immunoassays Flashcards
give some examples of immunoassays?
pregnancy tests
lateral flows
why is the use of antibodies in research, diagnostics and treatment widespread?
because of their ability to specifically bind to antigens and their ability to fix complement
how are antibodies produced?
theyre produced through matured b cells and are then clonally expanded and with the help of t cells, the b cells become plasma and start to secrete antibodies
how specific do you want antbodies to be for the purpose of recognising analyte if it has high avidity?
you want them to be really specific
what does analyte mean?
a substance whose chemical constituents are being identified and measured.
what tells the b cells what isotype of antibodies to make?
the signals around them
what does it mean if an antibody is specific and has high avidity?
this means that theyve got a high ability to bind
what does multiple immunisations increase?
it increases the amount and affinity of the antibody in the serum of immunised animals
what is the first isotype of antibody produced?
IgM
how do IgG antibodies get produced after immunisations?
the first type of antibodies produced are IgM then they undergo class switching to IgG which is highly specific and is produced after immunisations
if your making antibodies for use in humans, are they produced in humans first?
no, as they’ll target the own body, you make them in another animal
what is the process for producing antibodies for use?
o Once the amimals have been immunologically primed (mounted an antibody repsinse), they have high levels of specific antibody in the serum
o This means they can therefore collect the blood, separate serum and use this antiserum for future tests, treatments ect
o You can repeatedly bleed antimals, allowing a plentiful source of antiserum
You can collect several liters of antiserum from an immunised cow each month
o this is releatively cheap and easy and allows for the generation of polyconal antibosies
what is the problem with using serum as a way of making antibodies ?
it contains many other proteins which may cause problems to sensitive assays
why is it better to purify antibodies?
for improved storage and higher concentraion
it also allows you to only have helpful antibodies
what are the steps of the purification of antibodies from antiserum?
- the b cell binds the virus through the viral coat of the protein
- then the virus particle is internalised and degraded
- the peptides from internal proteins of the virus are presented to the t cell, which activates the B cell
- activated B cell produces antibody against viral coat protein
what does multiple immunisations do for the amount and affinity of antibodies in the serum of immunised animals?
it increases both the amount and affinity of antibodies in the serum of immunised animals
what does multiple immunisations also result in? [in terms of the type of antibody]
it results in class switching with the antibodies more likely to be IgG or IgA
what do you need to consider when generating antibodies for use?
antigen- so you make an antibody actually specific to the antigen
adjuvant - you want one thats effective at enhancing the body’s immune response to the antigen
kenetics- dunno why, just know theyre important
what does it mean when animals are immunologically ‘primed’?
they mount an antibody response
how do you get antiserum?
you immunise an animal until it gets an antibody response then collect the blood, separate the serum (antiserum)
what can antiserum be used for?
future tests
treatments
what is a relatively cheap way of producing polycolonal antibodies? and why?
antiserum
as you can repeatedly bleed animals allowing for plentiful source of antiserum and you can collect several litres of antiserum of an immunised cow every month
what are the cons of generating antibodies through serum?
the serum contains many other proteins which may cause problems in sensitive assays e.g. cross reactivity
therefore its better to purify improved storage and higher concentration
how do you purify antibodies for use?
the animal is immunised, then the serum is extracted which includes antibodies then the antibodies are bound to a protein and the serum proteins are washed off and elute the bound antibodies
what are the problems with polyclonal antibodies?
- theres lots of different classes and isotopes of antibodies present
- theres potentially different specificities against different parts of the molecule
- potentially contaminating antibodies that react with other proteins
- Sometimes need reagents that are much more specific & well characterised (e.g. for injecting into patients!)
how do you make monoclonal antibodies?
to make hybridomas, to fuse an antibody-forming cell with a tumour cell, then the antibody producing hybridomas are cloned and screened for antibody production, then monoclonal antibodies are isolated for cultivation
what are monoclonal antibodies used for?
they can be ised for many things such as experiments or tests for covid
what are the advantages of monoclonal antibodies?
- they can be produced in large quantities without the need for repeated bleeding of animals
- bind very specific epitope of an antigen
- exactly the same immunoglobulin class and isotype
- they can be highly purified
- allows for in depth analysis without batch variation to quantify specific analytes and to treat patients
what are the steps of using a bacteriophage to screen and produce monoclonal antibodies?
