Antibody techniques

Question 1

Antigen

Answer 1

Substance that binds to Ab or T cell receptor

Question 2

what part of Ag does Ab recognise ?

Answer 2

Epitopes

Question 3

Ab structure

Answer 3

4 polypeptide chains
Variable region: ABS
constant region

Question 4

Polyclonal

Answer 4

Mixture of Abs recognising Several epitopes of 1 Ag

Question 5

Monoclonal

Answer 5

1 clone Of Ab recognises 1 epitopes = highly specific

Question 6

polyclonal in serum come from?

Answer 6

activation of many B cells with specificity for diff epitopes on Ag

Question 7

Monoclonal come from

Answer 7

clones of individual B cells + have specificity to 1 epitope

Question 8

Monoclonal production Method

Answer 8

1) Animal Immunized with Ag
2) B cells removed from spleen
3) B cells + myeloma cells fuse = hybridoma
4) Cultured on HAT= only hybridoma grows
5) screen for specific Ab production
6) Desired Ab grown
7) Ab Isolated + purifed

Question 9

why myeloma cells?

Answer 9

cancer cell that grows continuously

Question 10

What HAT medium does

Answer 10

only hybridoma grows
unfused dies

Question 11

Hybridoma

Answer 11

B cell + myeloma cell fused together

Question 12

Screening

Answer 12

separated in microtiter plate t tested ability to produce specific Ab

Question 12

Polyclonal pros v cons

Answer 12

inexpensive, quicker
less specific, Batch variability

Question 13

monoclonal pros v cons

Answer 13

Expensive, longer, may be too specific
more specific, All batches identical

Question 14

Lab uses of mAb(x3)

Answer 14

Detect analyte in tissue (IHC)
Derect analyte in liquid (ELISA)
separation of analyte from mixture (immuno precipitation)

Question 15

what Immunoassay uses :

Answer 15

Detectable label attached to Ab E.g fluorescence, enzyme, Radioisotope

Question 16

what immunoassay does?

Answer 16

Quantify soluble Ag

Question 17

Non - competitive immumoassay Example

Answer 17

sandwich ELISA

Question 18

sandwich ELISA method

Answer 18

1) Well coated with capture Ab
2) sample Ag binds Ab
3) 2nd detection Ab with enzyme binds Ag
4) add substrate = colour change

Question 19

Sandwich ELISA detection

Answer 19

Spectroscopy

Question 20

HRP -linked AB

Answer 20

Binds detection Ab= amplified signal

Question 21

Competitive Immunoassay difference

Answer 21

Analyte competes for Binding site

Question 22

How Non / competitive measured

Answer 22

Non= occupied sites of labelled Ab
competitive= unoccupied sites

Question 23

competitive method

Answer 23

1) Sample + labelled Ag added to capture Ab
2) Either Ag or sample binds
3) Substrate added= colour change
4) measures Ab unbound with Ag

Question 24

Non competitive graph

Answer 24

directly proportional : increase conc= Increase absorbance

Question 25

competitive graph

Answer 25

inverse correlation : more signal less Ag in sample

Question 26

immunoassay to measures ab + Example

Answer 26

measure levels of Ab by coating wells with Ag E. g diagnose Coeliac

Question 27

Diagnosis of Cs

Answer 27

ELISA detects patients Ab against tissue Transglutaminase+ Gladin

Question 28

IHC benefits (x3)

Answer 28

Qualitative, Gives location + identify cell events

Question 29

ICC method

Answer 29

1) Tissue / cells on slide 2) Primary Ab binds 3) wash unbound 4) Secondary labelled Ab binds 5) wash unbound 6) substrate colour change

Question 30

Signal amplification : Biotinylated Ab

Answer 30

Avidin: 4 bunding sites bind Biotin Biotin: coenzyme binds Protein

Question 31

IHC / ICC example diagnosis

Answer 31

ANA detects auto antibodies using fluorophore Linked Ab =Specific staining pattern

Question 32

Biotinylation

Answer 32

using Biotin+ Avidin to amplify signal