BTC ch.26 assays

whats the 1st step involved in assay development, describe

sample prep - collecting a representative sample from the whole, processing it, and labeling it appropriately

whats the 2nd step involved in assay development, describe

method selection - following a standardized protocol or using a commercially available kit for the chosen assay

whats the 3rd step involved in assay development, describe

detection - comparing the sample to reference standards using instruments and collecting data about the analyte

whats the 4th step involved in assay development, describe

analysis and interpretation - analyzing the collected data and comparing it to standards to draw conclusions

define an assay

any test used to analyze a characteristic of a sample, such as composition, purity, or activity

define analyte

the specific substance being identified and measured in an assay

analytes attach to what?

substrates

define the control

a sample with known composition used as a benchmark for comparison

define negative control and describe

a sample lacking the analyte of interest, ensuring no response in the assay unless there’s an error

define positive control and describe

a sample containing the analyte, confirming the assay’s functionality

define direct dilution

its a method where controlled volumes of a concentrated stock solution are transferred directly to achieve desired concentrations, ensuring accuracy

define a reference standard

a highly purified substance with a known composition, used for comparison in assays

define replicate (in context of assays)

repeating an assay multiple times on the same sample to assess variability and ensure reliability

define serial dilution

its a dilution method where a substance’s concentration is progressively reduced in a series of steps, useful for creating highly diluted solutions

define specificity (selectively) in context of assays

the ability of an assay to accurately identify and quantify a specific analyte even in the presence of other substances .

define the standard method

its a documented procedure established by a recognized body to ensure consistency and reproducibility across different labs

what is an assay and why is it important?

it’s is any test used to analyze a specific characteristic of a sample such as composition, purity, or activity. They’re crucial because they provide trustworthy data used in decision-making and drawing conclusions across various fields like medicine, forensics, and food safety.

what are the key steps involved in assays?

sample prep, method selection, detection, and analysis/interpretation

why is sample prep important in assay development? (long answer question)

This is important because it directly impacts the assay's results. Biological samples often need preservation, treatment, incubation, or processing before analysis. These steps can affect the sample's components, causing degradation, analyte loss, or changes in enzymatic activity. To prevent errors, it is crucial to prepare all samples, controls, and standards consistently and test for potential losses or variations from different preparation methods.

what is the role of controls in assays and why are they important?

controls provide a reference point for comparison and help ensure accuracy. the negative control (the one missing the target analyte) helps confirm that the assay is not detecting something it shouldn’t. the positive control (containing the target analyte) should produce a specific response in the assay showing that the assay is functionary correctly.

what is method validation and why is it necessary?

its a process is showing that a lab procedure is robust, reliable, and reproducible. it ensures that the chosen method consistently produces accurate and meaningful results. validation involves evaluating various things like accuracy, precision, limit of detection, limit of quantitation, specifity, linearity, range, robustness, ruggedness, and system suitability

whats the difference between accuracy and precision in assay validation

Accuracy is how close a test result is to the true value, determined by comparing it to a known reference. Precision is how consistently a test gives similar results when repeated on the same sample. A test can be precise but not accurate if the results are consistent but far from the true value. Ideally, a test should be both accurate and precise.

what are the key considerations for developing a diagnostic assay?

cross reactivity (tendency of the assay to react with other substances other than the target analyte leading to false-pos results), sensitivity (ability o detect low concentrations of the target assay), specificity , and method validation

define robustness

capacity of a method to remain unaffected by small deliberate variations of method parameters

define ruggedness

degree of reproducibility of results obtained by the analysis of the same samples under a variety of conditions

what are the 2 types of diagnostic assays?

testing for cross-reactivity and a serological assay (testing for past infection to look for antibodies in a person’s blood)