Basic immunochemical methods. Immunoturbidimetry, ELISA, RIA.

Antigen refers to molecules of natural or synthetic origin. They can be proteins, polysaccharides, nucleoproteins. The methods explained here focus on interaction between antibodies and epitopes of antigens. Epitope is a part of the antigen that serves as binding for an antibody or lymphocyte receptor. A complete antigen should be differentiated from a Hapten. Hapten also known as incomplete antigen and doesn't posses the ability to elicit the production of antibodies like a complete antigen would be able to.

Immunochemical methods

Refers to a group of methods that employs the function of antibody where they form complexes with antigens.

They include methods such as :

• Precipitation methods in Gel : Single immunodiffusion, Double immunodiffusion, Immunoelectrophoresis/Immunofixation.
• Precipitation methods in Solution : Immunoturbidimetry and Immunonephelometry
• Agglutination : Direct or indirect, Agglutination inhibition test
• Enzyme immunoassay : Competitive and non competitive

Precipitation reactions

In Gel (agar gel) : Immunodiffusion

• The purpose of this type of method: to qualitatively and quantitatively analyze the presence of antigens or antibodies in patient sera based on the principles of diffusion of the component though the gel

Single immunodiffusion

• Single immunodiffusion : One component (Antibody or antigen) diffuses while the other component is evenly spread out on the gel
  • A variant of this : Radial immunodiffusion or Mancini method
    • Protocol
      • Where the sample is placed in circular wells through out the agar gel with antibodies that is evenly distributed on the gel
      • An important parameter : the diameter of precipitate ring is proportional the amount of the applied component (from sample)
      • Applications : Immunology, when trying to determine immunoglobulins in sera.
    • This test is replaced by more sensitive methods : nephelometry and ELISA

Double immunodiffusion :

• is similar to the aforementioned technique however it differs in one particular aspect, both antigens and antibodies diffuse.
• When the technique uses unidimensional, following the Oakley - Fulthrope procedure :
  • A layer of antibodies is sandwiched between two layers of agar on the top most layer you would have the application of the antigens. The principle of this test relies on the knowledge that they would diffuse during incubation through the layer of agar in between.
  • The reaction of precipitation would be seen in this middle layer of agar as band at the optimum concentration.
• When, two dimensional: Ouchterlony's double diffusion
  • A sample containing antigens would be applied in certain wells on a plate of agar gel and antibodies would be placed in other wells.
  • A positive test would be indicate on the presence on a line precipitation relying on the diffusion of both components out of their wells.
  • The line of precipitation can be interpreted in 3 ways :
    • Full identity : would be when the line of precipitation would take the shape of an arc which indicates that a common epitope of antigens are present in the sample
    • Non identity : when there are two lines that are formed are they intersect in the gel between in the wells which can be interpreted that the antigens in the sample do not share an epitope
    • Partial identity : you have two lines that are formed due to precipitation however they may represent the shape of gamma with a spur, the samples contain antigens with common epitope and additional epitopes.

Immunoelectrophoresis

Defined as method uses the principle of precipitation under an electric field. A hybrid method that combines both immunodiffusion and electrophoresis.

Protocol:

• The test can be divided into two steps, the first involves electrophoresis of the sample and antigens, which separates them according to charges/sizes in electric field
• In the second part of the test, antibodies applied to grooves which found parallel to the zone of migration in electrophoresis.
• The antigens that were seperated and the antibodies applied diffuse towards each other and becomes visible with a line of precipitation.
• Interpretation : antibody - antigen interaction with the line of precipitation and absence of which indicates non identity. The different antigens can be identified based on the intensity, shape and position of the precipitation lines.

Application :

• detection and typing of the antibodies found in samples obtained
• most typical samples : urine and CSF
• Clinical conditions this would be method of choice for : Monoclonal gammopathies, Multiple myeloma, Waldenstrom's macroglobulinemia.

Precipitation methods in solution: Immunoturbidimetry and Immunonephelometry

• a quantitive way to analyze antigen concentration found in samples
• Sample from patient (potentially containing target antigen) is applied to corvette which followed by the antibodies that target a particular epitope/antigen is added to determine its presence in the patient sample.
• Light passed through the sample is measured using a conventional spectrophotometer.
• In turbidemtery the intensity of the light that passes through the sample is measured. This based on the principle that the precipitates formed would obstruct light that reached through the wells, therefore allows a quantitative analysis.
• In nephelometry, the scatter of light is examined which analyses by a nephleomter which is specifically used to measure the scatter of light.
• The results are then compared to a calibration curve of know concentrations of the antigen.
• Errors may arise when in the range of antibody excess thus to identify the absorbance values obtained is in antigen/antibidy excess, it is recommended that measurements be repeated with dilutions of the sample.

Application

• Clinical uses: levels of CRP, Albumin and Immunoglobulins
• Rapid and simple
• Cost effective

Agglutination

Agglutination when simplified can be understood as a technique which specific antibodies bind to an antigen in the presence of , specifically where bivalent and polyvalent antibodies interact with antigens with multiple epitopes. Cross linking and the formation of the lattices would depend on polyvalent or bivalent nature of the antibodies. However, not all reactions result in agglutination, when such case arises the addition of the anti-Ig antibodies would be applied to achieve agglutination.

