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Questions and Answers

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?

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?

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?

$0.056$ Signup and view all the answers

For a competitive immunoassay, how is the Limit of Quantitation (LOQ) calculated?

Mean - 3 x SD Signup and view all the answers

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?

$0.31$ Signup and view all the answers

What factors influence the sensitivity of an ELISA?

Affinity of antibodies, capture efficiency, signal amplification. Signup and view all the answers

What is the significance of calculating the mean and standard deviation from a zero standard?

To assist in calculating the Limit of Detection. Signup and view all the answers

What is the primary objective of competitive immunoassays?

To compete for a limited amount of antibody. Signup and view all the answers

What is the final concentration of antibody after performing a 1 in 5000 dilution from a stock concentration of 100 mg/ml?

0.02 mg/ml Signup and view all the answers

How can the standard deviation (SD) be calculated on a Casio calculator?

By selecting 3 (1-Variable Calc) after pressing OPTN. Signup and view all the answers

What is the equation used to calculate the % CV for a competitive immunoassay?

%CV = SD / Mean x 100 Signup and view all the answers

In a competitive immunoassay, what does a lower concentration of bound label indicate?

More unlabelled test sample is present. Signup and view all the answers

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?

0.534 Signup and view all the answers

What is the best estimate of the dynamic range of the assay at < 30% CV?

Wider dynamic range than 20% CV Signup and view all the answers

Which of the following statements is true regarding precision in competitive immunoassays?

Precision improves at high log concentration values. Signup and view all the answers

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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Description

Enhance your understanding of competitive immunoassays and key data handling techniques in this quiz. Learn about antibody stock concentrations, %CV calculations, and precision profiles, as well as how to accurately measure recovery and linearity. This quiz is essential for mastering the concepts crucial for data accuracy in immunoassay procedures.

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