Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients

Our plain-English summary of Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients, by I. Raiko, I. Sander, D.G. Weber, et al (BMC Cancer, 2010).

The study

• Authors: I. Raiko, I. Sander, D.G. Weber, M. Raulf-Heimsoth, A. Gillissen, J. Kollmeier, A. Scherpereel, T. Brüning, G. Johnen
• Journal: BMC Cancer
• Published: 2010;10:242
• Cited by: 29 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Calretinin is one of the well-established immunohistochemical markers in the diagnostics of malignant mesothelioma (MM). Its utility as a diagnostic tool in human blood, however, is scarcely investigated. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for human calretinin in blood and to assess its usefulness as a potential minimally invasive diagnostic marker for MM.

Methods: Initially, attempts were made to establish an assay using commercially available antibodies and to optimize it by including a biotin-streptavidin complex into the assay protocol. Subsequently, a novel ELISA based on polyclonal antibodies raised in rabbit immunized with human recombinant calretinin was developed. The assay performance in human serum and plasma (EDTA/heparin) and the influence of calcium concentrations on antibody recognition were studied. Stability of spiked-in calretinin in EDTA plasma under different storage conditions was also examined. In preliminary studies serum and plasma samples from 97 healthy volunteers, 35 asbestos-exposed workers, and 42 MM patients were analyzed.

Results: The mean detection range of the new ELISA was 0.12 to 8.97 ng/ml calretinin. The assay demonstrated markedly lower background and significantly higher sensitivity compared to the initially contrived assay that used commercial antibodies. Recovery rate experiments confirmed dependence of calretinin antibody recognition on calcium concentration. Calcium adjustment is necessary for calretinin measurement in EDTA plasma. Spiked-in calretinin revealed high stability in EDTA plasma when stored at room temperature, 4 degrees C, or after repeated freeze/thaw cycles. Median calretinin values in healthy volunteers, asbestos workers, and MM patients were 0.20, 0.33, and 0.84 ng/ml, respectively (p < 0.0001 for healthy vs. MM, p = 0.0036 for healthy vs. asbestos-exposed, p < 0.0001 for asbestos-exposed vs. MM). Median values in patients with epithelioid and biphasic MM were similar. No influence of age, gender, smoking status, or type of medium (plasma/serum) on calretinin values was found.

Conclusions: The novel assay is highly sensitive and applicable to human serum and plasma. Calretinin appears to be a promising marker for the blood-based detection of MM and might complement other markers. However, further studies are required to prove its usefulness in the diagnosis of MM patients.

In plain English

This laboratory study described the development of a new blood test, an ELISA, for calretinin. Calretinin is a protein already used to help identify malignant mesothelioma in tissue samples. The authors first tried versions built on commercially available antibodies, then made their own antibodies in a rabbit. The new assay measured calretinin from 0.12 to 8.97 ng/ml. Its lower detection limit was about 46 and 4 times lower than the two earlier versions. In EDTA plasma, calcium had to be added for accurate measurement. Calretinin added to plasma stayed stable for 5 days at room temperature, 4°C or -80°C, and through 10 freeze/thaw cycles. In a preliminary comparison, median blood calretinin was 0.84 ng/ml in 42 mesothelioma patients, 0.33 ng/ml in 35 formerly asbestos-exposed workers without cancer, and 0.20 ng/ml in 97 healthy volunteers. All three differences were statistically significant. Levels were similar in epithelioid and biphasic mesothelioma. The authors concluded that calretinin might be suitable, possibly with markers such as mesothelin, for blood-based detection of mesothelioma. They noted the small numbers and called for larger studies.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory assay-development study (technical advance) with a preliminary comparison of blood calretinin levels between healthy volunteers, asbestos-exposed workers and mesothelioma patients
• Who was studied: Serum or plasma from three groups. 97 apparently healthy volunteers (54 men, 43 women; mean ages 45 and 41) recruited from employees and former employees of the IPA institute in Bochum, Germany. 35 workers formerly exposed to asbestos without evidence of neoplasia (34 men, 1 woman; mean age 69), some with pleural plaques or asbestosis. 42 malignant mesothelioma patients (31 men, 11 women; mean age 67) from French and German clinics or a commercial supplier; histology was known for 32: 19 epithelioid, 1 sarcomatoid, 12 biphasic
• Exposure: Former occupational asbestos exposure in one comparison group; fiber types, products and doses not reported. The main subject was the measurement of calretinin in blood
• Main results:
  • The new assay (variant 3) had a mean detection range of 0.12 to 8.97 ng/ml (n = 11). Its lower detection limit was approximately 46-fold and 4-fold lower than assay variants 1 and 2
  • Recovery of added calretinin in EDTA plasma without added calcium was low (21.3%, plus or minus 5.0%). With calcium added it was 96.1% (plus or minus 15.5%), comparable to serum (89.9%) and heparin plasma (92.2%)
  • Calretinin added to EDTA plasma was stable for 5 days at -80°C, 4°C or room temperature. Up to 10 freeze/thaw cycles had no significant influence on recovery
  • Median blood calretinin in mesothelioma patients was significantly higher than in healthy controls (0.84 vs. 0.20 ng/ml; p less than 0.0001) and than in asbestos-exposed workers (0.84 vs. 0.33 ng/ml; p less than 0.0001)
  • Asbestos-exposed workers had significantly higher median calretinin than healthy controls (0.33 vs. 0.20 ng/ml; p = 0.0036)
  • Median calretinin did not differ significantly between epithelioid and biphasic mesothelioma patients (0.87 vs. 0.90 ng/ml; p = 0.41)
  • Median calretinin showed no significant differences between serum and plasma within any group
  • Among healthy subjects, there was no significant difference by age, sex or smoking status (men vs. women 0.21 vs. 0.20 ng/ml; p = 0.56)
• The authors' conclusion: The authors concluded that, because calretinin levels were significantly increased in mesothelioma patients, it might be suitable, possibly combined with other markers like mesothelin, for blood-based detection of mesothelioma
• Limitations the authors state:
  • Results are based on relatively small numbers. Larger studies, including people with other tumors and non-malignant lung or pleural diseases, are needed to confirm them
  • Only one sarcomatoid mesothelioma was included, so this subtype could not be analyzed statistically

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Calretinin, Mesothelioma, Malignant Mesothelioma, Epithelioid, Biphasic Mesothelioma.

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• Mesothelin, Calretinin, and Megakaryocyte Potentiating Factor as Biomarkers of Malignant Pleural Mesothelioma — C. Jiménez-Ramírez, S. Casjens, et al. (2019)
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