PAD4 Autoantibody EIA Kit

Sample

Plasma, Serum

Species Reactivity

Human

Detection Method

ELISA

Intended Use

The PAD4 Autoantibody ELISA Kit is an immunometric assay which can be used to measure anti-PAD4 autoantibodies of any isotype (IgM, IgG, and IgA) in human plasma and serum without prior sample purification. Affinity-purified PAD4 autoantibody isolated from the plasma of a patient with RA is used as the standard. One unit is approximately equal to 1 ng of anti-PAD4 Ig protein. The standard curve spans the range of 15.6-1,000 U/ml, with an LLOQ of 15.6 U/ml.

Contents of Kit

1. Goat Anti-Human Ig(H+L)/HRP Conjugate, 2 vials/1.5 ml
2. PAD4 Precoated 96-Well Strip Plate, 1 plate
3. Anti-PAD4 (human) ELISA Standard*, 1 vial
4. Immunoassay Buffer B (10×), 2 vials/10 ml
5. Wash Buffer Concentrate (400×), 1 vial/5 ml
6. Polysorbate 20, 1 vial/3 ml
7. TMB Substrate Solution, 1 vial/12 ml
8. HRP Stop Solution, 1 vial/12 ml
9. 96-Well Cover Sheet, 3 covers
*The Anti-PAD4 (human) ELISA Standard was affinity-purified from human plasma.

Storage

This kit will perform as specified if stored as directed at 4°C and used before the expiration date indicated on the outside of the box.

Performance Characteristics

Sample Data
The standard curve presented here is an example of the data typically produced with this kit; however, your results will not be identical to these. You must run a new standard curve. Do not use the data below to determine the values of your samples. Your results could differ substantially. Development of the plate for 10 minutes typically results in an absorbance of >1.0 O.D. units for the 1,000 U/ml standard.

Precision

The intra- and inter-assay CVs have been determined for each point of the standard curve from the multiple measurements. This data is summarized in the table below.

Sample Precision:
Intra-assay precision was determined by analyzing 24 replicates of three matrix controls (human plasma) in a single assay.

Inter-assay precision was determined by analyzing replicates of three matrix controls (human plasma) in eight separate assays on different days.

Detection Range

15.6-1,000 U/ml

Detection Limit

15.6 U/ml

General Description

Protein arginine deiminase 4 (PAD4) catalyzes the conversion of arginine residues to citrulline within cellular protein substrates, resulting in the loss of a positive charge, which can alter protein structure and/or function. It is expressed in neutrophils, as well as a variety of tissues, including the brain, liver, lung, and kidney. PAD4 has a key role in NETosis, a lytic form of cell death characterized by the release of neutrophil extracellular traps (NETs). Upon neutrophil activation, PAD4 translocates to the nucleus where it citrullinates histones, initiating chromatin decondensation and the release of NETs. Neutrophils isolated from Pad4-/- mice exhibit decreased citrullination of histone H3 under both basal and LPS-stimulated conditions and are defective for NET formation in response to stimulation with LPS, phorbol 12-myristate 13-acetate (PMA), or hydrogen peroxide.4 Citrullination of PAD4 targets contributes to disease progression and the generation of autoantibodies in patients with autoimmune diseases, including rheumatoid arthritis, Hashimoto's encephalopathy, and multiple sclerosis. Additionally, PAD4 autoantibodies isolated from the serum of patients with rheumatoid arthritis are associated with disease severity.