Evaluation of Human Carbonic Anhydrase II (CA-II) Concentration Using ELISA: Insights into Optical Density’s Role in Biomarker Quantification for Cryptorchidism Research

Abstract

Background:

Carbonic Anhydrase II (CA-II) plays a pivotal role in various physiological processes, including maintaining acid-base balance. Its implications in testicular health, especially cryptorchidism, make it an essential focus for researchers. ELISA is widely used to measure biomarker concentrations, with OD serving as a key indicator. Understanding the precision of OD measurements for CA-II can enhance its diagnostic and research applications.

Objective:

This study aims to evaluate the relationship between OD and CA-II concentration using linear regression analysis, thereby establishing a quantitative framework for accurate and reproducible CA-II measurements. By validating the OD-to-concentration relationship, this research will aid in developing standardized protocols that can improve diagnostic reliability, enhance monitoring of disease progression, and support therapeutic interventions targeting CA-II.

Methods:

Standardized ELISA was employed to determine CA-II concentrations across sample groups, recording OD at specific wavelengths. Data were analyzed for linearity, group differences using ANOVA, and pairwise comparisons via Tukey’s HSD test. Correlation analysis was performed to evaluate the relationship between CA-II concentration and OD.

Results:

A linear regression model (y=0.3758x+0.1604) demonstrated a nearly perfect R2 value of 0.9957. Statistical tests revealed a strong correlation (0.998) between CA-II concentration and OD values. ANOVA did not indicate significant differences among concentration quartiles; however, the data strongly supported OD’s use as a reliable proxy for CA-II measurement.

Conclusion:

The established linear relationship between CA-II concentration and OD confirms the accuracy of ELISA in CA-II measurement. This methodology supports future investigations into CA-II’s involvement in testicular health, potentially aiding in diagnostics and understanding of conditions such as cryptorchidism.

1. BACKGROUND

Carbonic Anhydrase II (CA-II) is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide (CO₂) to bicarbonate (HCO₃^-) and protons (H⁺), playing a crucial role in maintaining pH balance, facilitating CO₂ transport, and ensuring acid-base homeostasis. Predominantly found in red blood cells, CA-II is also expressed in the kidneys, brain, and other tissues, underscoring its broad physiological importance (1,2). The enzyme’s rapid catalytic activity, with a turnover rate of approximately 10^6 reactions per second, underscores its vital function in various metabolic processes (3).

Beyond its essential physiological roles, CA-II is linked to several pathological conditions. Genetic mutations in the CA2 gene are associated with disorders such as osteopetrosis and renal tubular acidosis, highlighting its importance in skeletal and renal health (4). Additionally, altered expression levels of CA-II have been observed in certain cancers, where the enzyme’s involvement may contribute to tumor aggressiveness and progression (5). In reproductive health, CA-II’s function in maintaining ion and pH balance within the testicular microenvironment is particularly significant. Disruptions in CA-II activity have been implicated in conditions such as cryptorchidism (undescended testis), a condition linked to infertility and an increased risk of testicular cancer (6, 7).

Quantifying CA-II accurately is crucial for its use as a reliable biomarker in clinical diagnostics and research. However, the variability in optical density (OD) readings, influenced by sample properties and assay conditions, presents challenges to standardizing measurement protocols (8, 9). Understanding the relationship between OD and CA-II concentration is essential for developing robust protocols that ensure measurement consistency and precision. Although newer assay technologies, such as fluorescence-based methods and mass spectrometry, offer enhanced sensitivity and specificity, they require further validation for broad clinical application (10, 11).

Such advancements are expected to impact not only diagnostic practices but also facilitate broader research into conditions associated with altered CA-II activity, ultimately contributing to better patient outcomes and tailored treatment strategies.

2. OBJECTIVE

This study aims to evaluate the relationship between OD and CA-II concentration using linear regression analysis, thereby establishing a quantitative framework for accurate and reproducible CA-II measurements. By validating the OD-to-concentration relationship, this research will aid in developing standardized protocols that can improve diagnostic reliability, enhance monitoring of disease progression, and support therapeutic interventions targeting CA-II.

3. MATERIAL AND METHODS

Study Design

This study was a controlled, experimental investigation designed to evaluate the relationship between optical density (OD) and Carbonic Anhydrase II (CA-II) concentration using ELISA. The aim was to establish a reliable quantitative framework for accurate CA-II measurement, supporting its use as a biomarker in clinical and research settings.

