Last Bit of Stuff and Notes from Exams

purpose of ELISA

allows us to detect the presence and titer of antigens, antibodies, proteins/glycoproteins (all collectively referred to as analytes) in a solution

test traps analyte in solid phase (sticks them to an coated solid surface)

typically done in a 96-part well format

have to use a specially-prepped plate with wells coated either with the Ab to the analyte of interest or something else to promote antigen adherence

extremely sensitive test

elisa types

direct - antigen of interest adhered to plate, followed by Ab with marker substrate

indirect - antigen adhered, primary Ab to antigen, secondary Ab with attached marker binds the primary

sandwich - plate coated with holder Ab, antigen ends up sandwiches between the holder Ab and the marker-attached Ab

• can have plate-bound Ab binding the antigen then have a second Ab with an attached marker binding a different region of the antigen

• Plate Ab binds the antigen, another primary Ab binds the antigen and is bound by a secondary Ab with a marker

competitive elisa - competes for Ab-marker binding with another inserted antigen, good indicator of target antigen conc?

marker is often an enzyme that converts a substrate into a visible marker for binding (e.g. product has color)

direct elisa

direct analyte detection

requires an Ab conjugated to an enzyme for the indicator - fast but expensive

helpful when there are no commercial kits available for a specific protein target

advantages:

• quicker, fewer steps

• eliminates cross-reactivity risk with second Ab

disadvantages:

• immunoreactivity of primary Ab may be affected by conjugation with an enzyme or tag

• labeling of a primary Ab is time consuming and expensive

• less flexibility to swap out primary Abs

• may have low signal amplification

indirect elisas

indirect analyte detection

involved a primary, unlabeled Ab specific for the analyte and a secondary Ab specific for the primary with an attached enzyme/tag

commonly used

advantages:

• primary Abs are commercially abundant

• maximum immunoreactivity due to the primary being unlabeled - nothing gets in the way of primary-antigen binding

• sensitivity also maximal due to a primary Ab having many epitopes for the labeled secondary Ab to bind - strong signal amplification, easy to differentiate between antigen presence and absence

disadvantages:

• cross-reactivity may occur due to non-specific binding of the secondary Ab - more chance for mistakes with more moving parts

• takes longer since need extra incubation step for the secondary Ab

sandwich elisa

direct or indirect depending on design

uses matches Abs that are specific for different, non-overlapping epitopes on the analyte (so the plate-bound and primary indicator Abs can both bind the analyte simultaneously

have to coat the plate in “capture” Ab first before incubating with the analyte

advantages:

• high sensitivity due to analyte being captured by a specific Ab and detected by another - 2 checks of antigen ID

• can use less pure and more complex samples - no need to purify

• flexible and sensitive depending on whether direct or indirect is used

disadvantages:

• not an actual sandwich you can each (why did she put this on the slides lmao)

competitive elisa

competition for binding between patient antigen and enzyme-labeled antigen for binding

since it’s the competitor that’s labeled and not the antigen of interest, more signal here means a DECREASED conc of antigen

primary Abs are first incubated with patient antigen. Once they bind, these complexes are added to wells coated with the competitor. Another incubation period follows before unbound (not binding the competitor, since that’s the only thing tied down) Ab is washed away

less antigen in sample = more Abs bind to the plate = more signal

cardiolipin anti-human IgM elisa

direct elisa

measure intensity of added chromogenic substrate, read sample optical density

elisa is for enzyme-linked immunosorbent assay

semi-qualitative detection of cardiolipin IgG Abs

used in conjugation with other tests for diagnosis

elisa math

average the replicates

subtract negative control from all wells

optical density is y values, conc target is x

plot y=mx+b for standard curve to find unknown X values based on constants