L7 General and specific uses of mAbs

Question 1

What are three main applications of antibody technologies in research?

Answer 1

Purifying protein (affinity chromatography) Locating proteins in cells/tissue (IHC/ICC) Quantitating amount of protein present (ELISA)

Question 2

What are the advantages of pAbs over mAbs?

Answer 2

Easy to generate and are low tech.

Can use all conventional immunological techniques.

Often cross reactive (if desired)

Question 3

What are the disadvantages of pAbs over mAbs?

Answer 3

Heterogeneous even if specific to one antigen.

Limited supply of a set as from one animal, and cannot be reproduced in a new animal.

Sometimes difficult to even make monospecific.

Question 4

What are the advantages of mAbs over pAbs?

Answer 4

Single specificity and invariant in this.

Unlimited supply.

Impure immunogen can be used to produce pure antibody.

They are manipulable for alternative assay use (novel antibodies made such as chimerics).

Easy to purify.

Question 5

What are the disadvantages of mAbs over pAbs?

Answer 5

Costly to produce.

May not be easy to create.

Animal must die to produce them.

May be too specific (if cross-species reactivity is desired).

Question 6

What are the main applications of mAbs?

Answer 6

Diagnosis.

Immunoaffinity puririfcation (antigen purification)

Use as anti-toxins

Vaccines

Question 7

What techniques are antibodies used in diagnosis?

Answer 7

Immunoassays (competetive and non-competetive).

Histopathology.

Cell typing.

Imaging.

Question 8

What would an immunoassay when applied allow you to study?

Answer 8

Proteins/molecules of interest such as sterioid/peptide hormones, enzymes.

Identification of different isomer types in different tissues e.g. cardiac v smooth v skeletal muscle myosin types, muscle v brain creatine kinase, different interferons and interleukins.

Question 9

What are the two main types of immunoassay?

Answer 9

Competetive and non-competetive.

Question 10

What are two detection systems used in immunoassays?

Answer 10

Radio-immunoassay, ELISA

Question 11

Is the radioimmune assay an example of competetive or non-competetive immunoassay?

Answer 11

Competetive binding.

Sample with native unlabelled hormone, then labelled hormone isadded.

Both bind to immobilised antibody on a surface, competing.

The higher ratio of unlabelled antibody bound, with a defined amount of labelled added, the amount of native hormone can be quantified.

Question 12

What are the three standard ELISA methods?

Answer 12

Direct, Indirect and Sandwich.

Question 13

What is the difference in indirect vs sandwich ELISA in what they determine?

Answer 13

Indirect ELISA determines the amount of antibody to the specified antigen.

Sandwich ELISA, determined the quantitiy of the antigen in sample.

Question 14

What can immunoassays be used for in self-testing or in-office tests?

Answer 14

Management of chronic conditions e.g. diabetes (detection of glucose)

To detect pregnancy (detection of HCG)

Sore throat (detection of group A streptococcus)

To detect ovulation (leutinising hormone detection)

In drug testing (can detect controlled substances, 5-10 drugs tested in one assay)

For HIV diagnosis (either against HIV itself or to detect antibodies against HIV)

Question 15

What is ‘two site’ immunofiltration used for?

Answer 15

Separation of specific antibody from solution using immobilised antibodies and antigen of interst.

Used to confirm presence of an antibody (against specified antigen) in solution.

Question 16

What is enzyme immunochromatography used for?

Answer 16

Used for quantification and detection of drugs in a biological fluid.

Antibody immobilised on a strip. Antigen and hapten-enzyme conjugated move up strip based on spatial distribution and show presence when binding to immobilised antigen. Enzyme conveys colorimetric change on strip and height of this band conveys chromatographic data on molecule being investigated.

Question 17

What is a hapten?

Answer 17

Small molecules that cannot be detected by the immune system, and therefore antibodies cannot be generated against by themselves.

Question 18

How are carrier molecules used in parralel to haptens?

Answer 18

Haptens are embedded onto surface of carrier molecule (typically large protein) meaning antibodies can be produced agains the hapten as it is now large enough.

Question 19

What can histopathology be used for?

Answer 19

Cancer diagnosis and therapy

• Intermediate filament staining patterns
• Receptor types

Viral and bacterial organism identification

• Epstein Barr virus
• Herpes simplex virus I and II

Question 20

How can histopathology intermediate filament staining patterns be used in cancer diagnosis/treatment?

Answer 20

Different filament proteins presence can be used to identify tissue origin and type of tumour from a cancer biopsy sample.

e.g. int. filament protien cytokeratin indicates carcioma originating from epithelia.

Question 21

How can mAbs be used in to identify cell typing?

Answer 21

Using mAbs directed to cell surface markers.

T and B cell populations display characteristic CD markers. CD4 - T helper cell. CD8 - Cytotoxic T cell. CD20 - B cell.

CD4:CD8 ratio important in AIDS patients.

Very important in diagnosis of lymphonas and leukemias.

Use in FACS (fluorescence-activated cell sorting)

Question 22

What is fluorescence-activated cell sorting (FACS)

Answer 22

Specialised flow cytometry. Able to sort cells from a hetergeneous fluid into individual cell types based on their characteristic light scattering and fluorescence.

Antibody coupled to fluorescence marker, specific to certain cell marker. When hit by correct wavelength of light, targetted cells identified and can be fractioned off from others

Question 23

Can FACS be used to identify and sort multiple cell types in a solution?

Answer 23

Yes, using wide range of fluorophores that each attached to specific antibody targetting characteristic membrane CD markers. Laser targetted at fluid and wavelength can be varied to target each flurophore at a time.

Question 24

What is radioimmunoscintigraphy?

What can it be used for?

Answer 24

Using isotopically labelled mAb to provide image of an organ.

Detection of cancer, blood clots for example.

Antibodies are directed to tumour associated antigens, labelled with radioisotope. Isotope is then located using gamma camera imaging system to report on tumour.

Question 25

What cancer in particular is radioimmunoscintigraphy useful for?

Which two other cancers does it have some success with?

What antigens do they typically target?

Answer 25

Colorectal cancer. TAG 72, carcinoembryonic antigen

Breast cancer, CEA, human milk fat globule.

Ovarian Cancer: CEA, TAG 72