unit 3 immunochemistry

primary ab/ag reaction

the ab and ag bond

secondary reaction

precipitation, agglutination, and complement

tertiary reaction

opsonization, phagocytosis, chemotaxis

prozone

too little antigen and secondary reaction doesn’t happen

postzone

too much antigen and no secondary reaction happens

ouchterlony immunodiffusion

a secondary method - uses precipitation

ag in one well and ab in the other and migration will occur to the point of equivalence and precipitate

limitated by prozone/postzone, qualitative, cant do large molecules

counterimmunoelectrophoresis

uses electrical field so ag ab move toward each other and precipitate out when they meet

improves sensitivity over ouchterlony

immunofixation electrophoresis

same specimen is in 6 tracks, stained with different antiserum

like the protein gamma identification

radial immunodiffusion

antigen or antibody is INSIDE an agar so it preticipates right away

ring is where the antigen/antibody is equivilant

macinin technique: ring diamter squared vs std concentration

effected a lot by prozone and post zone

laurel rocket immunoelectrophoresis/electroimmunoassay

electrophorese into an antigen or antibody impregnanted agar and use electricity

makes a rocket shape precipitate which the length is proportional to concentration

faster but will denature proteins if too high

immunonephelometry

when a secondary reaction happen it scatters light before its even visible

detects the light scattered with a dectector at an angle

endpoint vs kinetic reaction

endpoint is you let the reaction go until it scatters as much light as it possibly can and it can no longer increase

or instead of waiting for that reaction to finish you take multiple measurements at specific points to make a curve and determine concentration with a slope

advantages vs disadvantages of immunonephlometry

sensitive, faster

but has prozone and postzone, large precipitations give false decreases because it doesnt scatter as much, colored solutions can decrease senstivity

advantages of looking at primary reactions vs secondary reactions

faster

ionic stregnth, pH etc not as important

can use just IgG which are easier to use

more sensitive because you dont need as much antigen

stoke’s shift

a fluorophor absorbs a wavelength and the emits light at a longer wavelength and the different between them is stoke’s shift

bigger shift = better

major limitation of using fluorescent labels

any background fluorescence

try and get around it with phosphors which emit light for longer after being excited without the light on it or using rare earth metals which have a giant stoke’s shift and emit longer

process of fluorescent instrument

a light source goes through the primary filter, which elects the excitation wavelength

that wavelength hits the specimen and it produces its emission wavelength

a secondary filter at an angle selects for that emission wavelength and it passes through to a dector

pros and cons of enzyme labels

enzymes are not consumed in the reaction

last a long time

safe

but denatued by metal and pH

can easily contaminate

enzyme label instrument

enzyme substrate is added in excess if you want to measure the enzyme itself

will either make color or fluoresce so measure with spec or fluorometer

radioisotopes

125 Iodine, 3 Hydrogen, 57Cobalt

uses scintillation counter to read radioactivity

sensitive and rarely contaminates

but hazardous and need a ton of special rules

chemiluminescent compounds

make light in chemical reaction

measured with photon counters which are like specs but specimen is light source, sometimes electrodes if a redox reaction

rare to have contamination, very sensitive

but needs a special instrument

order of label sensitivity

chemi > fluorescence > enzyme > radioisotope

avidin and biotin

biotin is a small ,water soluble vitamin that can bind to proteins like an antibody does but does not damage

avidin is a protein that “avidly” binds up 4 molecules of biotin almost irreversibly

little steric interference

since avidin binds 4 biotin it amplifies the reaction a lot

steric interference

big molecules can get in each other’s way

homogenous assays

a labeled substance gives a different reaction which unbound and bound so you dont need to a seperation step

everything is mixed together

heterogenous assays

bound and free label give off same reaction so they need to be seperated

non-competitive/sandwich assay

you make a sandwich of what you’re trying to measure

a capture molecule on bottom and a labeled molecule on top in order to measure

indirect vs direct sandwich assays

indirect adds another layer

so capture molecule, what you’re measuring, antibody to that, then another antibody that’s labeled

indirect has more non-specific reactions but is more sensitive than direct

types of special sandwich assays

antibody capture - a class of ABs has less than the others. not capturing with the antigen, because the other antibodies will bind that too, instead capture it with anti-Ig(whatever)

western blot - capture molecule created from sample going through electrophoresis

specific IgE/RAST - allergen is capture molecule, IgE in sample, then detected with labeled anti-E

immunometric assays - an antibody is labeled with an isotope or tag, where once the antibody binds to the analyte, that label comes off. increased signal from tag= higher analyte concentration

