Ab as diagnostic tools Flashcards
why are Ab used for diagnostic tests *
they have uniques specificity for their target antigens
they can be produced against almost any antigen, often but not always proteins, including Ig from other species = anti-Ab
why can you attach stuff to Fc *
normally binds to cells - doesnt need to so we can attach things
what can we attach to Ab *
enzymes - peroxidase, alkaline phosphtase
flurescent probes - dyes/beads of different colours - differnet bead size and colour bonded to different specificities mean you can multiplex and detect differnet things in the same sample
magnetic beads - purification of cell types
drugs - kadcyla (target breast cancer - anti-ER2 linked to emtansine)
describe indirect labelling using an anti-Ab *
primary Ab bound to Ag
secondary Ab binds to the Fc of the constant region - secondary is bound to the reporter eg dye
what is the benefit of using indirect labeling with anti-Ab *
you can use single secondary Ab to detect many primary Abs
what are Ab produced by the pt used for - clinically and in body *
autoimmune
defence against inffection
help diagnose infection
what are the 3 types of manufactured Ab *
antisera - from immunised animals - polyclonal - mix of different Ab, problem is there is a imited supply
monoclonal Ab
genetically engineered Ab - by using DNA segments that code specificity
describe the production off monoclonal Ab
immunise animal with Ag
take spleen cells as source of B cells - limited cell division
fuse with myeloma cells = hybridomas
culture in medium
select for positive cells
find clone that is prroducing Ab
so you can grow unlimited amounts - clone by limiting dilution
how can you produce Ab using recombinant DNA tec
make libraries of the DNA coding the Ab by putting them into bacteriophages - the codes are put on the surface of the phages - this is phage display library
have antigen immobilised on a plate - those with specificity bind - wash others away
advantage of using bacteriophage - grow very rapidly when exposed to bacteria
what are the therapeutic uses of manufactured Ab *
prophylactic protection against microbial infection eg IVIG (pt who immunodeficient give IV immunoglobulin - polyclonal with lots of different specificities) , synagis (anti-RSV)
anti cancer therapy - anti-HER2
removal of T cells from marrow grafts so they dont attak the host - Anti-CD3
block cytokine activity - anti-TNF-a eg for rheumatoid arthritis
anti-calcitonin gene-related peptide (CGRP) for migraine
anti-body against amyloid - for alzheimers
problem with using Ab therapeutically *
very expensive because of quality control and they are biological molecules
what are the diagnostic uses of Ab *
blood group serology
quantitative immunoassays for hormones, Ab, Ag
immunodiagnosis (Ab made by pt) - infectious diseases, autoummunity, allergy (specific IgE in allergy can be detected) IgE, malignancy (myeloma)
what does ELISA stand for *
enzyme linked immunosorbant assay
describe the process of ELISA *
have plastic wells coated in Ab against what you are looking for
if it is present the Ab binds
wash everything else away
Conjugated detection Ab bind to a different place - linked to enxyme that can causse production of colour taht can be detected - quantitive assay
the signal produced is related to the amount in the sample
describe lateral flow assay *
rapid testing, point of care
2 lines of immobilised Ab - antiAb to show the test is working (control line), line to detect what trying to detect
have another set of Ab detecting what want linked to gold nanoparticles
liquid sample on end
capillary action draws sample along strip
example of when you would have anti-HIV Ab but not beeen infected by virus
pregnancy - foetus not infected
what are the immunological concerns with vague aches and pains
immune complexes being deposited at various sites in body
what would be the immunological concern for loss of appetite and weight loss
effect of poor nutrition on bone marrow cells
what would be the immunological concern if there are enlarged lymph nodes
immune activation
what would be the immunological concern of fever, rash, small red patches, some lumpy
acute phase
activation
immune complexes deposited in the skin
describe the concerns of immune complexes *
depends on the size of the immune complexes - if there is antigen excess - immune complexes will be small - dont activate complement - but if bind to membrane - activate complement = inflammation
serum sickness - immune complexes in circulation
immune complex glomerulonephritis - if tey deposit here
problems if deposit in the skin, joints or lungs
describe how you would detect immune complex glomerulonephritis *
using immunoflurescence
or because the complexes activate complement by the classical pathway - can detect complement
what would you look for to check for immunodeficiency and how *
serum Ig levels - IgG/M/A/G1/G2/G3/G4 - using serum electrophoresis/ELISA/nephelometry
specific Ab (those that you have been immunised agaisnt to see if worked) - protein antigens (tetanus and haemophilus), polysaccharide antigens (pneumococcus) - use ELISA
look for different lymphocyte subtypes - CD3, 4, 8, 19, NK - use flow cytometry to look for these markers
describe serum electrophoresis *
albumin is the most prominant protein
smear for Ig - wide range
if elevated level of Ig (darker smear) - active immune repsonse
if there is a monoclonal expansion of B cells (one band on electrophoresis) - could be B cell malignancy - investigate for myeloma - abnormal precurser cell in marrow
describe flow cytometry to determine the lymphocyte subpopulations *
Ab have differnet colour markers - bind to cell marker: CD3+ (all T), CD4+ (T helper) 8+ (cytotoxic) 19+ B cell 56+ (NK)
single cell go through laser which detects the flurescence
qunatify relative proportion and number of cells
benefit of flow cytometry *
advanced and reliable infrastructure
disadvantages of flow cytometry *
need complex IT systems
continuous power supplies
skilled operators
high cost equiptment
what cell are you particular interested in for HIV
CD4 + T cell
describe the natural history of a HIV infection
immune system attacks virus to point - viral load down, CD4 up
clinical latency period - CD4 go down gradually and viral load doesnt go up by too much
then CD4 becomes low
allows opportunistic diseases - typical of each CD4 count
increased virus in circulation
describe the immune diagnositics for HIV *
pt tested for Ab to HIV
if positive perform CD4 count and viral load
if low CD4 and high viral load
commence ART
monitor CD4 and viral load - if get lower and higher respectively
second line ART needed
ART is combination of drugs - to prevent resistance
increased life expectancy
