Posts Tagged ‘ antigen ’

Diagnostic Tests Used for Viruses

Haemagglutination Assay

Introduction

A simple and rapid method of quantifying the amount of virus in a sample. Haemagglutination is the agglutination of red blood cells. Viruses with envelops or surface proteins are able to bind to the sialic acid, N-Acetylneuraminic acid found in the cell membrane of red blood cells. Because each agglutinating molecule (in this case each viral molecule) can bind to multiple red blood cells, a clump of cells begins to form, this is agglutination. This agglutination forms a lattice structure.

Haemagglutination Inhibition

Haemagglutination inhibition is the addition of an inhibitor of the virus. Antisera (a blood serum which contains antibodies) is used in this case. The antibodies will bind to the virus and thus prevent haemagglutination.

By creating multiple, increasing levels of dilution e.g. 1:2, 1:4, 1:8, etc. of virus to antiserum we can determine a haemagglutination titre, this is the highest value of dilution (i.e. smallest amount of virus) which inhibits agglutination.

A typical haemagglutination inhibition assay looks like this:

You can see that point of agglutination, where the contents of the well go from a homogenous cloudy red, to containing a red spot which is the agglutinated red blood cells. In the above example, sample E (the highlighted row) has a haemagglutination titre of 1:512 as this is the largest dilution which prevents haemagglutination.

By monitoring the haemagglutination titre, we can determine how the virus is progressing over time, for example:

  • 1st assay – an endpoint is reached at a dilution of 1:4
  • 2nd assay – an endpoint is reached at a dilution of 1:16
  • 3rd assay – an endpoint is reached at a dilution of 1:8

From this would we be able to see initially the virus count is low as it is inhibited at a low dilution, however at the next assay the haemagglutination titre is higher (greater dilution of virus reached before it is inhibited) this must mean the virus is replicating as the initial count of virus is higher (i.e. takes greater amount of dilution to reach inhibition). WIth the final assay, we can see that because the haemagglutination titre has decreased, the virus must be reducing in numbers, indicating recovery.

ELISA

Introduction

ELISA is an acronym for Enzyme-linked immunosorbent assay and is used to detect whether or not a certain antibody or antigen is present in a sample. To indicate the presence of an antibody or antigen, reporter molecules are used which are identifiable by a change in colour. Therefore, if an antigen or antibody is present, a colour change will be observed.

Method

A typical ELISA requires the use of a 96-well microtitre plate (as used in the haemagglutination assay), but the wells of the plate are coated with a known or unknown antigen. When diagnosing viruses, the antigen will be unknown as it is the sample which we are testing. To this an antiserum is added, if the virus antigen is present in the sample, then the antibodies will bind. If not, no binding will occur.

To determine whether or not an antibody-antigen complex has formed (i.e. a positive diagnosis of the virus), an antiglobulin is added. This is an antibody which binds to the antibodies used in the initial serum. For example, Goat anti-rabbit IgG is a rabbit antibody (Immunoglobulin G [IgG]) which will bind to goat antibodies. There are many possible variations of antiglobulins.

The antiglobulin will be ‘labelled’ with an enzyme (an enzyme is attached to the antibody which has a negligible effect on its binding capabilities).

Finally, the substrate of the enzyme using to label the antibody is added. This substrate will be broken down if the secondary antibody (antiglobulin) bound to the primary antibody. The break down of the substrate will be coupled with a colour change, for easy identification that this has occurred. The greater the intensity of the colour change, the higher the concentration of the initial antigen.

Sometimes a spectrophotometer (a device used to detect light intensity) will be used to determine the degree of change in colour, as this can be used to calculate reasonably accurate values for initial antigen concentration and thus virus concentration in the host.

Below is an example of the variations in colour which occur during an ELISA test. The coloured wells indicate positive samples:

This type of ELISA is known as an indirect ELISA test, however there are other forms of this test, such as sandwich ELISA, which is where the well is coated in antibody, to which an antigen binds and to this another antibody is added. The antiglobulin tests for the secondary antibody.

There are also competition ELISA tests, where the added sample is an antibody-antigen complex, this is added to antigen coated wells. If the concentration of initial antigen in the sample is high then there will be fewer available antibodies to bind with the antigen in the wells. The wells are washed to remove unbound antibodies and as with the indirect assay, enzyme-coupled antiglobulins are added with substrate that elicit a colour change. However in this case a high initial concentration of antigen in the sample will yield a low change in colour.

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Adverse Immune Reactions

Below is the introduction and summary to this article, the full article includes information about the types of hypersensitivity (types I-IV) you can view and download it now for free at www.jameswatts.co.uk

Introduction

The immune system has become adapted to ensure that ‘self’ cells are not subject to an immune attack. The body is able to do this because tolerance is developed towards self-cells, should this tolerance be broken down by some means, the host becomes subject to autoimmune attacks which can be potentially damaging.

Autoimmunity

Autoimmunity occurs when the body fails to recognise self-cells from non-self, this results in immune responses and damage to the tissue of the host. The variety of autoimmune responses can be split generally in to two groups; organ specific and non-organ specific. In autoimmune responses it is thought that either over reactive T-helper cells or deficient T suppressor cells are the cause. Autoimmunity can also be induced by reactions to a foreign antigen that then reacts with a self-antigen to invoke a response, for example infection with a minor bacteria (streptococcus) can lead to antibodies being produced against an antigen displayed on heart valves that would lead to cardiac problems. Autoimmunity is diagnosed by autoantibodies and the deliberate induction of autoimmunity has been used to control fertility and tumours (immunotherapy).

Summary

  • Autoimmunity – Inappropriate immune response to self antigens
  • Hypersensitivity – Overactive immune response to foreign and self antigens
  • Immunodeficiency – Ineffective immune response
  • Type I hypersensitivity – (IgE mediated, initiated in 2-30 minutes) Antigen induces cross-linking of IgE bound to mast cells with release of vasoactive mediators.
  • Type II hypersensitivity – (Antibody-mediated cytotoxic, 5-8 hours) Antibody directed against cell-surface antigens mediates cell destruction via ADCC or complement.
  • Type III hypersensitivity – (Immune complex mediated, 2-8 hours) Antigen-Antibody complexes deposited at various sites induces mast cell degranulation, neutrophil degranulation damages tissue.
  • Type IV hypersensitivity – (Delayed cell-mediated, 24-72 hours) Memory TH1 cells release cytokines that recruit and activate macrophages.