Immunotechnology 2

Question 1

Why are antibodies central to Immunotechnology?

Answer 1

Their high specificity for target antigens.
Enable the detection, localisation and quantification of proteins across a wide range of biological samples

Question 2

What is western blotting?

Answer 2

Antibodies identify and quantify specific proteins separated by the SDS-PAGE.
Primary antibodies bind to the target proteins
enzyme conjugated secondary antibodies enable detection through chemiluminescence

Question 3

What is immunohistochemistry?

Answer 3

Antibodies label proteins in preserved tissue sections
This allows visualisation of
protein distribution and localisation
within intact tissue structures

Question 4

What is immunofluorescece?

Answer 4

Antibodies tagged with fluorescent dyes localise proteins within cells
Reveals their intracellular position or colocalisation with other molecules
Under fluorescent microscope

Question 5

What is flow cytometry?

Answer 5

Uses fluorescently marked/labelled antibodies to
analyse the expression of surface or intracellular markers on thousands of single cells in suspension
Provides quantitative multiparametric data

Question 6

How do these methods work?

Answer 6

Uses antigen antibody interactions

Question 7

How does western blotting work?

Answer 7

Detects specific proteins after electrophoretic separation
Proteins transferred to membrane
On membrane antibody binding reveals presence and amount of a target protein
through light emitting enzymatic reactions

Question 8

How does ELISA work?

Answer 8

Uses antigen antibody binding in a plate
Quantifies protein or antigen concentration
using colourmetric or fluorescent signal

Question 9

How does immunohistochemistry work?

Answer 9

Preservers tissue architecture
uses enzyme linked or fluorescent detection
Visualises spatial expression

Question 10

How does Immunofluorescnece work?

Answer 10

Focuses on subcellular localisation
By using fluorophore tagged antibodies

Question 11

How does cytometry work?

Answer 11

Quantifies expression levels in individual cells
Measures fluorescent intensity as cells pass through a laser beam
Provides cell size and granularity data

Question 12

IHC workflow

Answer 12

Fix
Bed
Section
Please
Anti
Block
Anti
Dec
Fixation
Embedding
Sectioning
Peparrafinisation/rehydration
Antigen retrieval
Blocking
Antibody incubation
Dectection

Question 13

IHC fixation

Answer 13

Preserves tissue structure (commonly formalin)

Question 14

IHC bed - embedding

Answer 14

Dehydrate and embed in paraffin wax for sectioning

Question 15

iHC section - sectioning

Answer 15

Cut 3-5 um slices using G microtome and mount on slides

Question 16

IHC please - deparrafinisation/rehydration

Answer 16

Removes wax
Rehydrate tissue

Question 17

IHC anti - antigen retrieval

Answer 17

Unmask epitopes -heat or enzyme mediated

Question 18

IHC block - blocking

Answer 18

Prevents non specific antibody binding eg serum or BSA

Question 19

IHC anti - antibody incubation

Answer 19

Apply primary and secondary antibodies

Question 20

IHC deck - detection

Answer 20

Can be
Chromogenic
Fluorescent

Question 21

Chromogenic detection

Answer 21

HRP catalyses DAB to produce a brown precipitate
Visualised with blue haematoxylin counter stain

Question 22

Fluorescent detection

Answer 22

Fluorophore labelled secondary antibodies visualised with fluorescence microscope

Question 23

IF steps

Answer 23

Fixcells/ tissue eg paraformaldehyde

Permeabilise membrane if staining intracellular targets

Block with BSA or serum to reduce background

Incubate with primary antibody

Incubate with fluorophore tagged 2nd antibody (indirect)

Counterstain nuclei eg DAPI

Mount and image under fluorescence microscope

Question 24

Direct vs indirect If

Answer 24

Direct = fluorophore attached to primary antibody
Indirect = secondary antibody binds primary, providing amplification and flexibility

Question 25

What does western blotting provide

Answer 25

Provides semi quantative measurement of protein abundance Band intensity correlates with protein concentration when normalised against a loading control eg beta actin Detects post translational modifications such as phosphorylation

Question 26

Clinical examples of western blot

Answer 26

CFTR analysis in cystic fibrosis - distinguishes mature and immature glycoforms (F508del mutation disrupts maturation) HIV confirmation - detects antibodies against viral proteins

Question 27

What does flow cytometry provide

Answer 27

Fully quantative, single cell data Fluorescent intensity reflects abundance of target molecules Multiparametric capability allows simultaneous detection of different markers/cells Enables calculation of population frequencies, mean fluorescence intensities and cell phenotypes

Question 28

Example of flow cytometry

Answer 28

Monitoring CD4+ T cell levels in HIV p24 protein

Question 29

HER2 in cancer

Answer 29

Immunohistochemistry and immuofluorescence used to detect over expression of HER2 - a receptor tyrosine kinase in breast cancer HER2 +ve tumours show string membrane staining Determines eligibility for targeted therapies eg trastuzumab IF helps study receptor internalisation and trafficking

Question 30

HIV diagnosis with Immunotechnology

Answer 30

ELISAA - initial screening for anti HIV antibodies WESTERN BLOT- confirm true positives by showing specific band patterns for viral proteins eg gp160, gp120, gp41, p24 FLOW CYTOMETRY - monitors immune suppression via CD4+ T cell counts and detects intracellular HIV p24 in infected cells

Question 31

Applications of western blotting

Answer 31

Protein modification studies eg phosphorylation, glycosylation

Question 32

IHC/IF applications

Answer 32

tissue level pathology, biomarker localisation

Question 33

Flow cytometry applications

Answer 33

Immunophenotyping eg CD4/CD8 ratios Apoptosis Cell cycle analysis

Question 34

ELISA applications

Answer 34

Quantifying cytokines (IL-6, IL-8) or biomarkers in fluids