IEMA/IRMA

types of mmunometric label methods

IEMA uses enzyme, IRMA uses radiolabel

prone to hook effect

hook effect

a super high concentration of antigen makes it so capture and detection antibodies are completely saturated and the sandwich cannot form

so a super high concentration will seem falsely decreased because that signal is not being generated

how is hook effect avoided

add specimen to capture molecule first

wash all the excess away

add the labeled antibody to bind to the captures antigens

competitive immunoassays

unlabeled analyte competes with a labeled ligand already in the sample for sites on a limited amount of antibody/capture molecule

less color = more analyte if you’re measuring the bond ligand

RIA

radioimmunoassay

ligand is radiolabeled and will compete with analyte for sites on a limited antibody

decreased radioactivity = increased concentration

ELISA

enzyme linked immunosorbent assay

heterogenous

capture on a solid support, then an enzyme labeled detection molecule

can be competitive or non competitive

EMIT

enzyme multiplied immunoassay technique

homogenous

ligand and enzyme labeled ligand compete for an antibody

when the antibody binds to the labeled ligand, the enzyme is sterically inhibited

increased enzyme signal = increased analyte concentration

no seperation of the bound and unbound

pros and cons of EMIT

cant use too big of a molecule otherwise the enzyme wont be sterically inhibited

background interference can occur from color and endogenous enzymes in the sample since there’s no seperation

but fast, cheaper

FPIA

homogenous

uses fluorphor labeled ligand

polarized light excites the label and it emits

when its bound to an antibody, it rotates more slowly, and polarization increases

labeled ligand competes with analyte for sites on antibody

polarization decreases = more analyte

polarization increases = less analyte

CLIA

chemiluminescent

heterogenous

usually uses chemiluminescent tag in sandwich or competitive way

little specimen interference

CEDIA

cloned enzyme donor

homogenous

one enzyme piece bound to the analyte of interest

the analyte and an enzyme labled analyte compete for site

unbound enzyme label detected by uniting it by adding another piece of the enzyme to make it functional

what do you do if you think an assay is susceptible to the hook effect

dilution

biotin supplements

if someone is taking biotin supplements the high levels can inhibit immune complex formation when

AVIDIN is the BINDING AGENT

and FREE CAPTURE method is used, everything is in a tube TOGETHER

which will result in increase with competitive, decrease with sandwich

heterophile antibodies

someone has heterophile antibodies from an infection (mono), previous exposure or given therapeutic monoclonal antibodies, HAMA interfere with immunoassays because it was common for them to use animal antibodies

types of interfering antibodies

RF, autoimmune antibodies, heterophile antibodies, infectious antibodies

people at risk of having interfering antibodies

people who work with animals

therapeutic antibodies

women who’ve been pregnant a lot

autoimmune disease

transfused patients

infections

macromolecules

either type 1 - complex of Igs

type 2 - a non-antibody complex of an analyte or caused by lipids or drugs

macroamylase and macroprolactin are most common

problems with macromolecules

long half life but low biological activity

false increase, since they’re a big molecule, but biologically inactive

identifying macromolecules

will have an increased concentration in the lab but doesnt make sense clincically

so looking at other values

like macroprolactin making prolactin seem high, but otherwise has patterns of decreased prolactin

so treat a specimen with something to get rid of large molecules, can do PEG or filtration

detecting interfering antibodies

when you do a dilution the results make no sense, a negative control is positive, weird results when testing urine vs serum

so filter or precipitate out

blocking interference

heterophile antibodies bind up all the capture antibody or the detection molecule

so you can add excess animal serum to bind those hterophile antibodies and stop them, use a mammalian ab for one site and avian for another to see if theres a difference, or use chimeric antibodies which are made of parts from a human and mouse