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.
Direct particle count
(From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.8)
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)
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
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)
