Friday, January 23, 2009
Mechanisms of virus neutralization by Antibodies(Ab)
b) Ab inhibition of the interactions between the viral envelope protein and the cell-surface receptors is shown to occur after the virion has attached by binding via the receptors. Ab interference with any of these necessary links in a chain of events that lead to entry would constitute a neutralization mechanism.
c) Abs’ block of requisite interactions between viral and cell-membrane proteins would delay or prevent the penetration of the viral core into the target-cell cytoplasm. The virion may thus ultimately be destroyed through lysosomal degradation. In addition, Ab-mediated derouting of viruses that preferentially enter directly via the cell surface to a less permissive endosomal compartment may abrogate infectivity.
d) The intercalation of Ab in the fusion interface between the cell membrane and the envelope of a virus may block fusion at the cell surface, as illustrated, or in an endosome.
e) It has been conjectured that even a very low occupancy of antibody on the virion can cause global or internal changes by transmitting a signal across the viral envelope or outer layer. These hypothetical changes would allow the viral core to enter the cytoplasm but compromise further replicative steps
f) The neutralization of naked viruses could potentially differ from that of enveloped viruses.
Blogged @ 8:38 PM
Saturday, January 17, 2009
Direct electron microscopic particle count
Direct particle count
An electron micrograph of a spray droplet containing 15 latex beads (spheres) and 14 vaccinia virus particles (slightly smaller, brick-shaped particles).
(From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.8)
Blogged @ 8:45 PM
Monday, January 12, 2009
Haemagglutination Inhibition (HI)
Mechanism: The antigen in HI tests is simply a solution of the antigenic particles which is capable of inducing the reaction of haemagglutination when mixed with a suspension of red blood cells. The presence and concentration of antibody is measured by its ability to inhibit the agglutination at various dilutions.
Reagents:
The antigen is usually prepared by growing a naturally haemagglutinating virus in chick embryo and collecting allantoic fluid.
The only other reagent required for carrying out this test is a suspension of red blood cells. Most HI tests carried out in routine poultry serology use chicken erythrocytes.
Methods:
It is possible to carry out rapid haemagglutination tests and haemagglutination-inhibition tests on a plate, just as for the bacterial agglutination tests described above.
However HA and HI are generally only used in this way to confirm the presence and identity of a haemagglutinating antigen.
Identification and quantification of HI antibody, on the other hand, is nearly always carried out by the equivalent of the slow agglutination test, originally in tubes, now almost always in micro-titre plates.
Picture above-Haemagglutination-inhibition tests in a micro-titre plate. Wells with a non-agglutinated button of red cells are read as HI positive.
Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours.
Wells in the bottom row contain no virus.
Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity.
(From Fields Vriology (2007) 5th edition, Knipe,DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)
Blogged @ 8:26 PM
Thursday, January 8, 2009
Antibody detection
Western blot
Western blot analysis of HIV antigens and antibody. HIV protein antigens are separated by electrophoresis and blotted onto nitrocellulose paper strips. The strip is incubated with patient antibody, washed to remove the unbound antibody, and then
reacted with enzyme-conjugated antihuman antibody and chromophoric substrate. Serum from an HIV-infected person binds and identifies the major antigenic proteins of HIV. This data demonstrates the seroconversion of one HIV-infected individual with sera collected on day 0 (D0) to day 30 (D30) compared to a known positive control (PC) and negative control (NC).
(From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-7. )
Diagnostic methods for common human viruses
Blogged @ 9:16 PM
Tuesday, January 6, 2009
ELISA
ELISA - Enzyme-Linked ImmunoSorbent Assay
What is ELISA???
ELISA is a technique to detect the presence or a particular antigen and antibody in samples. It is commonly use in labs to determine diseases in humans, animals and plants.
The basic principle of ELISA is to use an enzyme to detect the binding of antigen and antibodies. The enzyme convert the colourless nsubstrate to a coloured product indication the antigen antibodies binding.
The ELISA experiment can be designed in many way to see if you are detecting antibodies or antigen. It is also one of the easier way to determine the presence of a particular antigen or antibodies.
What are the advantages of ELISA?
ELISA tests are generally relatively accurate tests. They are considered highly sensitive and specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus).
ELISA EXPERIMENT Materials
ELISA is a technique to detect the presence or a particular antigen and antibody in samples. It is commonly use in labs to determine diseases in humans, animals and plants.
The basic principle of ELISA is to use an enzyme to detect the binding of antigen and antibodies. The enzyme convert the colourless nsubstrate to a coloured product indication the antigen antibodies binding.
The ELISA experiment can be designed in many way to see if you are detecting antibodies or antigen. It is also one of the easier way to determine the presence of a particular antigen or antibodies.
What are the advantages of ELISA?
ELISA tests are generally relatively accurate tests. They are considered highly sensitive and specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus).
ELISA EXPERIMENT Materials
- Distilled water
- 37 C incubator
- Measuring Cylinder
- Micropippette
- Microplate reader
- Conjugate Solution
- Diluent buffer
- Coating Solution (10mM PBS pH 7.2)
- Blocking Solution (gelatin)
- Primary/Secondary AntibodySolution
- Wash Solution (PBS)
Blogged @ 6:24 AM
Saturday, December 27, 2008
Plaque Assay
Viruses, like other microorganisms are too small to be enumerated by direct counting. Plaque assay can be used to help in counting the viruses.
The theory of the plaque assay is very similar to the theory behind bacterial colony counting. This method requires that the virus infects a cell line that grows as a monolayer. 1 virus will infect one cell if the concentration of virus is low enough. The viral concentration can be determined by the following formula:
Virus concentration = (number of plaques) x (dilution factor(s)) pfu/ml
Plaque Assay: method
Typical Steps to conduct a Plaque Assay
Materials
- 45ml of viral diluent
- 1 vial of 0.5ml kunjin virus suspension
- Sterile test tubes
- Sterile pipette
- 2 6-well trays of confluent BHK-21 cell
- 96ml of viral diluent
- 60ml of 2times concentration maintaince medium
- 60ml of 2% CMC aquacide II Solution in ultrapure water
- Beaker for discard
Procedures
Blogged @ 8:20 PM
Tuesday, December 23, 2008
Viral Amplification
What is Viral Amplification???
Viral amplification is used to infect the cells and wait for multiple rounds of infection to grow the virus in large quantities. It is a useful method to get satisfying concentration of virus and to conduct further studies on the virus amplified.
The virus is first cultivated in cells in a small flask, the medium is then used to infect a larger flask to produces higher titres. To amplify the virus, the virus needed to be cultivated in a continuous cell line rather than primary cell line because primary cell line have a limited life span and needed to be regenerated every time. The multiplicity of infection (MOI), the ratio of infectious virus to infection cells must be at least 10 to get a better yield of virus.
The virus is first cultivated in cells in a small flask, the medium is then used to infect a larger flask to produces higher titres. To amplify the virus, the virus needed to be cultivated in a continuous cell line rather than primary cell line because primary cell line have a limited life span and needed to be regenerated every time. The multiplicity of infection (MOI), the ratio of infectious virus to infection cells must be at least 10 to get a better yield of virus.
Typical Steps to conduct Viral Amplication
Results
After viral amplification, the infected Vero cells which are high in titre do not divide but increasing in size and appear granular under microscope. However, the infected Vero cells which do not undergo viral amplification are low in titre, hence they continue to grow to confluency
Blogged @ 6:54 AM