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Anti-C4d Antibody (FITC) | BI-RC4D-FITC

Cat. No.: BI-RC4D-FITC

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Anti-C4d Antibody, FITC highlights:
Therapeutic Areas
Assay Characteristics
  • Method

    For detection of cell- or solid-phase bound C4d and C4d split product by flow cytometry using FlowPRA® Class I and II screening test beads from One Lambda.

  • Sample type

    Serum

  • Use

    Research use only

Product details

Anti-C4d Antibody (FITC) Product Overview

The Anti-C4d FITC antibody will identify human complement split product C4d. It is suggested to be a valuable marker for humoral rejection of transplants (kidney, heart, lung). The antibody is applicable for detection of cell- or solid-phase bound C4 and C4d split product by flow cytometry using FlowPRA® Class I and II screening test beads from One Lambda, Inc.

Anti-C4d Antibody (FITC) Components

100 µl  (125 tests) protein G purified polyclonal rabbit anti-human C4d antibody, FITC labeled

Storage instructions: for one month at 4°C. For prolonged period of time, aliquot and store at -20°C to -80°C. Avoid repeated freeze-thaw cycles.

Instructions for use

Anti-C4d Antibody (FITC) Assay Protocol

[C4d]FlowPRA Test Using FITC-conjugated Anti-human C4d Antibody (C4dpAb2)

The FlowPRA® Screening test (One Lambda, Inc., Canoga Park, CA, USA) consists of a pool of 30 different microbead preparations coated with either purified HLA class I or HLA class II antigens from different cell lines covering all common HLA antigens. According to the manufacturer´s protocol this assay is applicable for detection of anti-HLA class I or II IgG alloreactivities (Ref. 1). The [C4d]FlowPRA test was developed for detection of anti-HLA alloantibody-triggered C4d deposition to FlowPRA® beads by FITC-conjugated anti-human C4d antibody. As an adjunct to standard FlowPRA® screening, this test allows for selective detection of presumably more harmful classical complement-activating anti-HLA alloantibodies. The [C4d]FlowPRA test may represent a specific alternative to conventional complement-dependent cytotoxicity (CDC) PRA testing (Ref. 2).

Read the entire protocol before beginning the test.

1.

Incubate negative control serum or test serum (9 μl) with 0.5 μl FlowPRA® class I and 0.5 μl FlowPRA® class II beads*.

2.

Incubate for 30 minutes on ice.

3.

Add 30 µl serum obtained from a nonsensitized healthy male volunteer with normal
C4 levels, and normal CH50 activity.

4.

Incubate for 30 minutes on ice.

5.

Wash beads twice with cold phosphate-buffered saline (PBS).
6. Add 20 µL working dilution (1:25) of FITC-conjugated C4dpAb2 to 20 µl beads suspension (final dilution 1:50).

7.

Incubate for 30 minutes on ice.

8.

Wash beads twice with cold PBS and analyze by flow cytometry.

9.

The major bead population is gated on the forward-versus-side-scatter dot plot. Then, two gates are set on the FL2 histogram to separately analyze HLA class I-coated (FL2-negative) and class II (FL2 high-fluorescent)-coated populations. FITC conjugated anti-human C4d antibody binding to HLA class I or class II beads is evaluated on FL1
histograms. The marker is set according to staining with the negative control serum. Results are expressed as percentage of positive events.

C4d FlowPRA Test Using Anti-C4d FITC Antibody

[C4d]FlowPRA (HLA class I) screening of representative sera (serum A, serum B) obtained from two presensitized patients. A FL-1 histogram (FlowPRA® HLA class I beads are gated) is depicted. The marker is set according to C4d staining obtained with a non-binding control serum. The percentage of positive events, which represents %[C4d]FlowPRA reactivity, is indicated for the two test sera.

Additional Reagents Required

*FlowPRA® Class I Screening Test, Cat. No. FL1-30
FlowPRA® Class II Screening Test, Cat. No. FL2-30
One Lambda, Inc., 21001 Kittridge, Canoga Park, CA 91303, USA, www.onelambda.com  

Literature

Wahrmann, M., Exner, M., Haidbauer, B., Schillinger, M., Regele, H., Körmöczi, G., Böhmig, G.A., 2005. Hum. Immunol. 66, 526–534.
PMID: 15935890
 
Pei, R., Wang, G., Tarsitani, C., Rojo, S., Chen, T., Takemura, S., Liu, A., Lee, J., 1998. Hum. Immunol. 59, 313–322
PMID: 9619770
Background & Therapeutic Areas

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.

  • Organ transplantation
    • Kidney transplantation
    • Liver transplantation
    • Heart transplantation
    • Lung transplantation
Citations


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Cat. No.: BI-RC4D-FITC

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