Ab as diagnostic tools

Question 1

why are Ab used for diagnostic tests *

Answer 1

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

Question 2

why can you attach stuff to Fc *

Answer 2

normally binds to cells - doesnt need to so we can attach things

Question 3

what can we attach to Ab *

Answer 3

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)

Question 4

describe indirect labelling using an anti-Ab *

Answer 4

primary Ab bound to Ag

secondary Ab binds to the Fc of the constant region - secondary is bound to the reporter eg dye

Question 5

what is the benefit of using indirect labeling with anti-Ab *

Answer 5

you can use single secondary Ab to detect many primary Abs

Question 6

what are Ab produced by the pt used for - clinically and in body *

Answer 6

autoimmune

defence against inffection

help diagnose infection

Question 7

what are the 3 types of manufactured Ab *

Answer 7

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

Question 8

describe the production off monoclonal Ab

Answer 8

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

Question 9

how can you produce Ab using recombinant DNA tec

Answer 9

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

Question 10

what are the therapeutic uses of manufactured Ab *

Answer 10

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

Question 11

problem with using Ab therapeutically *

Answer 11

very expensive because of quality control and they are biological molecules

Question 12

what are the diagnostic uses of Ab *

Answer 12

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)

Question 13

what does ELISA stand for *

Answer 13

enzyme linked immunosorbant assay

Question 14

describe the process of ELISA *

Answer 14

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

Question 15

describe lateral flow assay *

Answer 15

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

Question 16

example of when you would have anti-HIV Ab but not beeen infected by virus

Answer 16

pregnancy - foetus not infected

Question 17

what are the immunological concerns with vague aches and pains

Answer 17

immune complexes being deposited at various sites in body

Question 18

what would be the immunological concern for loss of appetite and weight loss

Answer 18

effect of poor nutrition on bone marrow cells

Question 19

what would be the immunological concern if there are enlarged lymph nodes

Answer 19

immune activation

Question 20

what would be the immunological concern of fever, rash, small red patches, some lumpy

Answer 20

acute phase

activation

immune complexes deposited in the skin

Question 21

describe the concerns of immune complexes *

Answer 21

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

Question 22

describe how you would detect immune complex glomerulonephritis *

Answer 22

using immunoflurescence

or because the complexes activate complement by the classical pathway - can detect complement

Question 23

what would you look for to check for immunodeficiency and how *

Answer 23

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

Question 24

describe serum electrophoresis *

Answer 24

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

Question 25

describe flow cytometry to determine the lymphocyte subpopulations *

Answer 25

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

Question 26

benefit of flow cytometry *

Answer 26

advanced and reliable infrastructure

Question 27

disadvantages of flow cytometry *

Answer 27

need complex IT systems

continuous power supplies

skilled operators

high cost equiptment

Question 28

what cell are you particular interested in for HIV

Answer 28

CD4 + T cell

Question 29

describe the natural history of a HIV infection

Answer 29

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

Question 30

describe the immune diagnositics for HIV *

Answer 30

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