A variant : Hemagglutination

• in this variant would focus on the use of the red blood cells as particles that carry the antigen

Practically:

• They are considered to be more sensitive than immunoprecipitation reactions
• This group of techniques can be divided into direct and indirect methods

Direct

• Agglutination between antigen which bound usually to a cell surface on cells such as RBC (patient sample) and their respective antibodies
• Examples :
  • Bed side blood group identification tests,
  • quantitative test would involve examination of antibodies, particularly a titre of the antibodies (the highest dilution of sample in which agglutination would occur).
  • Detection of specific antibodies directed against antigens found on the surface of microbes : (Salmonella typhi : Widal test)

Indirect

• The test here would use antigens to detect the presence of antibodies in sample. Antigen would be carried by Red blood cells and other inert materials such as latex, colloidal gold and other substances
• Beware the use of tannic acid or chromium chloride would rewired for antigens bacterial or viral that don't adsorb easily.
• Application:
  • detection of autoantibodies such as rheumatoid factor.
  • reverse agglutination : to identify the presence of a soluble antigens (CRP, hCG)

Agglutination inhibition

• The mechanism used in the type of test is the competition of antigen present on particle surface and in solution. So antibodies are incubated with antigens present in sample and then followed by the addition of particles coated with antigens. A positive test would result in the lack of agglutination whereas a negative result would characterized by agglutination with the addition of the corpuscular antigen as the sample incubated prior to addition of corpuscular antigen lacked the particular antigen in question.

Enzyme immunoassay

The concept of enzyme immunoassay is the utilization of antigens/antibodies that is labeled in with enzymes in the test. An improvement in sensitivity is associated in this type of method as opposed to immunodiffusion. Both antibodies and antigens are often labelled with enzymes to increase the ease of detection.

Typically enzymes used:

• peroxidase
• alkaline phosphatase

Heterogenous vs Homogenous

You can have heterogenous enzyme immunoassays which are immunoassay where there is separation of free and bound components particularly to different free and bound labeled components. An example of which would ELISA. On the other hand, In homogenous immunoassays, such separation wouldn't occur. An example of which would be enzyme multiplied immunoassay technique (EMIT). EMIT have been used for analysis of hormones, drugs and certain metabolites. EMIT uses competition between labeled and unlabeled antigens (from patient sample) in a mixture with limited antibodies. Thus when the enzyme linked antigen binds to the antibody, there's decrease in enzyme activity which is typically seen when there is more conjugated antigens as opposed to unlabeled antigens.

Competitive enzyme immunoassay

• A prerequisite condition, the condition in which this test occurs in antigen excess
• The test uses the concept of competition between conjugated and unconjugated antigen for limited number antibodies. Thus when more conjugated antigen is present less unconjugated antibodies would be bound. This method is divided into two parts into a phase of incubation followed by a reaction to detect enzyme activity. Typically this reaction involves a color change due to the nature of the substrate that helps us which visualize the results of the test. The results obtained are used with help of a calibration curve.
• Application : when the test involves only one epitope such as test for hormones, r certain drugs

Non competitive enzyme immunoassay

In this type of method, the antigen is sandwiched between two antibodies. At first you have antibodies bound to solid phase subsequent to which the sample obtained would be added. In this method, there is antibody excess which means, that after a period of incubation, a second enzyme linked antibody against that particular antigen is applied to is added to the mixture/immunocomplexes formed earlier. This addition if antibodies is done so in excess. This step followed by a step involving rinsing of the excess second antibodies that remains free. After this which step, all that remain is sandwiched immunocomplexes. The results of the test is visualized by measuring the colour visible after the addition of chromogenic substrate. In this, the intensity here is proportional the amount of he antigen present in the sample.

• Application :
  • Microbiology : viruses and parasites : Detection of serum antibodies to
  • Immunology/Allergology: IgE antibodies in response to a particular antigen

Radioimmunoassay

This type of method uses radioisotopes to facilitate the determination of antigens and antibodies. This method is a culmination of three different principles : Ag-Ab binding, a competitive inhibition and measurement of radio emission.

This method is considered to highly sensitive and specific because of the competitive inhibition and measurement of radio emission. More specifically :

• The specificity is brought into this method due to competitive inhibition of the antigens
• Whereas, the sensitivity is conferred by the measurement of the radio emission.

Classical RIA

This method utilizes competitive binding, radio labelled antigens are applied to a sample containing unlabelled antigen and antibodies. After incubation, the amount of the radio-labeled that remains unbound usually reflects the amount of target antigen might be present in the sample.

Protocol :

(This an extremely simplified explanation: for more details -> https://microbenotes.com/radioimmunoassay-principle-uses-and-limitations/)

• Obtain patient sample
• Prepare radioactive labeled antigens and antibodies
• incubate the mixture after the addition of patient sample to the former two.
• Due to competitive inhibition you would see radioactive labeled antigens being displaced
• There is separation of unbound antigens from the sample and radio emissions measured.

Applications:

• Detection of peptide hormones
• Detect different viral antigens
• Detect of surface Hepatitis B antigen
• Detection of mycotoxins
• Detection in the early stages of cancer.