Immunology in the Clinic and Research Lab

Question 1

Describe the structure of antibodies.

Answer 1

Antibodies are fairly complex. They are made up of two heavy chains and two light chains held together by disulphide bridges.

We can split the antibody into two parts: the Fab region that binds to the antigen, and the Fc region which interacts with various aspects of the immune system to modulate various processes.

The Fab region can bind to specific epitopes on antigens via the complementarity determining regions (CDRs), also called the hyper-variable regions.

The Fc region, as mentioned previously, can interact with various aspects of the immune system to modulate processes like antibody-dependant cell-mediated cytotoxicity (ADCC), or antibody-dependant cellular phagocytosis (ADCP), or fixation of compliment. It is also related to the half-life of your antibody.

Question 2

Briefly, describe the polyclonal antibody response.

Answer 2

We have B-cells in our body that have specificities to particular antigens. The binding of the epitope on the pathogen to the B-cell activates it, causing it to proliferate. They form clonal B-cells that will then secrete the antigen-specific antibodies.

Question 3

Describe the method to making monoclonal antibodies.

Answer 3

We take the antigens (to which we want to make antibodies) and inject them into a mouse. After about a week or two, we harvest the B-cells that make the antibody.

We then take those B-cells and fuse them (in polyethylene glycol) with immortal myeloma cells. The myeloma cells are derived from a B-cell tumour but do not produce antibodies themselves. They lack the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene.

After this fusion process, we have a mixture of cells. We’ll have unfused B-cells and myeloma cells, then we have the fused cells called hybridomas.
We want to select these cells and get rid of the unfused cells.

We use Hypoxanthine-Aminopterin-Thymidine (HAT) selection.
HAT basically stops cells making DNA using the normal pathway. What can happen is that cells that contain the HGPRT gene can still make DNA using a different pathway.

The myeloma cells don’t have this gene so they can’t make DNA and die off. The B-cells do have the gene and can make DNA, but they are naturally short-lived, so they die off after a short time. The only cells that survive are the hybridomas.

We take the mixture of hybridoma cells and dilute them out to a single cell, then we culture that cell individually. The assay will then divide to form clones of the original cell. These cells will now produce an antibody of only one specificity. Those are called monoclonal antibodies.

Question 4

What are some features of monoclonal antibodies?

Answer 4

• Hybridomas can be stored indefinitely and grown to produce monoclonal antibody when required.
• Antibody genes can be cloned from the hybridomas which allows antibodies to be engineered for different applications.
• Polyclonal or monoclonal antibodies can be produced which bind to Fc regions of particular antibody classes e.g. to IgG’s, IgA’s etc. These are called anti-isotypic antibodies.

Question 5

Define immunoassays.

Answer 5

Using the antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection), we measure the amount/concentration of antibody or antigen.

It is very specific and sensitive, hence, it is widely used in research and analytical labs.

Immunoassays use polyclonal or monoclonal antibodies.

Question 6

What labels are used for immunoassays?

Answer 6

They were originally radioactive (radioimmunoassay, RIA), but now the antibodies are normally coupled to an enzyme (for e.g., horseradish peroxidase or alkaline phosphatase) which which convert substrate into a coloured substance. This can be detected using colourimetry.

This is called ELISA (enzyme-linked immunosorbent assay).

Other alternatives are luminescent.

Question 7

What are the two types of solid phase immunoassays, such as ELISA?

Answer 7

DIRECT/INDIRECT: often used to quantify an antibody

SANDWICH (CAPTURE): often used to quantify or detect an antigen

Using these assays, the concentration of analyte (antibody or antigen) in the sample can be calculated by comparison to analyte standards of known concentration.

Question 8

Describe how to perform a direct ELISA.

Answer 8

With a direct ELISA, we take an ELISA plate and we add antigen onto the plate. This is immobilised on the bottom of the well.

We then add our test antibody solution, which may be linked to an enzyme (like the ones mentioned previously), and we then add the enzyme substrate.

If the antibody binds to the antigen, when we add our substrate, the substrate will become coloured. We can then measure the amount of colour using a spectrophotometer.

Question 9

What are some uses for the direct ELISA?

Answer 9

• screen hybridoma supernatants

- detect exposure to infectious agent

Question 10

Describe how to perform an indirect ELISA.

Answer 10

With direct ELISA, it is similar to indirect ELISA, but there is one more step.

When we add the antigen onto the ELISA plate, we first add the primary antibody which is not labelled. Then, we add the secondary antibody, which is the one we are trying to detect. The secondary antibody binds to the Fc region of primary antibody.

Secondary antibody is often polyclonal and so may bind to different epitopes on a primary antibody. This allows multiple secondary antibodies to bind to the same primary antibody thereby amplifying the signal and increasing the sensitivity of the test

The amplification of the secondary antibodies is very helpful if you have a low amount of antibody that you are trying to detect.

