Anti-Eculizumab (SOLIRIS™) ELISA Kit

Sample

Serum and plasma

Detection Method

sELISA

Intended Use

The Anti-Eculizumab (SOLIRIS™) ELISA Kit is used as an analytical tool for quantitative determination of Anti-Eculizumab in serum and plasma.

Contents of Kit

1. Eculizumab Coated Microtiter Plate: 96 well polystyrene microplate (12 strips of 8 wells) coated with Eculizumab monoclonal antibody. 1 × 96 wells
2. Anti-Eculizumab Standard: Recombinant Eculizumab in a buffered protein base with protein stabilizer and preservatives 0.02% methylisothiazolone and 0.02% bromonitrodioxane - lyophilized, concentrated (1 ug/ml). 2 vials
3. Eculizumab-HRP Conjugate: Eculizumab conjugated to Horseradish Peroxidase with protein stabilizer and preservatives 0.02% methylisothiazolone and 0.02% bromonitrodioxane. 12 ml
4. Sample Diluent: Buffered protein base with protein stabilizer and preservatives 0.02% methylisothiazolone and 0.02% bromonitrodioxane. 2 × 50 ml
5. Standard Diluent: Buffered protein base with 1:100 dilution human serum and protein stabilizer and preservatives 0.02% methylisothiazolone and 0.02% bromonitrodioxane. 10 ml
6. Wash Buffer(20×): 20-fold concentrated solution of buffered surfactant with preservative thiomersol < 0.01%. May turn yellow over time. 25 ml
7. TMB Substrate: Stabilized Chromogen. 12 ml
8. Stop Solution: 2N Sulfuric Acid. 12 ml

Storage

1. All reagents should be stored at 2°C to 8°C for stability.
2. All the reagents and wash solutions should be used within 12 months from manufacturing date.
3. Before using, bring all components to room temperature (18-25°C). Upon assay completion ensure all components of the kit are returned to appropriate storage conditions.
4. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources.

Performance Characteristics

This kit has been validated as per EMA/FDA guidelines in line with ICH Code for Harmonization of Biological Assays.

Precision

Precision is defined as the percent coefficient of variation (%CV) i.e. standard deviation divided by the mean and multiplied by 100.
Assay precision was determined by both intra (n=5 assays) and inter assay (n=5 assays) reproducibility on two pools with low (20 ng/ml), medium (80 ng/ml) and high (640 ng/ml) concentrations.
While actual precision may vary from laboratory to laboratory and technician to technician, it is recommended that all operators achieve precision below these design goals before reporting results.

Sensitivity

Limit of Quantification: It is defined as the lowest concentration of an analyte that can be determined with an acceptable repeatability and the LOQ was found to be 18 ng/ml.
Limit Of Detection: It is defined as the lowest detectable concentration corresponding to a signal of Mean of '0' standard plus 2*SD. 10 replicates of '0' standards were evaluated and the LOD was found to be less than 12 ng/ml.

General Description

Eculizumab, sold under the trade name Soliris, is a medication used to treat paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). It is a humanized monoclonal antibody functioning as a terminal complement inhibitor. In people with PNH, it reduces both the destruction of red blood cells and need for blood transfusion, but does not appear to affect the risk of death. Eculizumab was the first drug approved for each of its uses, and its approval was granted on the basis of small clinical trials. Anti-Drug Antibodies (ADA) may induce unwanted side effects in biopharmaceuticals. Hence, ADA has been subjected to increase in scrutiny by the regulatory authorities using immunogenicity safety studies. ADA has been observed in pre-clinical and clinical studies, resulting in significant changes in toxicology, pharmacokinetics and efficacy. These effects result from the generation of drug-induced (neutralizing) autoantibodies against Eculizumab and can be responsible for allergic reaction, or even anaphylactic shock. This ELISA kit detects antibodies for Anti-Eculizumab and may be used for monitoring immunogenicity.

Standard Curve