BASIC ANTIBODY BASED IMMUNOASSAYS

how do precipitates form

cross linking of small/soluble antigens

how does agglutination occur

cross linking of large antigens

what are the 3 types of agglutination assays

direct agglutination

indirect agglutination

haemagglutination inhibition

describe direct agglutination assay

When RBC, bacteria and fungi are agglutinated by serum antibodies

determines if specific antibodies present in serum

describe indirect agglutination assays

soluble antigens are absorbed onto the surface of an RBC or particle

makes visible clumping easier to see and measure

what must be done if using RBC as inert particle in indirect agglutination

The serum must be absorbed by washing in RBC to remove any heterophilic antibodies

what is a common inert particle for indirect agglutination

latex particles

explain how haemagglutination inhibition occurs

serum containing suspected antibody is treated with an antigen before an agglutination reaction to remove the antibody

if the antibody is present then this will inhibit agglutination

explain haemagglutination inhibition using a virus as an example

some viruses cause haemagglutination

adding antibodies will bind to the virus

this prevents haemagglutination from occurring

what are the 2 ways a latex particle might be used

cover it in antigen → test if antibody present

cover it in antibody → test if antigen present

explain how a single radial immunodiffusion mancini plate works

it is a gel made with antibody in the agar

a hole is made in the gel and the antigen being measured is placed in this hole

the antigen will diffuse into the gel and come into contact with antibodies, forming a zone of precipitation

zone diameter is proportional to antigen concentration

explain how ouchterlony gels work

this is a double immunodiffusion

the gel has no antibodies in it

2 holes made in gel; one for antigen one for antibodies (can have several antigen wells around a central antibody well)

the 2 samples diffuse towards eachother and form a line of precipitationn

how would you interpret different ouchterlony gel results

identical → smooth line

non identical → lines crossing

partially identical → spur

describe immunofixation electrophoresis

an electrophoresis of serum is run in agarose gel

this is followed by overlaying with specific antisera

antigen-antibody complexes will precipitate in the gel

what does immunofixation electrophoresis test for

multiple myeloma → abnormal M protein on abnormal plasma cells

explain nephelometry

antigen is mixed with specific antibody in a tube, causing turbidity as aggregate forms

measurement involves passing light through the tube and detecting scatter

light scatter is directly proportional to antigen concentration

what might we label or conjugate an antibody to, with examples

fluorescent dyes → fluorescein isothiocyanate

enzyme → horse radish peroxidase

radioisotope → I¹²⁵

other → gold

define a conjugate

a labelled antibody