ANTIGEN – ANTIBODY INTERACTIONS

BY: RIA FAZULBHOY (MSIWM 031)

Antigen- antibody interactions are used to detect a number of immune diseases, check for humoral immunity and identify biological molecules. There is noncovalent interaction between the epitopes/antigenic determinants of antigens and the variable region (Vh & Vl) domain of antibodies. Noncovalent bonds include ionic bonds, hydrophobic bonds, hydrogen bonds and van der Waals forces.

Noncovalent bonds between antigen and antibody

Different Antigen – Antibody interactions:

1. PRECIPITATION REACTIONS:

Precipitation reactions occur between antibodies (Ab) and soluble antigens (Ag) present in aqueous solution. They bind by noncovalent bonds to give Ag-Ab complexes known as lattices, which are in turn seen as precipitate. The term precipitins is given to antibodies which aggregate with soluble antigens.

The formation of Ag-Ab lettuces depends on the valencies of both antigens and antibodies:

• Antibodies must be bivalent.
• Antigens must be bivalent or polyvalent.

Precipitation reactions in fluids form a precipitation curve:

1. Constant amount of antibodies is taken in a series of test tubes.
2. Soluble antigens are added in an increasing amount to each test tube.
3. Precipitates are formed in each test tube, and this precipitate is then centrifuged in order to form a pellet. Amount of precipitate is measured by the pellet.
4. By plotting the graph of the amount of precipitate against increasing antigen concentration, we get a precipitin curve.
5. Maximum precipitation occurs in the zone of equivalence where ration of antigen : antibody is optimum.
6. If there is an excess of antigens or antibodies, such extensive lattices are not formed and precipitation is not seen.

                                                                       Excess Antibodies   Equivalence   Excess Antigens

    2) AGGLUTINATION REACTIONS

Definition of agglutination states that it is the interaction between antibodies and particulate antigens which results in visible clumping known as agglutination. Antibodies participating in such reactions are called agglutinins. Agglutination reactions gave a principle similar to precipitation reactions (based on cross-linking of polyvalent antigens). Excess of antibodies inhibits agglutination and this effect is known as the prozone effect.

There are 2 types of agglutination reactions:

1.  Active (natural) agglutination
2. Epitopes of the antigen are naturally found on the test particle.
3. Eg. Antigens found on RBCs, bacteria, and fungal cells

• Passive (chemically fixed) agglutination
• Epitopes and soluble antigens do not occur naturally on the surface of the cells or particles
• They need to be chemically fixed onto either RBCs (with the help of tannins/ chromium chloride) or synthetic materials like latex beads and polystyrene.
• The synthetic materials offer more stability, uniformity and consistency.
• Eg: soluble antigens, viral diseases.

            Examples of agglutination reactions

1. Hemagglutination in blood typing

This is done to detect the blood group of patients and carry out proper blood transfusion. In typing for ABO antigens, red blood cells are mixed on a slide with antisera to the A or B blood group antigens. If antigen is present on the cells, they visibly clump due to agglutination taking place.

2. Agglutination inhibition

Agglutination inhibition is used to detect use of illicit drugs and also used in pregnancy tests. It is also used to detect viral infections in patients.

3) RADIOIMMUNOASSAY (RIA)

Radioimmunoassay is a technique used to detect the binding of antigen and antibodies in the given sample. It is based on the principle that there is a competition for binding between radio-labelled antigens and unlabelled antigens when they are in the same vicinity as high affinity antibodies. The antibody does not distinguish between labelled and unlabelled antigens, thus there is competitive binding between the two.

>The radio-labelled antigen is generally labelled with:

• Gamma emitting isotope like I125
• Beta emitting isotope like 3H (tritium)

>The test sample which contains unlabelled antigens is a complex mixture like serum or other body fluids.

How does Radioimmunoassay take place?

1. First the radio-labelled antigen (Ag*) is mixed with the antibodies at a concentration such that the antigens saturate the antigen binding site of the antibody. This concentration of antibodies which should bind to labelled antigens should be anywhere between 50-70%.
2. An increasing amount of unknown test sample of non labelled antigens is added to the mixture.
3. As the amount of non labelled antigens increase and bind to the antibody, the number of radio labelled antigens which bind to the antibody decreases. They compete to bind to the samples.
4. To determine the amount of labelled and non labelled antigens bound to the antibody, the Ag-Ab complex is precipitated to separate from unbound, free antigen.
5. Unbound antigens are separated by various methods like use of formalin killed S.aureus, other antibodies which react with free antigens, use of solid-phase RIAs, etc.
6. The precipitated Ag-Ab complex’s radioactivity is measured with the help of a radiation counter.
7. A standard curve can be obtained in order to plot and determine the amount of antigen present in the test sample.

ELISA

BY: SAI MANOGNA (MSIWM014)

ELISA stands for Enzyme Linked Immunosorbent Antigen assay. ELISA is an ANTIGEN-ANTIBODY response.
In 1971, ELISA was founded. By Peter Perlmann and Eva Engvall at Stockholm university in Sweden. This is a plate-based assay technique used to detect and quantify substances such as peptides, proteins, antibodies and hormones. An antibody conjugated enzyme interacts with a colorless substrate to create a colored substance. This substrate is considered as a chromogenic substrate. A variety of enzymes has been used in ELISA, such as alkaline phosphatase, beta galactosidase and horse-radish peroxidase. Some substrates such as ortho-phenylenediamine dihydrochloride (for peroxidase), p-nitrophenyl phosphate (for alkaline phosphatase), which are hydrolysed by the enzyme referred to above, are used to generate a coloured end product.