Question 11

Describe how to perform an Elispot immunoassay.

Answer 11

Another assay used in the labs, and is based on the ELISA, is called Elispot. It is used to detect cytokines that are given off by cells.

We have antibodies that are specific for the cytokine we are trying to detect, and they have been immobilised on the bottom of an ELISA plate. We add activated T-cells to the plate, so the T-cell will then secrete the cytokine. If it is the correct one, it will bind to the antibody.

We can then come in with a second antibody conjugated to an enzyme to detect a certain colour.

Question 12

Describe how to perform an SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) immunoassay.

Answer 12

We start off by taking the sample with the protein that we are trying to detect. Typically, we will boil it with sodium dodecyl sulphate. This binds to the protein and gives it a net negative charge.

We will then run that protein in a polyacrylamide gel. The protein will run through the gel according to its size. We will then take the gel and blot the protein onto nitrocellulose, which is like paper.

We can probe that nitrocellulose with an antibody that is linked to an enzyme that gives it a coloured product. Thus, when we add the substrate, we can see a band form on the nitrocellulose.

We will then compare the result to protein standards of known size. We can also tell, if there are smaller bands underneath where the protein should be, that it has been degraded into those smaller bands.

Question 13

Compare using ELISA and SDS-PAGE/WB (western blotting).

Answer 13

SDS PAGE/Western blotting often used alongside ELISA (to give us as much information as possible about protein).

In WB, protein concentration can be measured by comparing intensity of band we are detecting to band from a protein standard of known concentration.

If the protein is degraded, it may be more useful to use WB to calculate the protein concentration, as some of degradation fragments may contribute to the signal in ELISA if both coating and detecting antibody are able to bind to them.

Question 14

How can you use antibodies to purify immune cells?

Answer 14

The antibodies are covered with microscopic magnetic beads, and they will bind to certain proteins on the surface of the immune cell that you’re trying to isolate.

Once bound, you put the cells into a machine that has an iron wall mesh. When you apply a magnetic field, only the bound cells will stick to the iron mesh; the other cells will be washed out.

We can then remove the magnetic field, releasing the cells and collect them.

Question 15

Describe how the antibody-antigen interaction is used in flow cytometry.

Answer 15

Individual cells within a mixed population are tagged by treatment with monoclonal antibodies which bind to surface molecules and are labelled with fluorescent dyes.

Mixed cells are then forced through a nozzle to form stream of single cells.

Individual cells pass through a laser beam which scatters light and causes dye to fluoresce and provides information on bound antibody and cell surface protein.

Question 16

List some types of samples that are analysed by biomedical scientists.

Answer 16

• Blood serum
• Blood cells
• Urine
• Synovial fluid
• Saliva
• Mucus
• Cerebrospinal fluid

Question 17

List some types of diseases that we can test for.

Answer 17

• Transplant compatibility
• Immunodeficiency
• Autoimmunity
• Allergy
• Malignancy
• Infection

Question 18

How are antibodies implicated in the success of transplants?

Answer 18

Histocompatibility in transplants is important because the genetic differences between individuals are detected by the immune system, which leads to rejection of the non-self.

Major histocompatibility proteins (MHC) are major players in transplant rejection.

Best transplant results when donor and recipient MHCs are as similar as possible.

Question 19

Briefly, describe the genetics of MHC.

Answer 19

In humans, it is known as HLA (Human Leucocyte Antigens).

The MHC is located on chromosome 6 and contains 3 MHC class I proteins and 3 MHC class II proteins.

It is highly polymorphic - there are 100s of different variants.

Question 20

How would you identify MHC type for transplant compatibility?

Answer 20

MHC alleles of donor and recipient are identified by Polymerase Chain Reaction.

Question 21

How would you use antibodies to quantify cell populations?

Answer 21

We can use the technique of flow cytometry to quantify cell populations.

We can look for specific cell surface markers (using the antibodies against them).

Question 22

How can you use flow cytometry to monitor an HIV infection?

Answer 22

Lymphocyte subset estimations are performed using monoclonal antibodies to: CD3, CD4 and CD8 on whole blood and analysed by flow cytometry.

The percentages of cells in each subset is determined using a FACS machine.

The results are reported as percentages and absolute counts.

Typically, someone with HIV will have a decreased CD4 count, i.e. T-helper cells are often reduced.

Question 23

How can you use antibodies to track T cell responses?

Answer 23

Fluorescent MHC complexes are useful in helping us study T-cell responses.

At the C terminal end of the MHC, a vitamin called biotin is added. Biotin binds very strongly to another molecule called Streptavidin. Streptavidin is linked to another molecule that gives off light. When these are all mixed together, they form a tetramer (shown above).
The bond between TCR and MHC tends to be weak, so the fact that you have a tetramer increases the chances of binding.