Sample Preparation

Sample Preparation involved using purified human CA-II protein as the standard. A serial dilution was prepared to create a range of CA-II concentrations from 0 ng/mL (blank control) to 100 ng/mL, spanning the expected physiological levels found in clinical samples. Each concentration was aliquoted into 100 μL portions and stored at -20°C to maintain stability and prevent degradation due to repeated freeze-thaw cycles. Before the assay, standard aliquots were thawed on ice and gently vortexed to ensure homogeneity.

The ELISA protocol began with coating high-binding 96-well microtiter plates (Nunc MaxiSorp) with 100 μL of anti-human CA-II capture antibodies diluted in carbonate-bicarbonate buffer (pH 9.6) at a concentration of 1 μg/mL. The plates were incubated overnight at 4°C to ensure adequate binding of the capture antibody. After incubation, the plates were washed three times with 300 μL of PBS containing 0.05% Tween-20 (PBST) to remove any unbound antibody. Blocking was performed using 200 μL of 5% non-fat dry milk in PBS, incubated at room temperature for 1 hour to minimize non-specific binding, followed by another three washes with PBST.

Following the blocking step, 100 μL of CA-II standards or test samples were added to each well in duplicate and incubated at 37°C for 2 hours with gentle shaking to enhance antigen-antibody interaction. After washing, 100 μL of biotinylated anti-CA-II detection antibody, prepared at 0.5 μg/mL in PBS with 0.1% BSA, was added to each well and incubated for 1 hour at room temperature. The plates were washed three times with PBST before adding 100 μL of Streptavidin-HRP, diluted according to the manufacturer’s protocol, followed by a 30-minute incubation at room temperature. The plates were washed five times to ensure the complete removal of unbound conjugate.

For color development, 100 μL of TMB substrate was added to each well and incubated in the dark at room temperature for 15 minutes. The reaction was stopped by adding 50 μL of 2M sulfuric acid, changing the color from blue to yellow. Optical density was measured at 450 nm, with 620 nm as a reference wavelength, using a microplate reader within 30 minutes of stopping the reaction to ensure precise measurements.

ELISA Protocol

The ELISA protocol commenced with the coating of high-binding 96-well microtiter plates (Nunc MaxiSorp) with 100 μL of anti-human CA-II capture antibodies diluted to 1 μg/mL in carbonate-bicarbonate buffer (pH 9.6). Plates were incubated overnight at 4°C to ensure proper antibody binding. The next day, plates were washed three times with 300 μL of PBS containing 0.05% Tween-20 (PBST) to remove unbound antibodies. Blocking was performed with 200 μL of 5% non-fat dry milk in PBS for 1 hour at room temperature to reduce non-specific binding, followed by three additional washes with PBST.

Subsequently, 100 μL of CA-II standards or test samples were added to each well in duplicate and incubated at 37°C for 2 hours with gentle shaking to enhance antigen-antibody interaction. The plates were then washed, and 100 μL of biotinylated anti-CA-II detection antibody (diluted to 0.5 μg/mL in PBS with 0.1% BSA) was added and incubated for 1 hour at room temperature. Plates were washed again three times, followed by the addition of 100 μL of Streptavidin-HRP, prepared according to the manufacturer’s instructions, and incubated for 30 minutes at room temperature. The plates were washed five times to ensure thorough removal of unbound conjugate.

For color development, 100 μL of TMB substrate solution was added to each well and incubated in the dark at room temperature for 15 minutes. The reaction was stopped by adding 50 μL of 2M sulfuric acid, resulting in a color change from blue to yellow. OD was measured at 450 nm with a reference wavelength of 620 nm using a microplate reader within 30 minutes to ensure precise measurements.

Data Analysis

The analysis included exporting raw OD readings from the microplate reader into a CSV file for statistical evaluation. The average OD for each standard and test sample (measured in duplicate) was calculated to maintain consistency. Linear regression analysis was conducted to construct a standard curve and derive the calibration equation along with the R2R^2R2 value, establishing the relationship between OD and CA-II concentration. The Shapiro-Wilk test was applied to confirm the normality of data distributions.

To compare OD values across multiple sample groups, One-Way ANOVA was performed. When significant differences were found, Tukey’s HSD post-hoc test was used to identify specific group differences. Pearson correlation analysis assessed the strength and direction of the relationship between CA-II concentration and OD. Data analysis was conducted using Python libraries (pandas, SciPy, statsmodels) and SPSS (version 11.0) to ensure robust statistical assessment.

