practical MCQ - ELISA Flashcards
what is the practical about
detection of IgE and/or IgG against major Aspergillus fumigatus antigen Asp f1 in human serum using indirect binding and sandwich ELISA.
what is aspergillus
it is a fungus that is massively due to the degradation of plant material and waste and etc.
what does aspergillus cause
it causes opportunistic infections and infections in humans, predominantly in the lungs or other parts of the body like skin lesions in the eyes, nose, etc, depending on where they affect they show different signs and symptoms
how many aspergillus species are there and where do aspergillus species belong
Aspergillus is a genus of around 200 fungi, 18 of which are human pathogens.
Aspergillus species belong to the Ascomycota phylum
where is aspergillus found
Aspergillus are filamentous fungi found worldwide, whose spores are ubiquitously present in the air we breathe.
Aspergillus is commonly found in soil, food and air vents
in hospitals, it is found in the air, showerheads, hospital water storage tanks and potted plants.
what does aspergillus species have a role in
they have a significant role in the degradation of plant material as in composting.
what is the entire life cycle of aspergillus
1 - aspergillus releases spores into the environment
2 - people inhale spores
3 - either:
> nothing happens and you breathe them out again and thus your body gets rid of them
> or the spores can germinate, only when the spores germinate do you get an immune response
4 - when they germinate, they’ll have hyphal elongation, which is mould-like filaments coming out - this is like fingers, it spreads, looks for food and degrades material.
in aspergillus, it has dichotomous branching, which forks and separates very distinctly and you can look at it under a microscope.
5 - then the mass of hyphae occurs, which is mould in the body
it will spread into your tissue and alveoli or any other tissue depending on its location.
6 - then it will spread spores again
7 - then the cycle repeats
what happens when a healthy person inhales spores
the spores will go to the lungs, the spores will be attacked by antibodies.
you also get a mucosal cascade - mucus will push stuff out of your lungs.
this will work and get rid of the spores and you won’t get an infection.
what happens when an immunocompromised person (ICH) inhales spores
the spores will go into the lungs and germinate and this can cause an infection.
this may cause invasive aspergillosis. this is where hyphae spread across the lungs and then you get extensive fungal pathogen attack of your lungs and this causes a lot of damage.
what happens when aspergillus causes cavitary lung disease or chronic lung disease
aspergillus causes cavitary lung disease, this is where you already had TB or other diseases which damaged your lungs thus you already have pockets/holes in your lungs.
the fungus goes into your lungs and finds those holes and then it grows and creates aspergilloma which is a hard ball of fungi.
aspergillus causes chronic lung disease, which can result in CPA ( chronic pulmonary aspergillosis) or CNPA (chronic necrotising aspergillosis). this is where the tissue degrades.
what happens to a person with asthma that inhale spores
people with asthma have a hyper-responsive immune response, when they inhale spores, the body will react, which is called allergic bronchopulmonary aspergillosis (ABPA), or serve asthma with fungal sensitisation (SAFS).
this is where just breathing in spores, can cause an immune response which is an overreaction of the body.
the practical we are doing is about people who potentially have SAFS or ABPA
what drugs are used to treat fungus infection
fungi’s genetic makeup is very similar to humans, which means it’s really hard to kill fungi in our body because most drugs that are used to kill fungi could potentially kill us.
fungi have ergosterol, which is very similar to cholesterol and performs the same functions as cholesterol but their structures are a bit different, so a lot of drugs that are anti-fungal usually target ergosterol so they can kill fungi.
because ergosterol is very similar to cholesterol, the drugs need to be given in very low doses and over a long-term treatment plan.
how is covid associated with aspergillosis
covid has brought about a new version of aspergillosis.
Covid-19 associated invasive pulmonary aspergillosis (CAPA)
what 5 clinical manifestations are caused by aspergillosis
- Allergic aspergillosis
- Aspergilloma ( ball of fungus)
- Broncho-pulmonary aspergillosis ( lungs are affected)
- Disseminated, invasive aspergillosis ( spreads across the body)
- Toxicosis (fungus releases toxins and you get a toxic response)
what people get aspergillosis the most
aspergillosis occurs most in immunodeficient people, they usually get acute invasive aspergillosis
what happens when people with immune hyper-reactivity inhale spores
people with immune hyperreactivity are sensitive to spores and their responses to spores.
so, people who have asthma breathe in spores, they start to get difficulty in breathing and vaso-restriction of blood vessels that are associated with the lungs.
the more sensitive their body is to the spores, the more of a reaction they get.
what is allergic aspergillosis
it is an allergy to conidia (spores) of aspergilli - the major allergen of aspergillosis is Asp F1
aspergilli are the cause of asthma in 10-15% of patients
the ungerminated conidia in the bronchi cause the stimulation of IgE - this can lead to vaso-constriction
What is ABPA and how is it diagnosed
ABPA (Allergic bronchopulmonary aspergillosis) is an allergic disorder induced by aspergillus fumigatus.
it is diagnosed by the following tests:
- positive immediate skin reactivity
- elevated Afu-specific IgG and IgE levels in serum
- peripheral and pulmonary eosinophilia
what is ELISA and what is it used for
ELISA - enzyme-linked immunosorbent assay
ELISA is a test used to detect an antigen/antibody interaction.
the ELISA test involves an enzymatic reaction for the
detection of the interaction that is happening between an antigen and an antibody
and the quantification
ELISA is used as a diagnostic tool to detect the presence of specific antibodies or antigens in a sample
what is indirect ELISA used for and how is it carried out
Indirect ELISA is used to measure the presence of specific antibodies in a sample.