Once bound, the amount of fluorescence can be measured, and from that we can calculate the proportion of T-cells.

By creating MHC complexes loaded with HIV antigen we can calculate, for example, what proportion of CD 8+ T cells will bind to the antigen.

Question 24

Describe neutrophil deficiencies.

Answer 24

Neutrophils are found in acutely inflamed tissue. They ingest pathogens and kill using reactive oxygen species.
They then rapidly die after phagocytosis, which generates pus.

A deficiency in neutrophil numbers is called neutropenia, which leads to a high rate of infection.

A deficiency in phagocyte function is called chronic granulomatous disease (CGD) – patients cannot form reactive oxygen species, so they succumb to bacterial and fungal infections.

Question 25

How would you measure neutrophil function?

Answer 25

This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by reactive oxygen species (ROS), produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.

In people who have, for e.g. CGD, this reaction will not occur.

Question 26

Describe how you would quantify antibodies using nephelometry.

Answer 26

Nephelometry is an rapid automated system to measure serum immunoglobulin levels. It’s based on the principle of light scattering by antigen-antibody complexes.
Typically, it will be used to study the amount of antibody from different classes that are present in the serum.

For example, if you’re testing for the amount of IgG, then you would add the anti-IgG antibody in the serum. The complexes will be formed, and then we can shine a light through the serum, and the complexes will diffract the light.

The amount of diffraction will be measured and correspond to the number of complexes, which in turn will correspond to the amount of IgG complexes.

Question 27

Describe how you would diagnose allergies.

Answer 27

When exposed to a certain allergen, individuals who are allergic to that allergen will (for an unknown reason) produce IgE. This IgE will bind to receptors on mast cells of the immune system.
On second exposure, the allergen will bind to the IgE, which will cluster the IgE on the surface of the mast cells, which will send a signal through the cell. This signal causes the cell to degranulate, releasing the contents, giving rise to the allergic response.

One way in which we can measure this is the skin prick test. The arm of the patient is pricked with potential allergens. If the individual is allergic to it, a bump will form due to a localised allergic response.

Question 28

Other than a skin prick test, what are other ways in which you can diagnose allergies?

Answer 28

Another technique to diagnose allergies is the RAST (RadioAllergoSorbent Test) test.

The suspected allergen is bound to an insoluble material and the patient’s serum is added. If the serum contains antibodies to the allergen, those antibodies will bind to the allergen. Radiolabeled anti-human IgE antibody is added where it binds to those IgE antibodies already bound to the insoluble material. The amount of radioactivity is proportional to the serum IgE for the allergen.

Fluorescence is often used instead of radioactivity.

Question 29

Describe lupus, and how detecting antibodies is useful in it’s diagnosis.

Answer 29

Lupus, or Systemic Lupus Erythematous (SLE), is characterised by autoantibodies to nuclear antigens (e.g. DNA, RNA). These autoantibodies causing a range o symptoms, often dermatological.

Detection of these autoantibodies is useful for diagnosis and monitoring disease activity.

Question 30

Describe a test in which we can detect autoantibodies in lupus.

Answer 30

In immunofluorescence, we will take a human cell line, and incubate it with serum isolated from the individual. If they have lupus, we will expect to have antibodies in the serum that will bind to the nucleus of the cells.
We take the cells that have been bound and label them with another antibody that had a fluorescent tag.
We can then visualise the results using fluorescence microscopy.

Question 31

What are some applications for monoclonal antibodies in therapy?

Answer 31

Intravenous immunoglobulin (IVIG) is a blood product purified from the serum of between 1000-15,000 people/batch.

It’s used to treat patients with antibody deficiencies at 200-400mg/Kg/3 weeks.
At a high dose of 2g/kg/4 weeks, it is used as an immunomodulatory agent in a number of immune and inflammatory disorders.

It has applications in neurology, haematology, immunology, nephrology, rheumatology and dermatology.

Another example is with rabies. After a bite from a suspected rabid animal, polyclonal antibodies (Human Rabies Immunoglobulin (HRIG) are isolated from the serum of individuals who have been immunised with the rabies vaccine and injected into the wound site.

Question 32

What are some applications of monoclonal antibodies in therapies?

Answer 32

Approximately 45-50 monoclonal antibodies are licensed to treat disease: cancer, chronic inflammatory diseases, transplantation, infectious diseases and cardiovascular medicine.

Monoclonal antibodies can have their effects either by binding and blocking a process or by mediating immune responses such as initiation of complement or antibody-dependent cell mediated cytotoxicity (ADCC).

Molecules such as toxins or radionuclides can be joined to monoclonal antibodies: the antibody binds to the cancer cell which is then killed by toxin or radioactivity.