Antibodies as diagnostic tools

Question 1

Outline structure of antibody

Answer 1

Heavy and light chains

Classes differ by the constant part of their heavy chain

The top part has variable and constant domains. The variable part binds antigen

The bottom of the molecule is just the heavy chain, and this is the biological effector

Question 2

Where can molcules be attached to antibodies to aid in diagnostic

Answer 2

To the constant part of the heavy chain…. still allows antigen binding

Question 3

What molecules can be attached to the antibody

Answer 3

Enzymes, fluorescent probes, magnetic beads and drugs

Question 4

What enzymes can be conjugated to the antibody

Answer 4

perioxidase (gives brown colour), alkaline phosphatase

Question 5

Which fluorescent probes can be added to antibody

Answer 5

dyes

beads of different sizes (you can conjugate different coloured or different sizes beads to antibodies with different variable regions, so you can display presence of two antigens at the same time…. MULTIPLEX)

Question 6

What can antibodies conjugated to magnetic beads be used for

Answer 6

To draw out a particular cell type (i.e. purify cell type) so that this set of cells can be removed, proliferated in vitro and then put back into the patient….
basically allows you do draw a certain type of cell out of the patient

Question 7

Which drugs can be conjugated to antibody

Answer 7

Target toxic drugs to certain cell types

E.g. kadcyla= anti-HER2 linked to emtansine…HER-2 over expressed in certain types of breast cancer so this is cytotoxic to these cells

££££

Question 8

Why are antibodies powerful in diagnositcs?

Answer 8

Specificity to target antiigen

Question 9

Which molecules can ABs be made against

Answer 9

Any antigens include carbs, DNA and proteins

can even back antibodies against antibodies…..= anti-antibodies

Question 10

When can humans make antibodies against antibodies

Answer 10

E.g autoimmune disease such as rheumatoid…. IgM against constant part of IgG

Question 11

Outline indirect labelling using an antibody

Answer 11

primary antibody against an antigen can be detected using a secondary antibody which has a reporter molecule attached (e.g. enzyme of fluorescent)

Question 12

when are Abs produced in patient

Answer 12

In autoimmune disease, or for defence against infection

Question 13

How can antibodies be manufactured

Answer 13

Antisera from immunised animals (polyclonal)… deliberately infect animals

monoclonal antibodies (diagnostic)

“genetically engineered” antibodies (recombinant DNA)

Question 14

How are monoclonal antibodies manufactured

Answer 14

Give mouse antigen, take spleen cells

Combine spleen cells (limited cell devision, HGPRT+ve) with myeloma (immortal, HGPRT-ve) cells

This produced hybridomas

Then culture in HAT medium, which is a medium that only causes expansion of the HGPRT +ve cells (so you only get the hybridoma, and not the myeloma cells, dividing.

But bear in mind that this will not just contain the b cell specific for the antigen you injected it will contain b cells from all the cells in the spleen.

To make sure you only get the b cell with the Ab specific to the antigen you injected, you clone the cells by diluting them down to a single cell per well (clone by limiting dilution). So you have lots of clones of all the different b cells, then you screen the clones to find the one making the Ab you wanted

Harvest the monoclonal antibodies

Question 15

How can ABs be produced using recombinant DNA technology

Answer 15

You can take the parts of the human genome encoding variable regions of ABs and put them into bacteriophages. (Vl and Vh)

Then, the bacteriophages will start expressing ABs with all the different variable regions in the genome on their cell surface

Then you get an agar plate with the antigen you want an antibody against on it

Then you apply the ABs. The AB with the correct specificity for the antigen will bind, and you wash the others away. Then you can clone this one, and repeat the process with the bound antigen to get even greater specificity

BENEFIT: uses human AB from the beginning, so no immune response against (as there would be with foreign AB such as from animal)

Question 16

Outline therapeutic use of manufactured antibodies

Answer 16

Prophylactic protection against microbial infection

Anti-cancer therapy

Removal of T-cells from haematopoietic stem cell transpants (e.g. anti-CD3)

Block cytokine activity (e.g. anti TNFa)

Question 17

1. What is synagis
2. What is IVIG
3. Why are T cells removed from haemoatopoitic stem cell transplants

Answer 17

1. Therapeutic monoclonal antibody against RSV for prophylaxis
2. Some patients have to have IVIG (intravenous immunoglobulin if they can’t make their own)
3. To avoid graft versus host disease (i,e, so the donor transplant, which would contain T cells, don’t attack the host. Note that you don’t need the T cells in the bone marrow transplant, you are doing the transplant for the haematopoietic stem cells

Question 18

How can antibodies be used in diagnosis

Answer 18

1. Blood group serology
2. Immunoassays (to measure levels of the following)
   - hormones
   - antibodies
   - antigens
3. Immunodiagnosis
   - Infectious diseases
   - Autoimmunity
   - Allergy (IgE)
   - Malignancy (myeloma=plasma cell tumour)

Question 19

Give an example of immunoassay

Answer 19

Enzyme linked ImmunoSorbent Assay

Question 20

How does ELISA work

Answer 20

You put different antigen in different wells of a plate and this antigen is absorbed to the surface of the well

Then you pour antibody into the well….

