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. 2025 Oct 1;17(19):3206.
doi: 10.3390/cancers17193206.

Comparative Analysis of Manual ELISA and Ella, an Automated Instrument for ELISA, in Measuring Serum Galectin-3 Levels in Breast Cancer Patient Samples

Ella G Markalunas  1 , Shannon E Harold  2 , David H Arnold  2 , Julie C Martin  3   4 , W Jeffery Edenfield  3   4 , Anna V Blenda  2   3
Affiliations

Comparative Analysis of Manual ELISA and Ella, an Automated Instrument for ELISA, in Measuring Serum Galectin-3 Levels in Breast Cancer Patient Samples

Ella G Markalunas et al. Cancers (Basel). .

Abstract

Background: Circulating galectin-3 (Gal-3) levels have been indicated as a promising diagnostic, prognostic, and therapeutic target in breast cancer patients. Specifically, serum galectin-3 levels are traditionally measured using manual Enzyme-Linked Immunosorbent Assay (ELISA), but recent automated methods, such as Simple Plex assay by ProteinSimple™ run on an Ella instrument, have shown promising evidence of being more efficient and less error-prone than manual methods. This paper aims to assess whether there are differences in serum galectin-3 measurements between manual and automated ELISA methods. Methods: Serum galectin-3 levels were initially analyzed from one hundred and fifteen breast cancer samples using both manual ELISA and the Ella instrument. Following coefficient of variation (CV) and outlier analysis, ninety-five samples were analyzed further with JMP statistical software to perform Shapiro-Wilk, Spearman's correlation, Wilcoxon signed-rank, and regression analyses. Results: The Ella instrument resulted in significantly lower CV values, confirming that it is more precise and reliable than manual ELISA methods. There was a moderate correlation between ELISA and Ella measurements (r = 0.49, p < 0.0001), but a Wilcoxon signed-rank test revealed that serum gaelectin-3 measurements obtained with the Ella instrument were significantly lower compared to those obtained with manual ELISA, with a mean difference of -5.19 ng/mL (p < 0.0001). Regression analysis showed a significant increase in the difference between manual ELISA and Ella measurements as serum galectin-3 levels increase (p < 0.0001). This difference in measurements between manual and automated ELISA techniques remained consistent when analyses were performed within each breast cancer stage, immunophenotype, and histology. Conclusions: While the Ella instrument is a fast and reliable tool, the discrepancies between manual ELISA and the Ella instrument in quantifying serum galectin-3 levels are important to consider prior to widespread use.

Keywords: ELISA; Ella; Simple Plex assay; automated assay; breast cancer; comparative analysis; galectin-3.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
The three structural types of galectins, where CRD stands for Carbohydrate Recognition Domain. From Kamili, N.A. et al., 2016 [3].
Figure 2
Figure 2
The role of galectins in immune system regulation. From Dumic, J. et al., 2006 [21].
Figure 3
Figure 3
Best fit line of ELISA and Ella serum galectin-3 measurements with CV less than 10%. The Spearman’s correlation coefficient is r = 0.49 (p < 0.0001).
Figure 4
Figure 4
Bland-Altman Plot comparing the average serum galectin-3 levels between ELISA and Ella measurements, with the differences between the two techniques.
Figure 5
Figure 5
A fitted least-squares regression line between mean serum galectin-3 levels and the difference between ELISA and Ella values, with CV < 10%. Slope estimate = 0.92 ng/mL (p < 0.0001).
Figure 6
Figure 6
A fitted least-squares regression line between mean serum galectin-3 levels and the difference between ELISA and Ella values with CV < 5%. Slope estimate = 0.86 ng/mL (p < 0.0001).
See this image and copyright information in PMC

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