Calretinin
Characteristics
Sandwich ELISA with microtiter plate formate
Highly specific and sensitive rabbit anti-calretinin antibody
Good linearity between 0.25 – 4.0 ng/ml
Easy to handle, total assay time is 6 hours
Suitable for testing large series of serum or plasma samples
Validated in larger case-control studies
Involved in prospective studies
Procedure
Sample volume: 15 µl patient serum or plasma
1. Sample Preparation: 60 min shaking at room temperature
2. ELISA: 5 hours incubation at room temperature
Measurement in ELISA reader at 450 nm (620 nm reference wavelength)
6 Standards with range: 0.25 ng/ml – 4.0 ng/ml
2 Kit controls, lyophilized
Enzyme Conjugate 200 x concentrated; Wash buffer 50 x concentrated
All other reagents ready for use
For some time now, the production and application of asbestos has been banned in more than 55 countries in the world. However, the number of asbestos-associated cancers – mainly malignant lung cancer and mesothelioma – is still high.
Due to long latency of the disease and ongoing production and application of asbestos in several countries, no substantial improvement is expected. Detecting tumors early – preferably at stages without clinical symptoms – might improve the chances for a curative therapy. In collaboration with scientists of the Institute for Prevention and Occupational Medicine of the German Social Accident Insurance – Institute of the Ruhr-University Bochum (IPA) the DLD Diagnostika
GmbH developed a promising method based on calretinin as a biomarker for early detection – especially of mesothelioma – in plasma and serum samples.
Currently, calretinin is one of the best available markers for the detection of mesothelioma.
The calretinin ELISA is a sandwich enzyme immunoassay utilizing a purified rabbit polyclonal antibody. During incubation, calretinin from diluted patient samples binds to a calretinin antibody (capture antibody) immobilized on the surface of microtiter plate wells. After a washing step, a biotinylated calretinin antibody (detection antibody) is added that binds to the captured calretinin of the patient sample.
After a second washing step, conjugated streptavidin peroxidase is added, binding specifically to the biotinylated detection antibody. Following a third washing step, the bound amount of enzyme –
equivalent to the amount of calretinin – is quantified via the turnover of the substrate tetramethylbenzidin (TMB). During the enzyme reaction, a blue dye is generated. Addition of sulfuric acid stops the reaction and causes the solution to turn yellow.The extinction of the samples is measured using a microtiter plate reader at 450 nm (reference wavelength between 570 nm and 650 nm) and the concentration of calretinin is calculated using the standards and controls of the assay.
- Johnen G., Gawrych K., Raiko I., Casjens S., Pesch B., Weber D. G., Taeger D., Lehnert M., Kollmeier J., Bauer T., Musk A. W., Robinson B. W. S., Brüning T., Creaney J. Calretinin as a blood-based biomarker for mesothelioma
BMC Cancer 2017; 17: 386 - Casjens S, Weber DG, Johnen G, et al. Assessment of potential predictors of calretinin and mesothelin to improve the diagnostic performance to detect malignant mesothelioma:
results from a population based cohort study.
BMJ Open 2017;7:e017104.doi:10.1136/bmjopen-2017-017104 - Raiko I, Sander I, Weber DG, Raulf-Heimsoth M, Gillissen A, Kollmeier J, Scherpereel A, Brüning T, Johnen G. Development of an enzyme-linked immunosorbent assay for the
detection of human calretinin in plasma and serum of mesothelioma patients.
BMC Cancer 2010; 10: 242 - Pesch B, Brüning T, Johnen G, Casjens S, Bonberg N, Taeger D, Müller A, Weber DG, Behrens T. Biomarker research with prospective study designs for the early detection of cancer.
Biochim Biophys Acta 2014; 1844: 874-883 - Calretinin ELISA: A New Assay for the Detection of Mesothelioma in Blood Samples.
Georg Johnen, Irina Raiko, Ingrid Sander, Daniel G. Weber, Monika Raulf-Heimsoth, Adrian Gillissen, Jens Kollmeier, Klaus-Michael Müller, Arnaud Scherpereel, Thomas Brüning (data on file)