Indirect ELISA method:
1- you have a 96-well plate (this is to support the enzymatic reaction), we bind the Asp F1 peptide antigen ( this is signalling aspergillus) to the well plate
2- we then add the patient’s serum, purify the antibodies from it and we put it on the plate.
if the patient has antibodies against the Asp F1 peptide then it will bind to the antigen. but we can’t see this binding.
3- then we add a secondary antibody, which is a generic antibody that binds to a human antibody.
secondary antibodies are usually raised in other animals, they are usually given the antibodies.
when the antibodies are created they have an enzyme attached to them which is HRP (horse radish peroxidase).
the secondary antibody will only bind if the patient’s serum is there, and the patient’s serum will only bind if the Asp F1 is there.
you wash between each step, so nothing else can bind to the wells thus there is less non-specific binding.
4- the secondary antibody will only bind if the whole chain is there. you then add a substrate called ODP and the HRP changes the colour and produces an orangish colour.
we can measure how much antibody binding there is by the colour change that occurs.
in the practical what is measured in the 96-well and what does it show
the 96-well measures the presence of specific IgG and IgE antibodies in sera from 3 patients.
the highest concentration of Asp F1 peptide bound to antigen is at the top of the well. then we do 1:2 serial dilution down the well. so you get lower levels of antibodies present as you go down the plate. the last well (H) is empty - this is the negative control.
this serial dilution is done so we can detect the threshold of linear binding, so we can see at what point it saturated and calculate the number of antibodies present.
why is sandwich ELISA carried out in this practical and why is it better than indirect ELISA
Sandwich ELISA is used to measure the amount of antigen (Asp F1 peptide) between 2 layers of antibodies.
sandwich ELISA is more specific than indirect ELISA.
in indirect ELISA, non-specific binding can occur due to cross-reactivity between the layers and different antibodies.
in sandwich ELISA, we trap the antigen between the samples, this prevents non-specific binding.
what is the method of sandwich ELISA
1- we have 96-support wells
we bind human patients’ IgG against the wells.
2- we then add the Asp F1 peptide
they will bind if there are any antibodies against that peptide
3- we then add BSA, a generic protein, as a control
4- we will do serial dilution as you go down the well to quantify how good this reaction is
5- we then want to detect this, so we add antibodies that have HRP-conjugated IgE against the Asp F1 peptide
6- we get antibodies against antigens specifically and detect the colourimetric change and thus we can detect who’s got what (of the 3 patients)
Explain the rationale of the design of indirect and sandwich ELISA
Indirect ELISA
it is a 2-step ELISA which involves 2 binding processes of primary antibody and labelled secondary antibody
the primary antibody is incubated with the antigen.
however, this may lead to a non-specific signal because of the cross-reaction that the secondary may bring about.
Sandwich ELISA
it quantifies antigens between 2 layers of antibodies ( e.g. capture and detect antibodies). the antigen is only measured if it contains at least 2 antigenic epitopes capable of binding to the antibody since at least 2 antibodies act in a sandwich
what are the advantages of sandwich ELISA over direct binding ELISA
Sandwich ELISA is highly specific and suitable for highly complex samples. Direct ELISA has a much lower sensitivity as the signal is less amplified.
in direct ELISA the primary antibody must also be labelled which is a very costly and time-consuming process as you need a new one for each antigen.
what are the advantages of using a peptide marker instead of a full-length antigen in immunodiagnosis
it’s a cost efficient process
it can use a specific region as some antigens may have conserved regions
what are the other diagnostic methods to confirm allergic bronchopulmonary aspergillosis and invasive aspergillosis
-Direct observation
- allergic response test/ Scratching test
- A lot of people have asthma
- test IgE and IgG levels in serum
- take images of the lungs
- see which antibodies are precipitating
what are the pathological contributions of AspF1 of aspergillus fumigatus
Asp F1 produces ribo toxins
Aspergillus produces spores which can irritate immunocompromised individuals
how do the specificity and complementarity of immunodiagnosis compare with PCR-based diagnostic approaches in medical microbiology
PCR-based approaches are possible to detect A.fumigatus DNA but it requires extreme care to avoid false positive or negative results.
Conidia is usually present in the air and can induce false positive results by its transient presence in the respiratory tract. therefore it has higher false positives than ELISA tests.
however, combining PCR and ELISA can give reliable results even in the absence of symptoms
discuss how sometimes it is necessary to combine conventional microscopic identification with antigen detection
sometimes a combination of methods gives a definitive diagnosis.
conventional microscopy makes it possible to visualise fungal elements in very abundant samples,
to check against false positives from PCR-based approaches and inform on the use of immunodiagnostic approaches.
explain briefly how the sensitivity of these ELISA assays can be improved
Sensitivity can be improved by the implementation of new analytical platforms to
increase absorbance readings
and reduce background noise
what are the limitations of immunodiagnostics tests
they have the disadvantage of being reagent dependent, so you need to have well-characterised antigens available to get a clear diagnosis. thus a catalogue of immunological reagents is needed when testing samples
when false positives result occurs the test has to be repeated or combined with other diagnostic tests.
