Cell bio & physio ELISA

all done, as much as i can think of

advantage of a sandwich ELISA

high sensitivity and specificity for detecting target antigens - using 2 abs reduces background noise, ensures accurate results

function of a blocking buffer eg BSA with ELISA

prevents nonspecific binding

how to calculate values for elisa

convert all to the same unit (usually to ng/ml), find dilution factor by dividing amount per vial of reagent by their working [], then divide the amount that you need (amount per well x wells + 10% margin) by the dilution factor

general - how to detect proteins vs how to detect RNA/DNA

w/ affinity, DNA/RNA detected w/ primers/complementary strings

general steps of sandwich elisa + fxns of steps (15)

dilute in coating buffer and then add capture ab to wells of a microplate - so it adheres to the bottom, incubate overnight, wash plates w/ buffer 3x, add a protein rich blocking buffer (eg BSA), incubate, wash plates 3x, add sample/standards, incubate 2hrs, wash 3x, add detection ab, incubate 2hr, wash 3x, add streptavidin-HRP (substrate, reacts with the enzyme linked to the detection antibody, triggers a color change), add stop solution (usually an acid) to stop the reaction, determine optical density of wells using a microplate reader

why do you need to add stop solution after a short period of time

because otherwise even in the empty wells the rxn will happen, the enzyme decreases the rate of reaction but w/o it it will still happen eventually

why do you use antibodies from diff animals

to prevent false positives through cross reactivity

ELISA long

Enzyme-Linked Immunosorbent Assay

why is a standard curve necessary

it translates raw signal intensity (e.g., light absorbance or Optical Density) into a measurable concentration, it establishes a direct, mathematical relationship between known amounts of a target substance and the assay's output

optical reading wells descr

do correction by substracting readings at 540nm or 570 nm from readings at 450 nm, corrects for optical imperfections in the plate

what sort of antibody did we use

monoclonal - fuse ab producing B cell to tumor → can always keep producing more, only detects 1 epitope

why should you avoid placing elisa plate in direct light

because it causes photobleaching (decomposition) of the fluorophores and destabilizes the chromogenic substrates (like TMB) - causes premature color development, fading, or signal loss