Lecture 4: Immunoserology techniques

contributing factors to immunoserology techniques

affinity

avidity

affinity

initial force of attraction between a FAB site on an Ab and an EPITOPE on an antigen

avidity

overall strength of antigen/antibody binding. All of the affinities put together

precipitation general information

start with both antigen and antibody in soluble form. When they come together they become insoluble.

precipitation graph issues

prozone

zone of equivalence

postzone

prozone is when

too much Ab in patient serum means no PPT. dilute to fix false neg

postzone is when

too much antigen means no PPT

zone of equivalence is when

we can readily and accurately see PPT

techniques to measure precipitation in fluid

turbidimetry

nephelometry

turbidimetry measures

how much light comes through. How turbid?

nephelometry measures

how much light is scattered. Measured at 90 degree angle

techniques to measure precipitations in gel

radial immunodiffusion

ouchterlony immunodiffusion

basic premise of precipitation reaction in gel

antibody and antigen interaction. If they meet there is a visible line of PPT. Agarose stabilizes the reaction so we can visualize.

what is radial immunodiffusion

a single passive diffusion precipitation technique

what is the goal of radial immunodiffusion

look for patient Ag. gel contains Ab

how does radial diffusion work

Antibody incorporated into the gel itself

Punch holes in the gel and add Ag to the hole. Antigen diffuses into the gel

As the Ag diffuses it reaches the zone of equivalence and makes a PPT ring

how can we measure PPT in radial diffusion

Based on standards with known concentration Ag. create standard curve

Measure diameter and square. Compare to standard curve

pro of radial diffusion

can quantitate

con of radial diffusion

takes 3 days

what is the ouchterlony immunodiffusion

double passive diffusion technique

what is the purpose of ouchterlony immunodiffusion

compare the similarity of antigens

possible results of ouchterlony immunodiffusion

identity

non identity

partial identity

identity ouchterlony immunodiffusion looks like this

arc

non identity ouchterlony immunodiffusion looks like this

cross

partial identity ouchterlony immunodiffusion looks like this

spur

in a partial identity the middle antigen is ____________ similar to the underlined antigen

more similar to the underlined

methods of electrophoresis to measure precipitation

immunoelectrophoresis

immunofixation electrophoresis

purpose of immunoelectrophoresis

looks for antigen in patient serum

information about immunoelectrophoresis

double diffusion technique.

both Ag and Ab are being sent out

what does it mean when we see lines of PPT on immunoelectrophoresis

it means the patient has the target antigen

purpose of immunofixation electrophoresis

determination of identity of monoclonal gammopathy

could be used to diagnose multiple myeloma and waldenstrom's

immunofixation electrophoresis picture

nephelometry application

Immunoglobulins, complement, CRP, serum proteins

nephelometry principle

Light that is scattered is measured. ABS informs how much Ag/Ab is present

radial immunodiffusion application

Immunoglobulins and complement

radial immunodiffusion principle

Antigen diffuses out into the gel that is infused with Ab. measurement of diameter determines the amount of antigen present.

ouchterlony double diffusion application

complex antigens. compares which antigens are most similar

ouchterlony double diffusion principle

Both antigen and antibody diffuse out from wells in gel. Lines of PPT form and indicate the similarities between antigens

immunoelectrophoresis application

Differentiation of serum proteins

immunoelectrophoresis principle

Electrophoresis of serum is followed by diffusion of antibody into wells

immunofixation electrophoresis application

over or underproduction of antibodies

immunofixation electrophoresis principle

Electrophoresis of serum is followed by direct application of antibody to the gel. Tells us the identity of the Ab

what is agglutination

soluble + insoluble = looks for agglutination

what is the two step process for agglutination

sensitization

lattice formation

what is sensitization in relation to agglutination

coating particle/cell with something to help in agglutination

what is lattice formation in relation to agglutination

visualization of the agglutination reaction which is a product of crosslinking.

how much more effective is IgM than IgG

igM is 700X more effective than igG

why is IgM more effective than IgG

igM is a pentamer

what temp does igM work best at

room temp

what is coombs reagent

anti-human immunoglobulin. Helps IgG bind even though it is a monomer

techniques to measure agglutination

direct agglutination

passive/indirect agglutination

reverse passive agglutination

definition of direct agglutination

using known antigen attached to a particle to test for Ab in a patient.

what kind of test is a Wisal test

direct agglutination

describe the wisal test

detects antibody to salmonella typhi. All you need is the serum to test

hemagglutination

Involves RBC (sialic acid on RBC) binds viruses to form crosslinking agglutination

positive hemagglutination result

lattice

solid color

negative hemagglutination result

button

how should we read hemagglutination (titer)

the well where a positive reaction last happened

hemagglutination inhibition looks for _____________

patient antibody

steps for hemagglutination inhibition

Make dilutions of Pt serum and add to wells

Add virus to well

Incubate for 30 min

Add RBC

Incubate RBC for 30 min and interpret

what does the positive result for Hemagglutination inhibition look like

button

what does the negative result for Hemagglutination inhibition look like

solid color

lattice

when should Hemagglutination inhibition be used

Acute and convalescent serology example. Used for Rickettsia

hemagglutination inhibition assay is a type of

reverse passive agglutination

what is passive/indirect agglutination

employs particles coated with antigen not normally on the particle.

what is the use for passive/indirect agglutination

looks for virus ab in patient sample. Usually IgM

what is a common use for passive/indirect agglutination

cross reactivity

how can we fix cross reactivity for passive/indirect agglutination.

use as a screening test and do a confirmatory test after

what is a reverse passive agglutination

Uses carrier particles. Attached manufacturer Ab so we are looking for the patient Ag.

what is an example of reverse passive agglutination

rapid strep test

labeled immunoassays

competitive

non competitive

in competitive immunoassays everything is added _________________

everything is added at the same time

what is the goal of competitive immunoassays

look for patient antigen

competitive immunoassays

POS result _____________

NEG result _____________

POS result less color

NEG result more color

what is the goal for non competitive immunoassays

look for patient antigen

non competitive immunoassay

POS result________________________

NEG result _______________________

POS result more color

NEG result less color

formats of labeled immunoassays

radioimmunoassay

enzyme immunoassay

fluorescence immunoassay

chemiluminescence immunoassay

historical information about ratioimmunoassay

First kind of immunoassay developed in the 1950s

Radioactive labeled 125iodine

things needed for complement reaction

Pathogen/antigen

Antibody

Complement

complement fixation is a test where we add _____________ and ______________ and looks for __________ in a patient sample

adds RBC and C'

looks for antibody in pt

positive complement fixation reaction

button

intact RBC

negative complement fixation reaction

solid color

lysed RBCs

how do we quantitate complement fixation

titer

what is the common use for complement fixation

virology and dimorphic fungi

examples of dimorphic fungi

Coccidioides

Histoplasma

Penicillium marnefii

Paracoccidioides

Sporothrix