Immunology Pracs Flashcards
AA. What are the two viral proteins inserted in the virus envelope called and what do they do?
The capsid of a virus is surrounded by a host cell-derived lipid membrane envelope. Two viral proteins critical for VIRAL ENTRY and EXIT from susceptible cells are inserted into the envelope - the HAEMAGGLUTININ (H) and NEURAMINIDASE (N).
These proteins also largely determine the antigenic structure of influenza virus.
AB. What are the molecular methods for the detection of influenza virus?
Molecular methods - PCR, quantitative real time PCR and nucleotide sequencing.
Although serological methods are also commonly used, they usually only provide a retrospective picture of an infection as it takes time for the body to mount an immune response.
AC. What is PCR and what makes it useful?
Polymerase Chain Reaction
Able to exponentially increase small quantities of DNA to detectable levels by repetitive synthesis (amplification) by the action of a thermostable DNA polymerase (Taq).
This method is capable of detecting the presence of v small amounts of specific viral genomes.
AD. What are the 4 major disadvantages that conventional polyclonal antisera raised against both simple & complex antigens have that restrict usefulness as analytical & therapeutic agents?
- Ab’s produced are HETEROGENEOUS. Even when the antigen consists of a single molecular species it may contain multiple epitopes, each of which is capable of eliciting a range of Ab’s with different binding specificities.
- The SUPPLY is limited.
- Using animals to produce Ab’s should be limited for ETHICAL reasons.
- The combo of Ab’s produced by an individual animal is largely unique and cannot be exactly REPRODUCED in another animal.
AE. How is the problem of HETEROGENEITY solved in using conventional polyclonal antisera raised against both simple & complex antigens?
What is a problem that arises in turn from this solution and how is it resolved?
Using Ab derived from a single Ab-forming B Cell. This Ab has a singe specificity. Isolation and cultivation of a single Ab-forming B cell would give rise to a population of homogenous Abs.
Such cells have a v short life span when cultured in vitro, but the technique of cell fusion aka ‘somatic cell hybridisation’ overcomes this and ‘immortalises’ the B cell that produces a specific Ab.
The normal Ab-producing BC is fused with myeloma cells. Fused cells that secrete Ab of the desired specificity are then selected and are called ‘hyridomas’. They produce MONOCLONAL ANTIBODIES (mAbs).
AF. What are some of the common applications of mAbs (monoclonal antibodies) in immunology? (4)
- IDENTIFICATION of phenotypic markers unique to particular cell types.
- IMMUNODIAGNOSIS, detecting specific Ag’s/Ab’s in circulation/tissues using mAbs is helpful in diagnosis of many disease.
- Tumour Diagnosis & THERAPY. Tumour specific mAbs are used to detect & treat tumours.
- FUNCTIONAL ANALYSIS of cell surface & secreted molecules, e.g. mAbs that neutralise cytokines are used to find their functions in vitro & in vivo.
AG. What is theoretically the greatest advantage of a hybridoma derived mAbs?
a) they solve the problem of heterogeneity
b) you have an endless supply of mAb of identical antigen specificity of known isotype & binding avidity.
c) They avoid the ethical problems arising from sourcing Abs from animals frequently
d) none of the above
b) best answers the question: you have an endless supply of mAb of identical antigen specificity of known isotype & binding avidity.
a) They are the solution to this problem but not their greatest advantage.
c) this is helpful yes and avoids a disadvantage of using polyclonal antisera but is indirectly related to (b).
AH. Outline how monoclonal antibodies are produced and cultured in vitro.
Immune cells e.g. from spleen are isolated from specimen (e.g. from mouse) immunised with antigen X (B cells, some producing anti-X Ab). These are fused with a mutant immortal myeloma line. All cells are cultured in a selection medium, but only the fused cells (hybridomas) grow (unfused myeloma & spleen cells don’t survive). Isolated clones are derived from single cells. The supernatants of each clone are screened for anti-X Ab and positive clones are expanded.
This leaves us with hybridomas producing monoclonal anti-X antibody.
(see page 3 of U4P11)
AI. Define Humoral
Relating to the body fluids, especially with regard to immune responses involving antibodies in body fluids as distinct from cells.
Humoral responses to viral infections are of major importance in diagnostic virology. The titre (level) of specific Abs in serum can be determined using known viral antigens and conclusions can thus be drawn regarding the past contact of the patient w particular viruses.
AJ. What does the presence of IgM Abs to a specific virus in serum generally indicate?
a) A healthy immune system able to neutralise an infection if it occurred
b) nothing
c) IgG Abs are usually the indicative factor of a virus
d) Recent contact with that virus
d) The presence of IgM Abs to a specific virus in serum generally indicates recent contact with that virus, which can be of considerable significance.
(c) and (b) are false; (a) doesn’t answer the question regarding viruses
AK. Which of the following is not a serological test frequently employed in virology laboratories?
a) ELISA (enzyme-linked immunosorbent assay)
b) Haemagglutination Inhibition (HAI)
c) PCR
d) Virus Neutralisation
e) Western Blot
f) Immunofluorescence
g) All of the above are frequently employed
c) PCR is not a serological method
AL. How might viruses that attach non-specifically to receptors on RBCs be detected serologically?
Viral Haemagglutination - surface attachment protein on viruses, ‘haemagglutinin’ allows for viruses to bind to RBCs.
These RBCs will clump together and become visibly agglutinated, as opposed to normal unbound RBCs which will pellet at the bottom of a well due to gravity.
Haemagglutination titrations are commonly employed to measure the relative amount of free haemagglutinating virus present in a cell culture fluid.
AM. Which one of the following statements BEST describes the difference between cell lines & primary cells?
A. primary cells are always cancer cells, whereas cell lines are not
B. primary cells can be passaged a finite number of times, whereas cell lines can be passaged indefinitely
C. primary cells have been “immortalised”, whereas cell lines have not
D. primary cells grow as a monolayer, whereas cell lines cannot.
B. primary cells can be passaged a finite number of times, whereas cell lines can be passaged indefinitely
Cancer/normal cells which have been “transformed” by chemicals/viruses can be subcultured indefinitely and are said to have been “immortalised”. Once immortalised, these cells are called CELL LINES.
AN. Some lines have been derived from cells such as lymphocytes. These have been successfully utilised to culture…
a) cytomegalovirus
b) influenza viruses
c) lymphotropic viruses such as the human immunodeficiency virus (HIV)
d) all virus types
c) lymphotropic viruses such as the human immunodeficiency virus (HIV)
Re. Cytomegalovirus, these can be grown in HF (Human Foreskin) Fibroblast Cell Culture. The CPE observed: cells become enlarged; focal rounding & loss of cells.
AO. Which of the following viruses exhibits no distinctive CPE?
a) Polio virus
b) Adeno virus
c) Cytomegalovirus
d) Mumps virus
d) Mumps virus
Polio - Diffuse cytopathic effect; shrinking, rounding & loss of cells.
Adeno virus - Cells form grape-like clusters
Cytomegalovirus - Cells become enlarged (hence the name Cytomegalo). Focal rounding & loss of cells. Human CMV replicates only in diploid Human cells.
