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Interactions between antigen and antibody

Interactions between antigen and antibody

Interaction between antigen and antibody is a bimolecular association and it does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an antigen and antibody involves various non-covalent interactions between the antigenic determinant (epitope) of the antigen and the variable-region (VH/VL) domain of the antibody molecule. The specific association of antigens and antibodies is dependent on hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Walls interactions, which are all weak and non-covalent in nature.  So a large number of such weak interactions are required to form a strong antigen-antibody (Ag-Ab) interaction. These interactions can only take place if the antigen and antibody molecules are close enough for some of the individual atoms to fit into complementary recesses. The complementary regions of an antibody are its two antigen binding sites.  Like antibodies, antigens can be multivalent, either through multiple copies of the same epitope, or through the presence of multiple epitopes that are recognized by multiple antibodies. Interactions involving multivalency can produce more stabilized complexes.

Properties of antigen-antibody interaction

Affinity

A strong antigen-antibody interaction depends on a very close fit between antigen and antibody. The combined strength of the non-covalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope. Affinity is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody. There exists a weak association between low-affinity antibodies and antigen, which dissociates easily whereas high-affinity antibodies bind antigen more tightly and remain bound longer.  
The binding of an antibody (Ab) to its antigen (Ag) is reversible, so the binding reaction can be expressed as:
Ag  +  Ab  ↔  Ag-Ab ………………..(1)
Where k1 is the forward rate constant and k-1 is the reverse rate constant. The ratio k1/k-1 is the association constant Ka, a measure of affinity. Ka is the strength of the interaction and is expressed as:
Ka  =  [Ag-Ab]/ [Ab] [Ag]
In this equation, [Ag-Ab] is the molar concentration of the antibody-antigen complex, and [Ab] and [Ag] are the molar concentrations of the antibody and antigen, respectively. Affinity constants can vary widely between different antibodies and antigens, and are affected by pH, temperature, and solvent.
The dissociation of the antigen-antibody complex can be expressed as:
Ag-Ab  ↔  Ab  +  Ag……………………(2)
The equilibrium constant for the above reaction is expressed as Kd, and which is the reciprocal of Ka, and is given by
Kd  =  [Ab] [Ag]/ [Ab-Ag]
The affinity constants described above apply to single site interactions. However, all naturally occurring antibodies are multivalent and their functional affinity is dependent not only on their intrinsic affinity for antigen but also on the number of binding sites (2 for IgD,G and E and 10 for IgM). The association constant Ka, for binding of a univalent ligand to a multivalent antibody may be expressed as:

Ka = [Ab-Ag]/ [Ab] [Ag] =  r/c(n-r)
where, at equilibrium, c is the concentration of free ligand, r represents the ratio of the concentration of bound ligand to total antibody concentration and n is the maximum number of binding sites per antibody molecule (the antibody valence). This expression can be rearranged to give the Scatchard equation:
r/c = Kan - Kar
A set of values of r and c can be obtained from a series of experiments in which the concentration of antibody is kept constant with a varying concentration of ligand and from these a plot (Scatchard plot) can be constructed in which r/c is plotted against r. From a Scatchard plot. Both the equilibrium constant (Ka) and the number of binding sites per antibody molecule (n) or its valency can be obtained. If all antibodies have the same affinity, then a Scatchard plot gives a straight line with a slope of –Ka(figure-1) and if the antibody mixture has a range of affinities (polyclonal antibodies), a scatchard plot gives a curved line whose slope is constantly changing.

 

Figure 1: Scatchard plot of the antibodies having same affinity
Figure 1: Scatchard plot of the antibodies having same affinity

 

Avidity

When complex antigens containing multiple repeating antigenic determinants are mixed with antibodies containing multiple binding sites, the interaction of an antibody with an antigen at one site will increase the probability of reaction between those two molecules at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. Avidity is more than the sum of the individual affinities. Affinity describes the strength of interaction between antibody and antigen at single antigenic sites. Avidity is perhaps a more informative measure of the overall stability or strength of the antibody-antigen complex, which is controlled by three major factors: antibody-epitope affinity; the valence of both the antigen and antibody; and the structural arrangement of the interacting parts.

Specificity

Specificity of an antigen-antibody reaction refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. Antigen-antibody reactions are highly specific in nature, that means one antibody can react with its own antigen.  A strong antigen-antibody interaction depends on a very close fit between the antigen and antibody which requires high degree of specificity.  

Cross reactivity

The most striking feature of the antigen–antibody interaction is its high specificity and affinity.  But in some cases, antibody elicited by one antigen can cross react with an unrelated antigen, which is known as cross-reactivity. Cross reactions arise because the cross reacting antigen shares an epitope in common with the immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing antigen.