- isolate population of genes encoding antibody variable regions
- construct fusion protein of V region with a bacteriophage coat protein
- cloning a random population of variable regions gives rise to a mixture of bacteriophage - a phage-display region
- select phage with desired V regions by specific binding to antigen
what are the positives to phage display production of monoclonal antibodies?
- allows for rapid screening of many more V region expressing clones against the antigen of interest, increasing sensitivity and the likelyhood of success
- other subsequently use genes from positve clones and express into hybridoma (allows production of mammalian/ humanised monoclonal antibodies)
what negative is there to phage display production of monoclonal antibodies?
some versions of this method allow for the production of monoclonal antibodies by bacteria (meaning you get lots of proteins, cheap but not nessisarily corrcect folding)
theres also issues with morals as youre using antibodies from animals
how are nanobodies made?
theyre made through phage display but by using libraries from immunised camelids
what is the difference between convntional antibodes and nanobodies?
the conventional antibodies have heavy and light chains whereas the nanobody, also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family
what are the advantages to nanobodies?
Produced recombinantly: known sequence,
little batch-to-batch variation
Small size: better tissue, good for imaging and therapeutic antibodies
No heavy or light chains: less likely to generate an immune response
High stability: as smaller proteins
what are some applications for antibodies?
medical diagnostics, treatment of immune deficiencies and some types of cancer, and prenatal therapy specifically to minimize the risk of hemolytic disease of the newborn
why would antibodies be conjugated to an enzyme or flurochrome?
to allow for the detection of antibody
what types of things can be conjugated with antibodies to help them be read out?
enzymes
radiation
fluorescence
what are enzymes used for when theyre conjugated to antibodies?
they can be used to catalyse a reaction, often resulting in a colour change
what does radiation do when its conjugated to an antibody?
can link/ incorperate radioactivity emitters with the antibody
subsequently detect the radiation (alpha, beta or gamma) as appropriate
what does fuorecence do when conjugated to antibodies?
its an increasingly popular method as it avoids hazards associated with radiation but it can require expensive equipment to detect
what things can antibodies be linked chemically to which act as reporter molecules?
enzymes
fluorecence
radioactive molecules
enzyme beads
what is serology?
its the method used to detect or quantify antibodies in response to microbial infections
what can serology be used for?
it can determine if a person has experienced an infection as theyre produced during infections
it can also determine the strain of microbe the person has been infected with
the type of antibody that has been detected can also say if the infection is recent or historic and about the type of cellular immune response that was produced
what type of infection is IgM associated with?
a recently aquired infection
what type of infection is IgG associated with?
its associated with a historic or chronic infection
what can knowing the type of antibody present be useful for?
its useful in situations where the treatment, disease prognosis or other risks are affected by this consideration - pregnant people preventing the vertical spread of infections such as toxoplasmosa gondii
what are the steps of an ELISA for IgG?
- coat the surface with antigen
- add serum (this contains the IgG)
- add anti-IgG
- ass substrate
- stop the reaction and measure the level of antibody by measuring the absorbance
what is the difference between the ELISA for IgG and for IgM?
the only difference is the serum added and the anti-IgG is substituted for anti-IgM
why are samples serial diluted in ELISAs?
so that the endpoint can be determined
this is because there is a large concentration of antibodies found in serum and these arent all on the linear portion of the graph of absorbance Vs antibody conc
its more consistent between assays from day to day than simple absorbance as that can change with each ELISA
what type of determination allows for comparison of antibody content in ELISAs
endpoint determination
how can the principle of the ELISA be adapted?
by changing the label on the antibody
how can microbes be detected using antibodies?
immunological assays for pathogen detection are based on antibody-antigen interaction
what are the steps of an ELISA for pathogens?
