with 5% w/v defatted dry milk). Then membrane was washed tree times for 5 min with TTBS and antibody was visualized with ECL (Pierce) according manufacture protocol and detected by exposure to x-ray film (CEA).
Western immunoblotting pictures are in attached file