An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow’s Milk)
Abstract
:1. Introduction
2. Results
2.1. Validation of the Direct, Competitive Immunoenzymatic Method
2.1.1. Performance of the Method
2.1.2. Method Linearity (Calibration Curve)
2.1.3. The Minimum Detection Limit (Milk)
2.1.4. The Minimum Limit of Quantification (Milk)
2.1.5. Recovery (Recovery Percentage)
- -
- equation of calibration line: y = −3.4306x + 2.8249
- -
- standard error of regression line: ES = 0.1052
2.2. Determination of OTA in Human Milk Samples
2.3. Determination of OTA in Cow’s Milk Samples
3. Discussions
4. Conclusions
5. Materials and Methods
5.1. Equipment
5.2. Reagents
- Calibrators with OTA concentrations between 0.0–0.40 ng/mL: each kit contains multi-analyte calibrators with values attributed through a referential method that certifies that the values of the said analytes were established in accordance with WHO-certified reference materials for 6 consecutive levels of concentration, in a liquid conditioning medium, which can be used as such (6 bottles, 1.5 mL each, with OTA concentrations of 0.0, 0.02, 0.05, 0.1, 0.2, 0.4 ng/mL diluted in 70% methanol).
- Reagent kit for the quantitative detection of OTA, contained all the necessary items that enable the ELISA reaction for the detection of OTA: reactive diluent, enzyme conjugate containing OTA conjugated with horseradish peroxidase (HRP), an underlayer reagent containing stabilized tetramethylbenzidine (TMB), acid stop solution and washing solution as a lyophilized powder from phosphate-buffered saline (PBS) with Tween®20 (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween®20, pH 7.4); one sachet to be diluted in 1000 mL distilled water. Absolute methanol was purchased from Chemical Company, Romania.
5.3. Statistical Analysis
5.4. Sample Preparation
5.5. Method
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
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Tested Sample | %A0 Standard 0.02 ng/mL | %A0 Sample | %A0 Standard < 2 SD | Coefficient of Variation % | ng/mL |
---|---|---|---|---|---|
Colostrum | 86.5 | 92.3 | 89.3 | 1.6 | <0.08 |
Cow’s milk | 85.9 | 91.1 | 88.9 | 1.2 | <0.08 |
Nr. | Standard | Concentration (ng/mL) | Absorbance | r | Intercept | Slope |
---|---|---|---|---|---|---|
1 | STD 1 | 0.4 | 1.4800 | −0.9838 | 2.824 | −3.3430 |
2 | STD 2 | 0.2 | 2.0060 | |||
3 | STD 3 | 0.1 | 2.6070 | |||
4 | STD 4 | 0.05 | 2.7220 | |||
5 | STD 5 | 0.02 | 2.7410 | |||
6 | STD 6 | 0 | 2.7520 |
Regression Statistics | |
---|---|
Multiple R | 0.9838 |
R Square | 0.9681 |
Adjusted R Square | 0.9601 |
Standard Error | 0.1052 |
Observations | 6 |
ANOVA Statistical Analysis | |||||
---|---|---|---|---|---|
df | SS | MS | F | Significance F | |
Regression | 1 | 1.3426 | 1.3426 | 121.2284 | 0.0004 |
Residual | 4 | 0.0443 | 0.0111 | ||
Total | 5 | 1.3869 |
Coefficients | Standard Error | t Stat | p-Value | Lower | Upper | |
---|---|---|---|---|---|---|
95% | 95% | |||||
Intercept | 2.8249 | 0.0587 | 48.1313 | 1.11 × 10−6 | 2.6620 | 2.9879 |
Concentration (ng/mL) | −3.4306 | 0.3116 | −11.0104 | 0.0004 | −4.2957 | −2.5655 |
Sample Type | Mean Concentration (ng/mL, n = 20) | Standard Deviation (SD) | Limit of Detection (ng/mL) | Limit of Quantification (ng/mL) |
---|---|---|---|---|
Milk | 0.0079 | 0.0039 | 0.0197 | 0.0474 |
Tested Sample | Recovery Series I (%) | Recovery Series II (%) | Recovery Series III (%) | Recovery Mean (%) |
---|---|---|---|---|
Human milk | 96 | 110 | 95 | 100 |
Cow’s milk | 114 | 116 | 113 | 114 |
Procedure: | Blank | STD 1 | STD 2 | STD 3 | STD 4 | STD 5 | STD 6 | Sample |
---|---|---|---|---|---|---|---|---|
(0.4 ng/mL) | (0.2 ng/mL) | (0.1 ng/mL) | (0.05 ng/mL) | (0.02 ng/mL) | (0 ng/mL) | |||
a. Addition | x | 200 μL of reactive diluent was added to each well | ||||||
b. Addition | x | 100 μL | 100 μL | 100 μL | 100 μL | 100 μL | 100 μL | 100 μL |
c. Homogenization | x | 2–3 successive pipetting for homogenization were performed. | ||||||
d. Transfer | x | 100 μL of contents from each mixing well of microtiter plate was transferred. | ||||||
to corresponding Anti OTA antibody coated well from reaction microplate. | ||||||||
e. Incubation | x | 30 min at ambient temperature (22 °C). | ||||||
f. Wash | x | The content of the wells was removed and then wells were washed 3 times | ||||||
with washing buffer PBS-Tween (pause between washes was 60 s). | ||||||||
After the last wash the excess moisture was absorbed by blotting on filter paper. | ||||||||
g. Addition | x | 100 μL OTA-horseradish peroxidase (HRP) conjugate was added to each well. | ||||||
h. Incubation | x | 30 min at ambient temperature (22 °C). | ||||||
i. Wash | x | Three successive washes were carried out with washing buffer PBS-Tween. | ||||||
After the last wash the excess moisture was absorbed by blotting on filter paper. | ||||||||
j. Addition | x | 100 μL substrate solution TMB was added to each well. Being a chromogenic | ||||||
substrate for HRP, TMP produced a deep blue color during the enzymatic | ||||||||
degradation of hydrogen peroxide by HRP. | ||||||||
k. Incubation | x | 10 min at ambient temperature (22 °C). | ||||||
l. Homogenization | x | 2–3 successive pipetting for homogenization were performed. | ||||||
m. Reading | x | Using a spectrophotometer, optical density was read at λ = 450 nm with 630 | ||||||
nm reference filter. The intensity of the color was directly proportional with the | ||||||||
quantity of linked conjugate and indirectly proportional to the quantity of OTA | ||||||||
present in the standard or in the sample. |
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Cuciureanu, M.; Tuchiluș, C.; Vartolomei, A.; Tamba, B.I.; Filip, L. An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow’s Milk). Toxins 2021, 13, 673. https://doi.org/10.3390/toxins13100673
Cuciureanu M, Tuchiluș C, Vartolomei A, Tamba BI, Filip L. An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow’s Milk). Toxins. 2021; 13(10):673. https://doi.org/10.3390/toxins13100673
Chicago/Turabian StyleCuciureanu, Magdalena, Cristina Tuchiluș, Anca Vartolomei, Bogdan Ionel Tamba, and Lorena Filip. 2021. "An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow’s Milk)" Toxins 13, no. 10: 673. https://doi.org/10.3390/toxins13100673