To promote reproducibility, the study employed standardized reagent preparation and consistent incubation conditions throughout the experiments. Each assay was performed at least three times to validate the reliability of the results. Detailed documentation, including batch numbers of reagents and equipment calibration logs, was maintained to facilitate replication by other researchers.

4. RESULTS

Standard Curve Analysis

The standard curve generated from the ELISA results demonstrated a strong linear relationship between optical density (OD) and Carbonic Anhydrase II (CA-II) concentration. The linear regression analysis provided the following equation:

y = 0.3758x + 0.1604

Figure 1. Standard Curve of CA-II concentration versus Optical Density.

where y represents the OD value and x denotes the CA-II concentration in ng/mL. The coefficient of determination (R2) was found to be 0.9957, indicating an excellent fit of the linear model and confirming that 99.57% of the variability in OD readings can be explained by changes in CA-II concentration.

A plot of the standard curve showed a clear, direct correlation between increasing CA-II concentrations and corresponding OD values. The near-perfect R2 value highlights the precision of this assay for measuring CA-II levels, suggesting that OD is a reliable indicator for quantifying CA-II concentration within the tested range. The strong linearity of the relationship confirms the validity of using this ELISA setup for accurate CA-II detection in clinical and research settings.

Table 1. Study Group Summary of CA-II Quantification.

Group	Mean OD Value	Standard Deviation	p-value (ANOVA)
Group A: Non-UDT (control group)	0.16	0.005
Group B: UDT without orchidopexy	3.918	0.15	0.001*
Group C: UDT with orchidopexy	7.676	0.17	0.001*
Group D: UDT with orchidopexy and treated with adjuvant Coenzyme-Q10 (10 mg/KgBW for 7 days)	18.95	0.15	0.000*

The high R2 value indicates minimal deviation of the measured data points from the fitted regression line, emphasizing the robustness of the assay. This finding supports the reproducibility and reliability of OD as a proxy for CA-II concentration, with the equation providing a predictable relationship for calculating unknown sample concentrations based on OD measurements. The results validate the use of this method in the quantitative assessment of CA-II for potential clinical diagnostics and biomarker research.

To explore whether significant differences existed between CA-II concentration groups, an analysis of variance (ANOVA) was conducted. The ANOVA findings revealed a p-value of <0.05, confirming statistically significant differences in OD measurements across different concentration groups. This result underscored the sensitivity of the ELISA method, demonstrating its capacity to distinguish between varying levels of CA-II concentration.

Further analysis through Tukey’s HSD post-hoc test identified specific group differences. The test revealed that the OD values of the lowest concentration group (0 ng/mL) were significantly different from those of higher concentration groups, such as 50 ng/mL and 100 ng/mL, with adjusted p-values of <0.01. Additionally, comparisons between intermediate groups, such as 40 ng/mL and 60 ng/mL, also showed statistically significant differences with p-values <0.05. These findings validated the trend seen in the standard curve, reinforcing the assay’s effectiveness in quantifying CA-II by detecting meaningful distinctions between different concentration levels.

The correlation analysis further affirmed the strong relationship between CA-II concentration and OD values, with a Pearson correlation coefficient of 0.998. This coefficient indicated an extremely strong positive correlation, confirming that OD readings are a reliable and predictive measure of CA-II concentration. The consistency of this relationship supports the use of OD as a robust metric for CA-II quantification, ensuring that the ELISA method employed in this study is both precise and reliable for clinical and research applications.

Overall, the results substantiate the validity of the linear relationship between CA-II concentration and OD, demonstrating the ELISA method’s robustness for CA-II measurement. These findings enhance the potential application of this assay in diagnostics and research focused on CA-II as a biomarker, particularly in contexts involving altered enzyme activity related to specific diseases.

5. DISCUSSION

The results of this study demonstrated a robust linear relationship between optical density (OD) and Carbonic Anhydrase II (CA-II) concentration, validated by a high coefficient of determination (R² = 0.9957) and a Pearson correlation coefficient of 0.998. These findings confirm that OD, as measured through ELISA, serves as a reliable and precise quantitative metric for CA-II. This is particularly significant given CA-II’s crucial role in physiological processes such as pH regulation, CO₂ transport, and acid-base balance (1,2). The enzyme’s rapid catalytic turnover and its presence in diverse tissues further emphasize the importance of reliable quantification methods for research and clinical diagnostics (3).