Then you wash it away…. if the antigen that the AB is specific for is present, then the AB binds….
if not then AB is washed away….

then you add enzyme, and then a solution which contains a substrate which, if the enzyme is present, will be broken down by the enzyme to make a colour

THen you measure absorbance of light… light is proportional to the maount of AB and thus amount of antigen…

QUANTITATIVE ASSAY

Question 21

What is ELISA sandwich

Answer 21

When you add AB, which is absorbed to surface, then add antigen, then add AB with reporter

Question 22

Outline how point of care rapid testing works

Answer 22

You use lateral flow on test

You put the sample on the plate

Then there is an strip of ABs with a coloured bead linked to it on the plate

If the antigen that the AB is specific for is present in the sample, then the antigen will bind to the AB.

Whether or not the antigen binds, the ABs containing the beads will be washed down the plate

At the bottom of the plate there are 2 strips containing immobilised ABs.

The first strip contains immobilised ABs against the antigens. If antigens are present, they will bind, then a coloured line will appear at this strip (because the antigens are now bound to an AB which is conjugated to a reporter molecule). If there is no antigen, then there will be no colour at this strip

The second strip contains immobilised ABs against the ABs which have the coloured beads conjugated to them. The AB will bind here if the test is working

Question 23

What are the immunoligcal concerns associated wieth the following symptoms:

Vague aches and pains

Loss of appetite
Weight loss

“Glands” up in his neck

Fever, rash, small red patches, some lumpy

Answer 23

Vague aches and pains= Immune complexes

Loss of appetite
Weight loss = Effect of poor nutrition on bone marrow cells

“Glands” up in his neck=
Immune activation

Fever, rash, small red patches, some lumpy= Acute phase, activation, immune complexes

Question 24

What is an immune complex

Answer 24

Antigen + AB

Question 25

What influences the size of an immune complex

What is the disadvantagfe of small immune complexes

Answer 25

The ratio of antigen to AB….

If there is large amount of antigen and only a small number of ABs, immune complexes will be smaller.

Smaller immune complexes are worse at activating complement and are harder for the body to clear

Larger ones directly activate complement

Question 26

What is serum sickness

Answer 26

Immune complex mediated disease….. hypersensitivity type reaction

Question 27

What problems do immune complexes cause

Answer 27

They can be deposited in a range of sites such as glomerulus= glomerulonephritis

but also other areas such as in the skin, causing aches and pains

Question 28

What can large immune complexes do in the glomerulus capillary

Answer 28

Large immune complexes attach to the basement membrane
Activate platelets and granulocytes, activating vasoactive mediators affecting endothelial cells

This increases gaps between endothelial cells, allowing smaller immune complexes to pass through endothelial barrier

Smaller immune complexes attach to epithelial layers and activate complement/ cause recruitment of granulocytes

This all causes damage and glomerulonephritis if in the kidney

Question 29

What would be tested if immunodeficiency suspected

Answer 29

Serum Immunoglobulin levels (next card for how)
IgG, IgM, IgA and IgG1, IgG2, IgG3, IgG4

Specific Antibodies (ELISA)

Lymphocyte subsets (Flow Cytometry)

Question 30

How are serum immunoglobulin levels tested

Answer 30

Serum electrophoresis / ELISA / Nephelometry (turbidity)

Question 31

Which specific antibodies may be tested when looking at whether somebody has immunodefficiency

Answer 31

YOU TEST TO SEE IF PEOPLE MAKE CORRECT ANTIBODIES AGAINST THINGS THAT THEY HAVE BEEN VACCINATED WITH

Protein antigens – Tetanus & Haemophilus

Polysaccharides antigens – Pneumococcus

Question 32

How is electrophoresis used to detect AB

Answer 32

Big albumin band at bottom

In healthy individual should be a smear so colour across lots of immmunologbulin

In an individual undergoing immune response, this band would be darker

In an individual with B cell malignancy ther ewould be a very dark band at one point…. shows monoclonal expansion

Question 33

How does flow cytometry work and what is it for

Answer 33

It identifies different types of cell

So basically you have different antibodies with different coloured beads on them

Then you put the cell in an the antibodies bind if their antigen is there….

Then you but through a machine which measures cell size, cell granularty and the flouresecens so you can work out if CD3+, etc etc

work out what type of t cell

Question 34

When might flow cytometry be used

Answer 34

Youc an count number of CD4+ T cells in HIV paitent

Question 35

Draw the natural history of HIV infection

Answer 35

Look at CD4+ T cells and the HIV RNA

Question 36

What is masured in HIV lab

Answer 36

HIV RNA and CD4+ t cell count

allows monitoring of treatment

Question 37

What diseases can HIV infected people hae with very low CD4 count

Answer 37

Very low= CMV/lymphoma

Bit higher PCP

Bit higher Haposi’s sarcoma

Bit higher bacterial skin infection and herpes, zoster etc

Hugher still lymphadenopathy and thryombocytopenia

Question 38

Give an example how detection of specific antibodies can diagnose immunodeficiency

Answer 38

You can give vaccination of an antigen (or the ABs to this antigen)

Then do blood sample

Do ELISA where you coat the well in antigens and see if any ABs attach by treating it with an enzyme

If they are immune deficieny tey won’t be able to make any of their own antibodies against the antigen

Question 39

Which antigen speficiies are used in flow cytometry to detect the different types of lymphocyte cell

Answer 39

CD3+- T cells (pan T cell marker)

CD4+- helper T cells

CD8+ cytotoxic T cells

CD19+ B cells

CD56+ Natural Killer cells

Question 40

Outline natural history of HIV infection

Answer 40

HIV primary infection

Lots of replication so very high HIV load, and CD4+ve T cells begin to decrease

However, the HIV load falls initially as the immune system controls the infectin

Then over time there is increase in viral load and reduction in CD4+ve T cells, putting person at risk of opportunistic infection infections