In ELISA, an antigen must be immobilised on a solid surface and complicated with an antibody associated with an enzyme. Detection is achieved through an incubation with a substrate to create a measurable product by measuring the conjugated enzymes activity. A highly specific antimicrobial interaction is the most important aspect of the detection strategy.

Principle : Usually an ELISA test is performed in a multi-well plate of 96 or 384 well plates. The multi-well plate provides the stable surface for the antigen to stabilise. The immobilisation of the analyte makes it possible to isolate the antigen from the other components in the sample. This function makes ELISA one of the easiest experiments to conduct simultaneously in the multiple samples.

Types of ELISA :

ELISA assays can be found in various formats, based on the binding interactions between antigen-antibody.

They are :

1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
   
   

• Direct Elisa : the antigen is immobilised and detected on the surface of the multi-well plate with an antigen specific antibody, directly conjugated to HRP or other detection molecules.

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• Indirect ELISA :  With an indirect ELISA, an antibody can be detected or quantitatively determined.
  
  1. An antigen-coated microtiter well is applied to the serum or any other biological sample containing primary antibody (Ab1) and allowed to react with antigen attached to the well.
  2. The presence of an antibody bound to the antigen is identified after washing away any free Ab1 by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody.
  3. Any free Ab2 antibody is then washed away, and an enzyme substrate is added.
  4. Specialized spectrophotometric plate readers calculate the amount of colored reaction product that forms and can measure the absorbance of all the wells of a 96-well plate in seconds.
  4. This method of choice is for detecting the presence of serum antibodies against Human immunodeficiency virus (HIV), the causative agent of AIDS.
  
  

• 
  Reaction combinant envelope and core HIV proteins are adsorbed as solid-phase antigens to microtiter wells in this assay. HIV infected individuals can develop serum antibodies to these viral proteins in the epitopes. HIV serum antibodies will usually be detected by indirect ELISA within 6 weeks of infection.
  

• Sandwich ELISA :  The antigen can be detected or measured using sandwich ELISA
  
  1. In this procedure, the antibody is immobilised on a microtiter well (rather than the antigen).
  2. An antigen containing a sample is added and allowed to react antibody being immobilised.
  3. A second enzyme linked antibody specific to different epitopes on the antigen is added after the well is washed and allowed to react with the bound antigen.
  4. After washing, substrate is added after any free second antibody is removed and the coloured reaction product is detected.
  

• Competitive ELISA : Competitive ELISA is another variation for measuring the amounts of antigen in the sample.
  
  1. In this procedure, antibodies are first incubated in a solution with an antigen-containing sample.
  2. To an antigen-coated microtiter well, the antigen-antibody mixture is then added.
  3. The more antigen present in the sample, the less free antibody will be available for binding to the well coated with antigen
  4. The addition of an enzyme-conjugated secondary antibody (Ab2) unique to the primary antibody isotype can be used to calculate the quantity of primary antibody bound to the well. (as well as in Indirect ELISA).
  5. Nevertheless, in the competitive assay, the higher the concentration antigen, the lower the absorbance i.e present in the original sample.
  
  

Chemiluminescence : During certain chemical reactions, the measurement of light emitted by the chemiluminescence provides a convenient and highly sensitive alternative to absorption measurements in ELISA that use chemiluminescence. For Example, the oxidation of the luminol compound by H2O2 and the horse-radish peroxidase (HRP) enzyme produces light.

Enhanced sensitivity is the value of chemiluminescence assays over chromogenic ones. In general, by switching from a chromogenic to luxogenic substrate and with the addition of enhancing agents, more than 200 times, the detection limit can be increased at least tenfold. In fact as few as 5moles (5 attomoles) of the target antigen have been detected under ideal conditions.

ELISPOT Assay :  ELISPOT Assay is the modification of the ELISA assay.  This assay makes it possible to quantitatively determine the number of cells present in a population that contain antigen-specific antibodies or an antigen for which a specific antibody is present.

1. In this method, the plates are coated with the antigen recognised by the antibody of interest or with the antigen-specific antibody which is being tested for production. These specific antigen and antibody are known as capture molecules.

2. The coated plates are then supplemented with a suspension of the cell population under examination and incubated.

3. The cells settled on the plate surface. The secreted molecules that react with the captured molecules, will create a ring of antigen-antibody complexes around each cell that produces molecules of their interest.

4. The plate is then washed, added and allowed to bind an enzyme-linked antibody specific to the secreted antigen or specific to the species ( eg; goat anti-rabbit) of the secreted antibody.

5. Subsequent production of the assay shows the location of the each antigen or antibody producing cell as a point of colour or light by introducing an appropriate chromogenic or chemiluminescence-producing substrate.

Advantages and Disadvantages :

ELISA	Advantages	Disadvantages
Direct ELISA	– Saves time and reagents (Short protocol) – No cross reactivity from secondary antibodies	– In sample all proteins bind to the surface – Primary antibody must be labelled individually. – No signal amplification – Low flexibility
Indirect ELISA	– Has signal amplification – High flexibility	– Long protocol compared to direct ELISA – Potential cross reactivity from secondary antibody.
Sandwich ELISA	– High specificity – Suitable for complex samples. – High flexibility and sensitivity	– Finding two antibodies to the same target that identify different epitopes and function well together even at difficult times.
Competitive ELISA	– Suitable for small antigens.	– Depends on base ELISA selected.