- coat the surface with pathogen antibody
- add the sample which contains the pathogen
- add anti-pathogen
- add substrate
- stop the reaction and measure
what is an ELISPOT?
its based on an ELISA but it allows for quantification of cytokine producing cells
what is fluorescence?
its the stimulated emissionof light from a substance which has absorbed radiation of another wavelength
how is fluorescence useful in immunoassays?
its useful when flurochromes are conjugated onto antibodies
allows for the measurement of compounds in the sample including drugs
what are some commonly used flurochromes?
green fluorescent protein (GFP)
fluorescein (FITC)
phycoerythrin (PE)
how can fluroscence microscopy be used?
and what are the steps involved in this?
you can use it to visualise thin slices of tissue
stain with enzyme linked of fluorescence-linked antibodies to detect the cells or molecules of interest
you then visualise under light microscpoy to see colour changes or with laser-scanning fluorescence microscope
what does forward scatter determine?
cell size
defracted light i.e shadow of the cell which is related to cell size
what does side scatter determine?
granularity of the cell
the reflected light i.. light reflecting from cellular components related to cell granualrity and complexity
what does a single parameter histogram show in flow cytometry?
the horizontal axis shows the level of fluorescence - the brighter the cell, the further right it is
the vertical axis show the number of events per channel number and allows you to look at expression of the marker gene
what does a two parameter dot plot show for flow cytometry?
one axis shows the first colour
the second axis showa the second colour
this allows you to look at individual populations of cells based on two different markers on the cells
what is the downside to measuring immune cells with flow cytometry?
LOCATION LOCATION LOCATION
you dont know where the immune cells are from in the cell and the most important thing for immune system is location
how can you measure immune cells in vivo?
using two photon microscopy - which allows you to image living organs
what are the pros and cons of the ‘immunology toolbox’?
- lots of in vitro assays provide good info in a reductionist, controlled experiment
- BUT theyve lost the structure, cellular interactions and compexity fo the system as well as requiring manipulation to reveal function
- it also has caveats e.g. complexity of revealing fucntion, cost, technical expertise and ethics
what do in vivo experiments provide?
they provide lots of potential for understanding the compexity of the immune response
e.g. is a vaccine protective and what is the mechanism of protection
what does the use of genetic engineering in in vivo experiments allow?
it allows you to add/ remove specific genes
they provide lots of models of different diseases ranging from arthritus, athsma, TB and HIV
allows us to develop theraputics by identifying specific molecules important in a given disease so we can target them
what can indentifing the mechaninsm of disease allow you to do?
it allows you to identify specific molecules to inhibit in patients using targeted theraputics
e.g. in arthritus - studies have identifyed an important role for TNF alpha- now you can use specific antibody to inhibit in patents
why is it important to know stuff about certian cells?
response to vaccination
effects of drugs
prescence of infection
diagnosis of disease
what might we want to know all about a cell?
isolate cells
evaluate number of cells
are cells proliferating
are cells viable/ dying
can cells carry out their functions
what steps would you have to carry out to isolate cells?
source - peripheral blood lymphocytes or post-mortem or during surgery
you need to determine the type of cell and then purify them as its a mixed population of cells
how do you separate out white blood cells from peripheral blood?
density centrifuging
how can you separate out cells apart from centrifusion?
by using antibodies or magnets, this can be more specific and can isolate a certian type or subset of cells for further analysis
what are the steps to evaluating the number of cells?
get the total number of cells
get the proportion of specific cell type
how do you quantify the number of cells?
by counting them with a haemocytometer
if you have quantified the number of cells, how would you identify how many of them are t cells?
by flow cytometry
what causes T cells to divide?
antigen specific responses
mitogen-induced reposnes- mitogens activate cells in non-specifc manner, driving proliferation
what are the ways to investigate the number of cells proliferating?
by enzymes, radiation or fluorescence
what is the enzymatic approach to measuring cell proliferation?
- this relies on enzymes in the mitochondria converting salt into coloured product
- only living cells have active mitochondria
- amount of colour is proportional to the number of cells which is proportional to the amount of cell proloferation
what is the radioactive approach to measuring cell proliferation?
you need to add a source of radioactive thymidine to the cell culture
as the cells proliferate, they incorperate this into the DNA of daughter cells, resulting in radioactive cells.
you then measure the amount of cell-associated, proportional to the amount of cell division
what is the fluorescent approach to measuring cell proliferation?
you can label proteins of cells with fluorescent dye e.g. CFSE - carboxyfluorescein succinimidyl ester
if the cell divides, each daughter cell is half as bright
this allows for accurate quantification of the number of cell divisions within the population
how can you measure the levels of cell death in a sample?
when cells die, they lose their membrane integrity meaning that vital dyes are able to enter the cell and stain them e.g. tryptan blue - can see down a conventional light microscope or propidium iodide which becomes fluorescent when it binds DNA
how do you measure different cell functions?
you can measure lots of functions using antibody based technologies such as ELISAs, ELISPOTs, flow cytometry ect
cell killing is also an important immune function and can be measured by fluorescence or radioactivity
what hormone spike at week 8 of pregnancy?
