Immunology Testing Methods and Assays

demonstration of ___________ are considered to be diagnostic

4-fold increase or decrease in titer

what is sensitivity?

how much antibody is present before it is detectable

examples of sensitive tests

EIA, RIA, IFA

what is specificity?

a test’s ability to detect only true positives, less false positives

what is precipitation?

soluble antigens combined with specific antibodies produce a solid precipitate

precipitation only occurs at—

optimal antigen-antibody ratio (zone of equivalence)

excess antigen causes—

(post zone) small complexes to form with no precipitation

excess antibody causes—

(prozone) large complexes to form with suboptimal precipitation

the precipitin curve shows—

the amount of precipitation with varying antigen concentration and constant antibody concentration

how do you set up an ouchterlony double diffusion test?

• agar lawn with wells carved in circle and one central well

• central well contains specific antigen

• patient sample and controls are in surrounding wells

what is the main principle of the ouchterlony double diffusion test?

• antigen and antibody diffuse from wells into agar

• antibody-antigen complexes form and precipitate

• bands show patterns of identity

pattern of identity indicates—, looks like—

two antibodies are identical and share all antigenic epitopes

• continuous curved band between two outer wells and central well

pattern of partial identity indicates—, looks like—

two antibodies are similar and share some antigenic epitopes

• two bands present between outer wells and central well with a “spur” that continues past intersection of the bands

pattern of non-identity indicates—, looks like—

two antibodies are unsimilar and do not share any antigenic epitopes

• two bands present between outer wells and central well with clear continuing intersection

what is the principle of radial immunodiffusion?

antigen is within the agar and serum precipitates outwards

• diameter of ring is compared to the serially diluted controls to determine concentration

agglutination reactions are more—

sensitive than precipitation reactions

• specifically hemagglutination

what is an agglutination reaction?

visible agglutination of particulate antigen or antigen coated inert particles

direct agglutination uses—

antigens that are naturally on the indicator particle

passive agglutination uses—

an indicator particle coated in antigen to detect antibody

reverse passive agglutination uses—

an indicator particle coated in antibodies to detects antigen

what Ig is a better agglutinator and why?

IgM is better because it has 5x the binding sites as IgG

what Ig is a better precipitator and why?

IgG is better because it is smaller and can diffuse better

the complement system is—

a series of serum proteins that bind in sequence to antibody coated cell membranes leading to cell lysis

an increase in serum complement indicates—

acute phase response or inflammation

a decrease in serum complement indicates—

decreased synthesis of serum proteins/complement or increased consumption of serum proteins/complement

an immune complex syndrome may occur as a direct result of—

complement activation

what is the mechanism of immune complex syndrome?

circulating soluble antigen react with complement fixing antibodies to form soluble antigen-antibody complexes that activate complement

immune complex syndrome complexes can have clinical importance in—

SLE, serum sickness, and other autoimmune disorders

what is direct IFA staining?

antisera is conjugated with FITC is added to the antigen

direct IFA staining is used to detect—

immune complexes or antibodies deposited in tissue or fixed to a patient specimen by using antisera directed against human Igs

what is indirect IFA staining?

patient antibody is layered on an antigen substrate and FITC labeled antisera against human Igs is layered on top to form a complex (sandwich)

indirect IFA staining detects—

circulating antibodies

how is patient serum pretreated when testing for IgM with IFA?

remove IgG

why do we remove IgG from patient samples tested for IgM using IFA?

false positives and negatives can be caused by

• IgG binding to antigenic sites, causing decrease in IgM binding and decrease in FITC binding and a false decrease in signal

• IgM RF can bind to IgG bound on antigenic sites leading to increase in FITC binding and a false increase in signal

enzyme immunoassays are extremely —

sensitive to antibodies

what is the sandwich technique of EIA testing?

• known antigen or antibody is coated into a plastic well where patient serum is added

• if the antigen or antibody is present in the serum, it binds to the well

• an enzyme tagged antibody to the reaction is added

• an enzyme substrate is added and a spectrophotometer is used to detect and quantify or visualize the reaction

what is the multiplex flow immunoassay methodology?

simultaneous detection and identification of antibodies in one sample by using a combination of flow cytometry and EIA by identifying different colored dyed beads

an example of multiplex flow immunoassay is—

the bioplex 2200 test system

immunocap is based on—

extremely high binding capacity

how is the high binding capacity in immunocap achieved?

cellulose in each solid phase in an enclosed capsule provides an ideal microenvironment to bind allergens irreversibly and maintain native structure

immunocap is designed as a—

sandwich immunoassay

• specific allergen or antigen is present and binds with patient IgE against it

• enzyme labeled antibodies to human IgE form a complex

• a developing agent is added and fluorescence if measured

flow cytometry measures—

physical and chemical characteristics of individual cells

how does flow cytometry establish cell lineage?

fluorescent dyes attach to cell CD markers which become excited and emit light of a certain wavelength which is detected

chemiluminescence has —

high sensitivity and high specificity

what are the two parts of a chemiluminescence immunoassay?

immunoreaction technique and chemiluminescence technique

what is the basic principle of chemiluminescence?

an enzyme in the immunoreaction acts on a luminescent substrate causing a chemical reaction and releasing energy and photons

in chemiluminescence, the photon yield is proportional to—

the amount of substance measured in a sample