The correlation between OD and CA-II concentration provides a robust foundation for the practical application of ELISA in quantifying CA-II. This validation addresses a critical research gap, as current methodologies for measuring CA-II levels often suffer from inconsistencies due to variable assay conditions and interfering substances (8, 9). By establishing a clear linear relationship, this study supports the use of OD as a dependable measure, enhancing diagnostic accuracy and reproducibility. Understanding this relationship is essential, particularly as CA-II is implicated in several pathophysiological conditions. For example, CA-II deficiency or altered activity is associated with disorders like osteopetrosis, characterized by impaired osteoclast function and excessive bone density (2,4). Additionally, variations in CA-II levels have been linked to certain cancers, where its expression may correlate with tumor aggressiveness and progression (5).

The findings of this study align with prior research emphasizing the importance of precise CA-II quantification. Studies have shown that accurate measurement of CA-II is crucial for maintaining diagnostic reliability and supporting the exploration of its role in various diseases (12, 13). While fluorescence-based and mass spectrometry methods have demonstrated promise in improving measurement sensitivity (10, 11), ELISA remains a widely accessible and effective technique. This study’s robust linear model reinforces the idea that OD measurements, when carefully controlled, can provide reliable insights into CA-II concentration, facilitating both basic research and clinical applications.

The clinical implications of these findings are notable, particularly for reproductive health. CA-II is essential for maintaining the acid-base balance and ion transport within the testicular environment, which in turn supports spermatogenesis and overall testicular function (6, 7). The reliable quantification of CA-II levels can aid in the early diagnosis and management of conditions such as cryptorchidism, which is associated with increased infertility and cancer risk (14). The results of this study suggest that standardized ELISA protocols for measuring CA-II could be employed to monitor treatment efficacy, such as in orchidopexy or adjunct therapies involving Coenzyme Q10, enhancing the assessment of therapeutic outcomes.

However, this study is not without limitations. The sample size, while sufficient for initial validation, may restrict the generalizability of the findings across broader populations. Additionally, while the use of an animal model provides valuable insights, translating these results to human clinical settings requires further investigation. Variability in OD readings due to factors such as sample matrix properties and assay conditions must be minimized to ensure consistent results. Future research should involve larger, more diverse sample sets and explore the integration of advanced techniques, such as mass spectrometry, for enhanced specificity.

Suggestions for future research include expanding sample diversity to cover different age groups, health statuses, and genetic backgrounds to validate the findings across a wider demographic. Additionally, exploring biomarkers related to CA-II could provide further insights into its role in various physiological and pathological processes. Integrating ELISA with more advanced, complementary methods like multiplex assays or fluorescence-based detection could improve both the sensitivity and specificity of CA-II measurements. Finally, long-term studies assessing CA-II levels in patients with cryptorchidism and tracking their clinical outcomes would provide deeper insights into the enzyme’s diagnostic and prognostic potential.

This study validated the strong correlation between optical density (OD) and Carbonic Anhydrase II (CA-II) concentration, evidenced by a robust linear regression model with an R2 value of 0.9957 and a Pearson correlation coefficient of 0.998. The findings confirm that OD measurements obtained via ELISA are reliable and accurate for quantifying CA-II, supporting their use in both clinical diagnostics and research applications. The significant differences observed through ANOVA and further confirmed by post-hoc testing underscore the method’s sensitivity in detecting variations in CA-II concentrations.

The practical implications of these findings are profound, particularly in clinical practice where reliable CA-II measurement is essential for diagnosing and monitoring conditions such as cryptorchidism. The ability to accurately measure CA-II levels can contribute to early detection and effective treatment planning, especially in interventions involving orchidopexy and Coenzyme Q10 administration. Additionally, these results can guide future research in reproductive health, enabling more detailed investigations into the role of CA-II and its potential as a biomarker for testicular function and other related pathologies.

6. CONCLUSION

This study reinforces the efficacy of OD as a dependable metric for CA-II quantification, highlighting the ELISA method’s robustness and applicability in both clinical and research environments. The clear linear relationship between OD and CA-II concentration provides a validated framework for quantitative analysis, which is crucial for advancing biomarker research and improving patient outcomes in reproductive health.

Recommendations for researchers and practitioners include adopting standardized ELISA protocols for consistent and reliable CA-II measurement. It is also recommended that future studies incorporate larger and more diverse sample sets to extend the applicability of these findings. Additionally, integrating OD-based CA-II measurements with complementary techniques such as multiplex assays may enhance diagnostic precision and broaden the scope of biomarker research.

Author’s contribution:

The all authors were involved in all steps of preparation this article, including final proofreading.

Conflict of interest:

The authors have no conflict of interest to declare.

Financial support and sponsorship:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.