HCG
pregnancy is associated with a blood and unrine increase in what hormones?
HCG (human gonadotrophin hormone)
oestrogen
progesterone
what are the cons of using historically serium or urine in biological assays using animals?
- animals are expensive to keep
- these tests arent very reliable
- youve got ethical considerations as
- youre using the animals as the test
- it takes a while to get an answer for these tests
what are the three main types of immunoassays for pregnancy?
inhibtion of latex agglutination - clinical
radioassay - clinical
dip-stick test - home and clinical
expalin what happens in the inhibition of latex agglutination pregnancy test?
rabbit anti-HCG is mixed with urine or serum
- if HCG is present it will be bound and thus removing anti-HCG from further reactions
- if HCG isnt present, the mixture will contain free-anti-HCG
the latex beads coated with HCG are added to the sample
- the negative test would show agglutination and the positive test wouldnt
what are the steps of a radioassay for a pregnancy test?
the arbbit anti-HCG is mixed with a sample of urine or serum
- if there is HCG present, it will be bound and the anti-HCG will be removed
- if there isnt any HCG, the mixture will still contain free anti-HCG
then the radiolabelled HCG is added to the sample and if the person isnt pregnant then the test will read out as highly reactive and if the person is pregnant then the test will read out as not reactive
how does the dip-stick pregnancy test work?
Rabbit anti-HCG is bound to coloured latex beads and dried onto filter paper
When mixed with urine
* If HCG is present it will be bound by Ab
* If HCG is not present -it is not bound!
The Latex beads travel up the filter paper where they pass a line of immobilised anti-HCG
* If HCG is present on the latex beads, they will be bound by Ab and the beads stopped AND FORM A LINE
* If HCG is not present on the beads, they continue to move up the filter
Some Latex beads travel further up the filter paper where they pass a line of immobilised anti-rabbit IgG
* They are bound by Ab and the beads stopped AND FORM A LINE as a positive control
what are the advantages and disadvantages to the diferent pregnancy test?
inhibition of latex aggragation - time consuming and relies on visula interpretation and isnt absolutely quantiative
radioassay- produces radioactive wastes
dipstick test- not quantative
what are digital pregnancy tests?
theyre the same tests as the sip-stick test but it has a spectrophotometer which measures the absorbance of the test area, the control area and a refrence area, then a computer programme calculated HCG concentraion and displays the appropriate message and can tell you how pregnant you are from how strong the read out is
what are the problems with measuing HCG as a way of testing for pregnancy?
- the levels can also be raised in other conditions such as tremours
- it can also be raised if theres been an incomplete abortion
- the levels of the hormone wont raise if theres an ectopic pregnancy
how can flow cytometry be used to measure the progression of HIV to AIDS?
HIV infects CD4+ T cells, the infection is associated with a decline in the percentage and absoulte no. of CD4+ T cells.
monitoring the number of CD4+ t cells can be used to follow the progression of the disease towards AIDS and can therefore also be used to measure the effectiveness of chemotherapy
what can fluorscent microscopy count in a known volume?
it can count lymphocytes from blood samples and can be used to quantify them
you can count the number of CD3 and 4+
What are the problems with fluorescent microscopy?
its time consuming
you can only count small numbers of cells at a time
its dependant on the operator to the interpretation of each cell
what does a flow cytometer do?
it analyses cell suspensions
how does a flow cytometer work?
it passes cells though a laser beam, then the light emitted from each cell is collected and analysed
it can use one colour or multiple colours can measure different types of cells by splitting them into different populations and identified by surface markers
what are examples of immediate hypersentivity reactions? and their symptoms?
