Kap 7: Immunochemical Techniques

Question 1

Regarding recombinant antibodies which two methods are most commonly used to produce these?

Answer 1

1. DNA libraries –> bacteriophage expressing antibody
   fragments on the surface
2. From B cell DNA (used for DNA extraction), the
   antibody-coding genes are then selectively amplified
   by PCR and transferred into the genome of a
   eukaryotic cell line

Question 2

Blood samples are taken throughout the immunisation program and the serum is tested for specific activity to the antigen by

Answer 2

ELISA

Question 3

____ blot analysis of the serum can confirm the specifi city of the antibodies produced
(see Section 7.7

Answer 3

Western

Question 4

What is the rapid method for IgG purification from serum?

Answer 4

by the addition of chaotropic ions, typically ammonium sulfate (60 % saturated) in solution giving a precipicate

Question 5

by using chaotropic ions for antibody purification does not work well in tissue culture supernatant, why?

Answer 5

since media components such as ferritin are coprecipitated

Question 6

ammonium sulfate precipitation may be used as a:

Answer 6

preparatory method prior to further

chromatographic purification.

Question 7

Regarding antibody purification what purifcation method is used after a preperatory method (ammonium sulfate) has been used?

Antibody-containing fractions are identified by spectrophotometry using absorbance at ___ nm (specific wavelength for protein absorbance; see Section 13.2.4 ) and are pooled

Answer 7

affinity chromatography

280

Question 8

Name one antibody labelling protocol used for detection,

and in the actual qunatification of antigens, one uses labels, there’re two types, which two?

Answer 8

ELISA

direct and indirect

Question 9

Antibodies can be labelled by the addition of a marker enzyme such as (2):

Answer 9

HRP and AP

Question 10

How is antobodies labelled using a marker enzyme?

in the preparation labelling reagents is also avaliable, what derivate is used to get fluorescent conjugates?

Answer 10

by a linkage achieved by cross-liking chemistry –> stable antobody-enzyme conjugates

FITC

Question 11

streptavidin–____ bridge as a very high affinity protein–ligand interaction and often utilised to link proteins in immunoassays (ELISAs). In typical applications, the antibody
is conjugated to biotin, and streptavidin is conjugated to a fluorophore (see Section 13.5.1 ) or enzyme, thereby providing signal amplification and increased sensitivity
so that antigens that are expressed at low levels are more likely to be detected

Answer 11

biotin

Question 12

Regarding immunoblotting, this technique is also known as:

Explain the steps in this technique

Answer 12

western blot analysis

1. electrophoresis in reducing/non-reducing conditions
2. staining with general protein stain (sec. 5.2 & 6.3)
3. transferred onto nitrocellulose or polyvinyl membrane
   by using an electroblotter.
4. membrane is then treated with a protein-blocking
   solution to prevent non-specific binding of antibody to
   the membrane itself. with either casein or BSA
5. indirect labelling, in antigen specific primary antobody
   solution, then washed and treated with a solution of
   secondary enzyme-linked antibody – in order to
   regognize species of the primary antibody, with
   peroxidase or alkaline phosphatase
6. the emitted light is detected by exposure to
   photographic film or a CCD