       Cross-reactivity is often observed among polysaccharide antigens that contain similar oligosaccharide residues. The ABO blood group antigens are the glycoproteins expressed on red blood cells. Subtle differences in the terminal residues of the sugars attached to these surface proteins distinguish the A and B blood group antigens. An individual lacking one or both of these antigens will have serum antibodies to the missing antigens. Thus, anti-A antibodies are found in the serum of group O and B individuals and anti-B antibodies are found in the serum of group O and A individuals. Group AB individuals are believed not to have anti-A nor anti-B antibodies because they express both antigens on their red cells. The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestinal bacteria. The blood-group antibodies, although elicited by microbial antigens, will cross react with similar oligosaccharides on foreign red blood cells, providing the basis for blood typing tests and accounting for the necessity of compatible blood types during blood transfusions.

       A number of viral and bacterial antigens elicit antibody that cross reacts with the host-cell components, which results in a tissue damaging reaction. Cross-reactivity is also exhibited by some vaccines.


Types of antigen-antibody reaction

Precipitation reactions

Precipitation reaction is the reaction, in which a soluble antibody reacts with a soluble antigen to give an insoluble product or the precipitate. Soluble antibodies that aggregate soluble antigens are called precipitins. Soluble antigen that induces the formation of a specific precipitin is called a precipitinogen.

       When the antigens, which must have at least two epitopes per molecule are cross-linked by the bivalent antibodies, a lattice formation occurs which ultimately develops into a visible precipitate. For the lattice to be formed, the bivalent antibody will bind to epitopes on two different antigens. A second antibody molecule combines with the second epitope on one of the antigen molecules and a third epitope on another antigen molecule, so that the complex is formed.  The complex continues to grow and when it is sufficiently large, it becomes insoluble and can be visible as a precipitate. Thus, formation of the visible precipitate takes sufficiently longer time than formation of the soluble antigen-antibody complexes.

       Precipitation reaction can occur using polyclonal antibodies or mixture of monoclonal antibodies. Polyclonal antibodies can form large aggregates, that precipitate out of solution. If the antigen is monovalent or a single monoclonal antibody is used, the antibody can link only two molecules of antigen and no precipitate is formed.

       There is a marked effect on the precipitation reaction by changing the concentration of the antigen. A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amounts of antigens to the tubes. Plotting the amount of precipitate against increasing antigen concentrations yields a precipitation curve.  A precipitation curve for a system of antigen-antibody in the figure (figure-2) shows three zones, among which the first one is the zone of antibody excess or prozone, in which the antigen concentration is very low and that of the antibody is relatively high, as a result of which precipitation is inhibited, formation of small complexes occur and residual antibodies will remain in the supernatant.

 

Figure 2: A precipitation curve showing three zones
Figure 2: A precipitation curve showing three zones

       

       The second zone is the equivalence zone, also the zone of maximal precipitation in which antigen and antibody form large insoluble complexes and there is neither antigen nor antibody present in the supernatant. The third zone is the zone of antigen excess or postzone, in which the antigen concentration is very high, and therefore with increasing the amounts of antigen, the lattice size becomes too small to precipitate as a result of which precipitation is inhibited and binding of antigen-antibody is absent in the supernatant.

Types of precipitation reactions

Precipitation reactions occur both in solution and in gel phase, where antigen-antibody forms a precipitate. Similar to the precipitation reaction in fluid, visible precipitation occurs in the region of equivalence and no visible precipitate forms in regions of antibody or antigen excess in gel phase.  

Fluid phase precipitation

Fluid phase precipitation is a double diffusion method, where in a capillary tube an antigen solution is layered over an antibody solution. Both antigen and antibody will diffuse towards each other and  at the interface, when antibody recognizes antigen, precipitate forms. The amount of the precipitate is proportional to the concentration of both the antigen and antibody. This method is used to identify unknown antigen or unknown antibody.

Gel phase precipitation

In gel phase precipitation, instead of solution, gel is used as a semisolid medium. The gel contains “pores” that allow the movement of molecules. In immunoprecipitation reactions, the gel is a derivative of agar and is called agarose. Agarose gel allows soluble antigen and /or antibody to diffuse through the pores until the antigen and  antibody reach the optimal concentration for lattice formation. Smaller molecules move through the gel faster than larger molecules.

       When antibody is incorporated into the agar, and antigen diffuses into the antibody-containing matrix, or when antigen and antibody diffuse toward one another in agar, a visible line of precipitation will form unlike a precipitation curve in fluids.  Two types of immunodiffusion reactions can be used to determine relative concentrations of antibodies or antigens, i.e. radial immuno-diffusion and double immuno-diffusion.