Hayfever- pollens - Symptoms – sneezing, mucus secretion, itchy and teary eyes
Asthma pollems, house dust mites, animal fur - Symptoms – coughing, consitriction of airways
- Insect bites - Symptoms – wheal and flare, itch
Food allergy – peanuts, shellfish -Swelling of lips and tongue
why are type I hypersensitivity reactions associated with irritibility?
because it causes neurochemical changes in the body
what type of response are delayed allergic reactions associated with?
T cell responses
what type of response are immediate hypersensitivity responses associated with?
IgE responses
what symptoms can a systemic response cause?
- Increased vascular permeability
- Swelling of lips, tongue and larynx
- Smooth muscle contraction in lungs
- Fall in blood pressure
- Death (its a maybe)
what are the benifits of allergy testing?
- Identifies allergens for a person so that they can be avoided
- May be especially impotnat in the industrial setting
- Monitor desensitisation treatment
how do you do allergy testing by passive cutaneous anaphylaxis?
you inject rats with evans blue then with the patient serum, any present IgE will bind to the Fc receptors on the mast cells of the rats
each rat is intradermally injected with a different allergen and if the IgE specific for the allergen is present, then it binds to the allergen and cause mast cell degranulation, where this occurs, a blue area around the injection site forms due to the increased vascular permeability
what are the steps of the passive cutaneous alaphalaxis test?
- Evans blue is injected into the IV
- Serum from subject is injected into the IV
- Antibodies diffuse into tissue
- IgE binds the Fc epsilon R on mast cells
- Test allergens is injected intradermally
- Allergen is bounf by mast associated IgE
- IgE cross-linking occurs
- Mast cell degranulation
- Increased vascular permeability
Evans blue escapes into skin-obious wheal and flare reaction
what are the steps of skin testing for allergies?
a persons back is marked into grids and small doses of allergens are applied and the skin is scratched then its examined after 30 mins for the characteristic wheal and flare reaction
what is a RISK assay?
it measures the total IgE
what are the steps to a RISK assay?
paper disks are coated with IgE and is incubated with the serum sample then any IgE present binds to the disk, then theyre washed and the amount of radioactivity present is recorded
what is the problem with a RISK assay?
it only measures IgE levels, not the actual allergen you’re reacting tio
what does a RAST assay measure?
it measures the specific levels of IgE
what are the steps to a RAST assay?
paper disks are coated with known allergens and incubated with serum samples
any IgE specific for the specific allergen binds to the allergen then the disks are then washed and incubated with anti-IgE-radiolabelled antibody. theyre then washed again and the radioactivity present is recorded
what is the future of allergy testing?
miniturisation and testing hundreds of different antigens on a microarray
the radioactive assays are replaces with fluorscent bead arrays
what allergy therapies are there?
antihistamines - to block the release of histamines
increasing the dose of antigen injected subcutaneously results in a shift from IgE to IgG
densensitisation of the allergen can be dine through continuously immunising with the allergen
what are the advantages of bead arrays?
its similar to protein arrays
you can mix and match arrays to make custom arrays for individual patients
theyre also easily quantifiable
why is it important to achieve the correct dose of drug in patients?
- to gain the max theraputic benifit
- to prevent unwanted side effects
why is it important to monitor digoxin therapy?
8-20% of patients on digotoxin therapy were administered too much of the drug
50% of those could die
and others wouldnt be getting adequate levels of drug for the max theraputic benifit
how do you measure the amount of drug in a patient?
- measure drug in serum by direct method e.g. HPLC
- measure drug in serum by immunoassay e.g. FPIA
what is fluorescent polarisation immunoassay?
its based on principle describes by danliker and feigen
- polarised light is used to excite a fluorescent label on a drug
- its a homogenous, competitive assay
- use restricted to molecules with a molecular weight of <20,000 - this includes insulin, cortisol, hCG and drugs such as digoxin, theophylline and gentamycin
what can fluorescent polarisisation immunoassay do?
it can measure drug concentrations in serum accuratelt, quickly and easily
its responsible for success of theraputic drug monitoring
what is the quality of an immunoassay dependant on?
it hinges on the level of affinity and specificity of the antibody for the antigen
in the case of a whole microbe, the antibody must bind to an epitope on its cell surface
