Podcast
Questions and Answers
What happens to the dynamic range when the coefficient of variation (CV) increases from 20% to 30%?
What happens to the dynamic range when the coefficient of variation (CV) increases from 20% to 30%?
- It remains the same.
- It becomes undefined.
- It becomes wider.
- It becomes narrower. (correct)
What is the Lower Limit of Detection (LLD) also known as?
What is the Lower Limit of Detection (LLD) also known as?
- Minimum Detectable Concentration (correct)
- Mean Detectable Concentration
- Maximum Detectable Concentration
- Limit of Quantitation
How is the Limit of Quantitation (LOQ) for a non-competitive immunoassay calculated?
How is the Limit of Quantitation (LOQ) for a non-competitive immunoassay calculated?
- Mean + 2 x SD
- Mean + SD
- Mean + 3 x SD (correct)
- Mean - 3 x SD
What is the Limit of Quantitation (LOQ) for the non-competitive ELISA with a mean OD450 of 0.050 and SD of 0.002?
What is the Limit of Quantitation (LOQ) for the non-competitive ELISA with a mean OD450 of 0.050 and SD of 0.002?
For a competitive immunoassay, how is the Limit of Quantitation (LOQ) calculated?
For a competitive immunoassay, how is the Limit of Quantitation (LOQ) calculated?
If the mean of zero standard (B/Bo) is 0.45 and the SD is 0.048 for a competitive ELISA, what is the calculated LOQ?
If the mean of zero standard (B/Bo) is 0.45 and the SD is 0.048 for a competitive ELISA, what is the calculated LOQ?
What factors influence the sensitivity of an ELISA?
What factors influence the sensitivity of an ELISA?
What is the significance of calculating the mean and standard deviation from a zero standard?
What is the significance of calculating the mean and standard deviation from a zero standard?
What is the primary objective of competitive immunoassays?
What is the primary objective of competitive immunoassays?
What is the final concentration of antibody after performing a 1 in 5000 dilution from a stock concentration of 100 mg/ml?
What is the final concentration of antibody after performing a 1 in 5000 dilution from a stock concentration of 100 mg/ml?
How can the standard deviation (SD) be calculated on a Casio calculator?
How can the standard deviation (SD) be calculated on a Casio calculator?
What is the equation used to calculate the % CV for a competitive immunoassay?
What is the equation used to calculate the % CV for a competitive immunoassay?
In a competitive immunoassay, what does a lower concentration of bound label indicate?
In a competitive immunoassay, what does a lower concentration of bound label indicate?
What is the mean calculated from the standard values 0.555, 0.525, 0.533, 0.522, 0.515, 0.550, 0.540, 0.545, 0.510, and 0.546?
What is the mean calculated from the standard values 0.555, 0.525, 0.533, 0.522, 0.515, 0.550, 0.540, 0.545, 0.510, and 0.546?
What is the best estimate of the dynamic range of the assay at < 30% CV?
What is the best estimate of the dynamic range of the assay at < 30% CV?
Which of the following statements is true regarding precision in competitive immunoassays?
Which of the following statements is true regarding precision in competitive immunoassays?
Flashcards
Competitive Immunoassay
Competitive Immunoassay
A type of immunoassay where both labelled and unlabelled analyte compete for a limited amount of antibody.
Calibration Curve
Calibration Curve
A graph that shows the relationship between the concentration of an analyte and the signal produced by an immunoassay.
Antibody Stock Dilution
Antibody Stock Dilution
The process of diluting a stock solution to a desired concentration.
Precision
Precision
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Coefficient of Variation (%CV)
Coefficient of Variation (%CV)
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Precision Profile
Precision Profile
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Dynamic Range
Dynamic Range
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Accuracy
Accuracy
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ELISA Sensitivity
ELISA Sensitivity
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Lower Limit of Detection (LLD)
Lower Limit of Detection (LLD)
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Limit of Quantitation (LOQ)
Limit of Quantitation (LOQ)
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LOQ Calculation - Non-competitive
LOQ Calculation - Non-competitive
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LOQ Calculation - Competitive
LOQ Calculation - Competitive
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Mean OD450 of Zero Standard
Mean OD450 of Zero Standard
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Standard Deviation (SD) of Zero Standard
Standard Deviation (SD) of Zero Standard
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Interpolated LOQ Concentration
Interpolated LOQ Concentration
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Study Notes
Immunoassay Data Handling - Part 2
- Learning Objectives: Review competitive immunoassays, data handling, antibody stock concentrations, %CV calculations, data handling for precision profiles, data handling for measures accuracy, recovery, and linearity.
Competitive Immunoassays
- Mechanism: Either labelled antigen (Ag) or antibody (Ab) and unlabelled specimen Ag (or test sample analyte) compete for a limited amount of antibody.
- Principle: Less label measured means more unlabelled Ag is present. A concentration of bound label is plotted against analyte concentration, creating an inverse standard curve.
Calibration Curves
- Non-competitive ELISA: Standard curve displays a gradual increase in optical density/fluorescence/lumens with increasing control concentration.
- Competitive ELISA: Standard curve shows a decreasing trend in optical density/fluorescence/lumens with increasing control concentration.
Antibody Stock Concentration
- Stock Concentration: 100 mg/mL
- Dilution: 1 in 5000
- Final Concentration (calculation): 0.02 mg/mL
- Lab Dilution (calculation): 0.2 mL antibody required; 0.8 mL buffer required for 1 mL final volume.
Calculating Standard Deviation (SD)
- Calculator Method (Casio):
- Enter data into the variable column.
- Press = after each entry followed by OPTN.
- Choose 1-Variable Calc -> Standard Deviation.
- The relevant SD value (σx) will be displayed
Precision – SD and % CV
- Standard Values: 0.555, 0.525, 0.533, 0.522, 0.515, 0.550, 0.540, 0.545, 0.510, 0.546
- Calculation: % CV = SD / mean x 100
- Mean: 0.534
- SD: 0.0147
- %CV: 2.75%
Precision Profiles
- Dynamic Range: The range of analyte concentrations where the assay exhibits acceptable precision (usually under 30%CV). Graphs illustrate different precision profiles as concentrations increase
Sensitivity
- Factors: Affinity of antibody to analyte, capture efficiency of the plate, signal amplification, buffer/wash conditions, and the detection method.
- Lower Limit of Detection (LLD/MDC): The lowest measurable value statistically different from zero.
Limits of Quantitation (LOQ) - Non-competitive
- Formula: LOQ = Mean + (3 * SD) (where Mean and SD are from zero standard)
- Method: Interpolate the calculated Absorbance value from the graph to find the corresponding concentration on the x-axis.
Limits of Quantitation (LOQ) - Competitive
- Formula: LOQ = Mean – (3 * SD) (where Mean and SD are from zero standard)
- Method: Interpolate the calculated B/B0 value from the graph to find the corresponding Log concentration on the x-axis.
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