Antibodies as diagnostic tools Flashcards
Outline structure of antibody
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
Where can molcules be attached to antibodies to aid in diagnostic
To the constant part of the heavy chain…. still allows antigen binding
What molecules can be attached to the antibody
Enzymes, fluorescent probes, magnetic beads and drugs
What enzymes can be conjugated to the antibody
perioxidase (gives brown colour), alkaline phosphatase
Which fluorescent probes can be added to antibody
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)
What can antibodies conjugated to magnetic beads be used for
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
Which drugs can be conjugated to antibody
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
££££
Why are antibodies powerful in diagnositcs?
Specificity to target antiigen
Which molecules can ABs be made against
Any antigens include carbs, DNA and proteins
can even back antibodies against antibodies…..= anti-antibodies
When can humans make antibodies against antibodies
E.g autoimmune disease such as rheumatoid…. IgM against constant part of IgG
Outline indirect labelling using an antibody
primary antibody against an antigen can be detected using a secondary antibody which has a reporter molecule attached (e.g. enzyme of fluorescent)
when are Abs produced in patient
In autoimmune disease, or for defence against infection
How can antibodies be manufactured
Antisera from immunised animals (polyclonal)… deliberately infect animals
monoclonal antibodies (diagnostic)
“genetically engineered” antibodies (recombinant DNA)
How are monoclonal antibodies manufactured
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
How can ABs be produced using recombinant DNA technology
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)
Outline therapeutic use of manufactured antibodies
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)
- What is synagis
- What is IVIG
- Why are T cells removed from haemoatopoitic stem cell transplants
- Therapeutic monoclonal antibody against RSV for prophylaxis
- Some patients have to have IVIG (intravenous immunoglobulin if they can’t make their own)
- 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
How can antibodies be used in diagnosis
- Blood group serology
- Immunoassays (to measure levels of the following)
- hormones
- antibodies
- antigens - Immunodiagnosis
- Infectious diseases
- Autoimmunity
- Allergy (IgE)
- Malignancy (myeloma=plasma cell tumour)
Give an example of immunoassay
Enzyme linked ImmunoSorbent Assay
How does ELISA work
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
What is ELISA sandwich
When you add AB, which is absorbed to surface, then add antigen, then add AB with reporter
Outline how point of care rapid testing works
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
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
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
What is an immune complex
Antigen + AB
What influences the size of an immune complex
What is the disadvantagfe of small immune complexes
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
What is serum sickness
Immune complex mediated disease….. hypersensitivity type reaction
What problems do immune complexes cause
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
What can large immune complexes do in the glomerulus capillary
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
What would be tested if immunodeficiency suspected
Serum Immunoglobulin levels (next card for how)
IgG, IgM, IgA and IgG1, IgG2, IgG3, IgG4
Specific Antibodies (ELISA)
Lymphocyte subsets (Flow Cytometry)
How are serum immunoglobulin levels tested
Serum electrophoresis / ELISA / Nephelometry (turbidity)
Which specific antibodies may be tested when looking at whether somebody has immunodefficiency
YOU TEST TO SEE IF PEOPLE MAKE CORRECT ANTIBODIES AGAINST THINGS THAT THEY HAVE BEEN VACCINATED WITH
Protein antigens – Tetanus & Haemophilus
Polysaccharides antigens – Pneumococcus
How is electrophoresis used to detect AB
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
How does flow cytometry work and what is it for
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
When might flow cytometry be used
Youc an count number of CD4+ T cells in HIV paitent
Draw the natural history of HIV infection
Look at CD4+ T cells and the HIV RNA
What is masured in HIV lab
HIV RNA and CD4+ t cell count
allows monitoring of treatment
What diseases can HIV infected people hae with very low CD4 count
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
Give an example how detection of specific antibodies can diagnose immunodeficiency
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
Which antigen speficiies are used in flow cytometry to detect the different types of lymphocyte cell
CD3+- T cells (pan T cell marker)
CD4+- helper T cells
CD8+ cytotoxic T cells
CD19+ B cells
CD56+ Natural Killer cells
Outline natural history of HIV infection
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