Radial immunodiffusion (The Mancini method)

In radial immunodiffusion (figure-3), an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antiserum. The antigen diffuses in all directions from the well, and accordingly the region of equivalence is established and a ring of precipitation (precipitin ring) forms around the well. The area of the precipitin ring is proportional to the concentration of antigen. The diameter of the area of precipitation (including the well diameter) is measured to determine the concentration of antigen.

 

Figure 3: Radial immunodiffusion
Figure 3: Radial immunodiffusion

 

Double immunodiffusion (The Ouchterlony method)

In double immunodiffusion (figure-4), if antigen to be detected, a known reagent antibody is placed in the center well and the unknown samples are placed in the surrounding well. If antibody is to be detected, unknown antigen is placed in the center.

 

Figure 4: Double immunodiffusion
Figure 4: Double immunodiffusion

      

       After each of the samples and reagents have been added to the appropriate wells, diffusion occurs and both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. A line of precipitation forms at the zone of equivalence.

 

Immunoelectrophoresis

Immunoelectrophoresis is a gel electrophoretic technique which uses both electrophoresis and double diffusion. The samples that contain the proteins (the antigen mixture) to be analyzed are added to the wells on the gel plate. The mixture of samples could contain serum from healthy individuals as well as from infected persons. In this process, the antigen mixture is first electrophoresed to separate its components by charge. A trough is created parallel to the length of the electric field, into which a single purified species of antibody or known mixture of antibodies is added. The antibody molecules diffuse outward from the trough solution into the gel. When an antigen is encountered by an antibody, formation of a visual precipitate occurs. Precipitin arcs form at the zone of equivalence between the antigen and specific antiserum (figure-5). The pattern of precipitation can reveal antigenic differences between the normal serum and the serum from an infected person. If a nonspecific antiserum is placed in the trough, then only one arc will be formed if the particular serum component is present.

 

Figure 5: Immunoelectrophoresis
Figure 5: Immunoelectrophoresis

      

       Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the serum. This process separates the various proteins in a sample in an electric field and then probes the separated proteins using the desired antiserum. In the clinical laboratory setting, immunoelectophoresis is used to examine alterations in the content of serum, especially changes concerned with immunoglobulins. Change in the immunoglobulin profile can be the result of immunodeficiencies, chronic bacterial or viral infections, and infections of a fetus.

       Another electrophoretic precipitation technique, used primarily in research and coagulation laboratories, is the rocket, or Laurell technique.  Rocket electrophoresis is used to quantitate antigens other than immunoglobulins. In this process, a negatively charged antigen is electrophoresed in a gel containing antibody. As the antigen migrates through the gel, it combines with antibody, precipitation occurs. The precipitate formed between the antigen and antibody has the shape of a rocket, the height of which is proportional to the concentration of the antigen in the well. 

 

Agglutination reactions

The interaction between antibody and a particulate antigen results in visible clumping, called agglutination. The general term agglutinin is used to describe antibodies that agglutinate particulate antigens. Agglutination is a serological reaction and is very similar to the precipitation reaction. Both reactions are highly specific because they depend on the specific antibody and antigen pair.  As an excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions, this inhibition is known as prozone effect. The main difference between these two reactions is the size of antigens.  For precipitation, antigens are soluble molecules, and for agglutination, antigens are large, easily sedimented particles. Agglutination reactions can be used to type blood cells for transfusion, to identify bacterial cultures, and to detect the presence and relative amount of specific antibody in a patient’s serum.

Prozone effect  

At high antibody concentrations, the number of antibody binding sites may greatly exceed the number of epitopes. As a results, most antibodies bind antigen only univalently instead of multivalently. Antibodies that bind univalently cannot crosslink one antigen to another. Prozone effects are observed by performing the assay at a variety of antigen or antibody concentration. Higher levels of agglutination can be seen at optimum dilution of antibody concentration. When using polyclonal antibodies incomplete antibodies (class IgG) also causes prozone effect.  The antibodies present in high concentration in the antiserum, which bind to the antigen but do not induce agglutination, are known as incomplete antibodies.

Types of agglutination reactions
 

Quantitative agglutination (Bacterial agglutination)

Agglutination has been commonly used to determine whether a patient had or has a bacterial infection. This type of agglutination reaction is also known as quantitative agglutination test as here the measurement of level of antibodies to particulate antigens is done. The presence of serum antibodies in a person specific for surface antigens on the bacterial cells can be detected by bacterial agglutination reactions. If a patient is suspected of having typhoid fever, the patient’s serum is mixed with a culture of Salmonella typhi. If an agglutination reaction occurs, shown as clumping of the bacteria, the patient either had or has an S. typhi infection.  Since certain antibodies can persist in a patient’s blood for years after the patent has recovered from the infection, a positive reaction does not mean that the patient currently has the infection.  To determine whether a patient is currently suffering from typhoid fever, the amount or titer of the antibody will be determined at the onset of illness and two weeks later. The serum from the suspected patient is serially diluted in an array of tubes to which the bacteria is added. Visible agglutination can be seen in the last tube which will reflect the serum antibody titer of the patient. The agglutination titer is defined as the reciprocal of the greatest serum dilution that elicits a positive agglutination reaction. Naturally, the higher the titer, the greater is the antibody response of the individual to the disease. The patient currently suffering from suspected typhoid fever shows a significant rise in the agglutination titer to Salmonella typhi. Agglutination reactions also help to type bacteria.

