Download biomedica product list 2018Anti C4d
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Method
Immunohistochemistry, indirect immunoflourescence
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Sample type
Paraffin embedded tissue sections, frozen sections
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Use
CE marked – for IVD use in the EU
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Regulatory status
CE marked – for IVD use in the EU
Product Overview
The antibody will identify human complement split product C4d in kidney, lung and heart transplants.
Components
250 µl protein G purified polyclonal rabbit anti human C4d antibody.
Storage instructions: All reagents of the soluble Semaphorin 4D ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.
Assay Protocol
Read the entire protocol before beginning the test.
Immunohistochemical staining of paraffin sections using the C4d polyclonal antibody
This protocol has been developed for staining of paraffin embedded tissue sections (tissue fixed with 4.5% neutrally buffered formalin).
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1. |
Deparaffinize and rehydrate tissue section |
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2. |
For antigen retrieval, pressure-cook slides for 5 minutes at +125°C in TRIS/EDTA buffer (pH 8.5) (in Pascal Pressurized Heating Chamber, Dako or equivalent) and cool down to room temperature (20 minutes) TRIS/EDTA Buffer: 10 ml 1M TRIS (pH 9) + 20 ml 0.05M EDTA (pH 7.5) + 970 ml H2O bidest. |
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3. |
Wash sections in PBS. |
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4. |
Block endogenous peroxidase with 3% H2O2 in PBS for 10 minutes |
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5. |
Wash sections in PBS |
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6. |
Apply 5 minutes UltraVision Block (LabVision) |
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7. |
Wash sections in PBS. |
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8. |
Apply anti C4d antibody 1:30-1:50 in PBS including 1% BSA for 60 minutes at room temperature |
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9. |
Wash in PBS |
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10. |
Apply Value Primary Antibody Enhancer (LabVision) 20 minutes at room temperature |
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11. |
Wash in PBS |
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12. |
Apply Value HRP Polymer (LabVision) 30 minutes at room temperature (dark chamber) |
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13. |
Wash in PBS |
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14. |
Apply DAB or AEC chromogen (adjust time according to positive control) |
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15. |
Wash sections in tap water for 10 minutes |
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16. |
Counter stain sections with haemalaun acc. Mayer for 1 minute |
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17. |
Coverslip sections using an aqueous mounting media |
Indirect immunofluorescence on frozen sections using the C4d Polyclonal Antibody.
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1. |
Frozen sections are incubated with rabbit anti-C4d pAb in a dilution of 1:20-1:30 in phosphate buffered saline (PBS) for 30 minutes at room temperature. |
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2. |
Wash 4 times with PBS |
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3. |
After washing with PBS, bound antibody is detected by incubation with an appropriate FITC-labeled secondary anti-rabbit antibody (dilution according to the respective supplier’s protocol). |
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4. |
Wash 4 times with PBS |
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5. |
Coverslip sections using an aqueous mounting media |
Additional reagents required:
Detection System: LabVision™ Corporation, Cat. No.: TL-060-HLS or TL-125-HLS Lab Vision™ UltraVision™ LP Detection System HRP Polymer (ready to use) http://www.labvision.com/
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Information on the Analyte
C4d Function
Circulating alloantibodies encounter the grafted endothelium as the first target. Living endothelial cells can rapidly eliminate bound antibodies from the cell surface by “capping”, “shedding” or “internalisation”.
C4d is the degradation product of the activated complement factor C4, a component of the classical complement cascade, which is typically initiated by binding of antibodies to specific target molecules. Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response against the allograft. The majority of publications describe C4d as an important biomarker in kidney transplantation but also in heart, liver, and other transplants.
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Literature
1. Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection. Regele H et al.,
Nephrol Dial Transplant 2001, 16:2058-20662. C4d-positive acute humoral renal allograft rejection: effective treatment by immunoadsorption. Böhmig GA et al.
J Am Soc Nephrol, 2001 Nov;12(11):2482-24893. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. Böhmig GA et al.
J Am Soc Nephrol. 2002 Apr;13(4):1091-10994. Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular an glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection. Regele H et al.
J Am Soc Nephrol. 2002, 13:2371-2380 -
Therapeutic Areas
Organ transplantation
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Veverka V et al., J Biol Chem, 2009; 284:10890-10900.
PMID: 19208630
PMID: 19208630