Analytical Chemistry

Immunoassay

What is immunoassay

method that uses an antibody or antigen as a reagant

Ab and Ag Binding is

non-covalent

Ab and Ag binding is related to what four things

specificity

affinity and avidity

concentration

environmental conditions

Ab and Ag binding is specific 

pAb: mixture of Abs

mAb: a single Ab clone

cross reactivity 

Ab and Ag binding has a high

affinity- strength of a single Ab-Ag bond

avidity- sum of the attractions of different Abs toward a particular Ag

Ab and Ag binding is concentration dependent or independent?

dependent

What are the three zones during a precipitan reaction

Prozone: Ab excess

Zone of equivalence- optimal ratio of antibody concentration to antigen concentration

Postzone: Ag excess

The two categories of Immunoassay methods are

Unlabeled

labeled 

What is measured during an unlabeled method of immunoassay?

Direct measurement of Ag-Ab immunocomplex

In unlabled IA methods Turbidimetry and nephlemetry have

limited sensitivity- using mainly serum proteins

nephlometry has better sensitivity

What kind of reagents do Labeled IA use

labeled Ag or Ab

What IA has the highest analytical sensitivity

labeled IA

What are three types of labels used in Labeled IA

Enyzymes/substrates

flurophores

chemiluminscents

What is the most common label used in Labeled IA

Enzymes substrate, becase it is more specific and sensitive which leads to an amplified detection, but it also is versatile

What are advantages to the flurophores label in Labeled IA

Specificity and sensitivity

What are advantages to chemiluminescents

sensitivity, and its simple design

In heterogeneous assays what isnt changed uring the binding process

physical propoerties of the label

In heterogeneous assays what is required and what is measured

washing and seperation steps are required

remaining signal is measured 

(WITH A VERY HIGH SENSITIVITY, DIFFICULT TO AUTOMATE, AND WORKS WITH BOTH BIG AND SMALL MOLECULES)

What does solid-phase absorption mean

Ab or Ag can be attached to a solid surface

solid surfaces like microtiter plate wells like we used in ELISA

Tubes, because they are easy to automate

and large/microscopic beads to seperate through filtration

In Homogenous Assay what behaves differently when bound

The labeled reactant

WHAT IS DIFFERENT ABOUT HOMOGENOUS ASSAY

SEPERATION STEP IS NOT REQUIRED

The principle of competitive IA

An Ag excess method where AG* competes with Agx(analyte) for limited Ab binding sites

what is a competitive IA limitation

Analytical sensitivity and precision decreases at higher sample concentrations, BUT is better for smaller analytes

The Two types of competitive IA

Heterogenous: requires seperation, better for sensitivity and specificity

homogeneous: DOES NOT require seperation, better for speed and adaptability

low enzymatic product in competitive heterogenous ELISA means

Higher uknown [Agx]

Competitive homogenous IA is best for

small antigens

-therapeautic/abused drugs

ie- EMIT, FPIA

How does EMIT work

Uses steric hinderance to distinguish bound Ag* from free Ag*, soo when the enzyme labelled Ag* is bound to Ab, ab blocks substrate from reaching the enzyme active site (steric hinderance) leading to a decrease in colored enzymatic product formation, but when Agx competes for Ab, it frees Ag* which increases the colored enzymatic product.

The calibration curve of EMIT is

Non-linear w enzyme activity increasing as Agx increases

What is the primary application of EMIT

Measures ONLY Low MW analytes

In FPIA the Difference between unpolarized and polarized light

Unpolarized light- XZ plane and XY plane

Polarized light- One plane only 

During FPIA, polarized light emmitted through small molecules cause 

reorientation, making polarization lost 

How is FPIA appplicable

Measures ONLY low MW analytes

Noncompetitive IA aka

Sandwich assay, has the highest sensitivity and specificity, good for large molecules

Non competitive IA principle

An ab excess, where labeled reagent Ab* is used to detect Agx

Reaxtion components to Non competitive IA

Ab= capture molecules- immobolized on a solid phase

Ab*=conjugate molecule- signal ab

What are the steps of noncompetitive heterogenous IA

Agx binds to []-Ab to form []-Ab-Agx

Ab* then binds to []-Ab-Agx to form sandwich

excess free Ab* is removed by washing

Bound Ab* (conjugated Ab*) is measured

Interference with Noncompetitive IA and its remedies

Hook effect, a rapid increase in signal

remedy is to sample dilute using a 2-step method

What is microparticle enzyme IA

Non competitive heterogenous (sandwich assay)

Ab bound to microparticles like Glass fiber matrix 

Microparticle enzyme IA rxn steps

Analyte(Agx) is added to capture Ab-microparticles—>

Agx-Ab-microparticle complexes 

The Agx-Ab-microparticle-matrix is then washed to remove and unbound material

Labeled conjugate (Ab*) is added to form Ab*-Agx-Ab-microparticle-matric

excess Ab* is washed away

How is CMIA different from MEIA

CMIA uses a chemiluminescent compound as its label

its seperation step uses magnetic particles

measured using a PM tube

And has higher sensitivity 

What are the twp types of ELISA

Sandwich ELISA- measures Agx

Indirect ELISA- measures Abx (signal is proportional to [Abx]

common error- improper washing—> false High

What is the immunoblot Western blot

A transfer technique used to detect specific protein

sample is denatures and seperated by SDS-PAGE

the seperated proteins are transferred to a membrane when Abs are added to the membrane to form Ag-Ab

Unbound Abs are removed by washing

Secondary Ab* is added making Ag-Ab-Ab* complexes where they are visualized

Western blot is used to detect !

HIV Abs

What are some interferents in IA

Serum pigments- interferes more with homogenous

compounds of simlilar structure

autoantibodies

heterophile antibodies

What are heterophile antibodies

Human Abs that develop against animal Ig’s, which cause atypical IA results