 

Qualitative agglutination (Hemagglutination)

Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody.  The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen. Hemagglutination is a specific form of agglutination that involves typing of red blood cells. The ABO blood group antigens are intrinsic red blood cell antigens, with the ‘A’ and ‘B’ signs referring to proteins on the surface of red blood cells. Individuals expressing the ‘A’ antigen are designated as blood type ‘A’. Similarly those express ‘B’ antigens, are designated as blood type ‘B’. Individuals expressing both ‘A’ and ‘B’ antigens are designated as blood type ‘AB’, and those not expressing either ‘A’ or ‘B’ antigen are designated as blood type ‘O’. Blood type can be determined by using antibodies that bind to the A or B blood group in a sample of blood. In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood-group antigens. If the antigen is present on the cells, they agglutinate forming a visible clump on the slide. For example, if antibodies that bind the A blood group are added and agglutination occurs, the blood is either type A or type AB. To determine between type A or type AB, antibodies that bind the B group are added and if agglutination does not occur, the blood is type A.  In blood grouping, the patient's serum is tested against RBCs of known blood groups and also the patient's RBCs are tested against known serum types. In this way the patient's blood group is confirmed from both RBCs and serum.
 

Passive agglutination

Passive agglutination is like agglutination reaction but performed with soluble antigens. It is defined as the agglutination of particles that have been coated with soluble antigen, by antiserum specific for the adsorbed antigen. Passive hemagglutination is a kind of passive agglutination in which erythrocytes, usually modified by mild treatment with tannic acid or chromium chloride, are used to adsorb soluble antigen onto their surface, and which then agglutinate in the presence of antiserum specific for the adsorbed antigen.  Serum containing antibodies is serially diluted into microtiter plate wells and the antigen-coated red blood cells are then added to each well. Agglutination is assayed by the size of the characteristic spread pattern of agglutinated red blood cells on the bottom of the well. Passive agglutination can be performed with tanned erythrocytes or synthetic particles, such as latex beads. The use of synthetic beads offers the advantages of consistency, uniformity and stability. The initial step in the test is the linking together of the latex particle by the antibody molecules that specifically attach to the antigenic determinants on the surface of the particles. There is a formation of large lattices through these cross links and these large lattices sediment readily due to the large size of clumps and are visible to the unaided eye within minutes. The degree of agglutination can be determined by plotting the agglutinant  concentration which gives a bell shaped curve. The antigen-antibody complexes can be magnified using the latex particles.
 

Agglutination inhibition

Agglutination inhibition is the modification of the agglutination reaction. If the antibody is incubated with antigen prior to mixing with latex, agglutination is inhibited; this is because free antibodies are not available for agglutination. In agglutination inhibition, the absence of agglutination is diagnostic of antigen, provides a high sensitive assay for small quantities of antigen. One example of which is the home pregnancy test kits included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG. When urine of a pregnant woman containing HCG, added to it, agglutination of the latex particles inhibited when the anti-HCG was added. The absence of agglutination is the indication of pregnancy. If the urine contains no HCG, then visible clumping occurs and agglutination can be observed which indicates no pregnancy (figure-6).

 

Figure 6: Agglutination inhibition
Figure 6: Agglutination inhibition

      

       Agglutination assays can be employed for detecting the presence of any illegal drugs in a person’s blood sample or urine sample. Agglutination inhibition assays are also widely used in clinical laboratories to determine whether an individual has been exposed to certain types of viruses that cause agglutination of red blood cells. This technique is commonly used to determine the immune status of women with respect to the rubella virus.

 

Complement fixation

The complement fixation test is an immunological medical test that can be used to detect the presence of either specific antibody or specific antigen in a patient's serum. Complement is the activity of blood serum that completes the action of antibody. The complement system is a system of serum proteins that react with antigen-antibody complexes.

The basic steps of complement fixation test (figure) includes:

  • Isolation of serum from the patient.
  • The complement proteins in the patient's serum must be destroyed and replaced by a known amount of standardized complement proteins. This is done to negate any effect on the test as patient’s serum naturally has different levels of complement proteins.
  • The serum is heated in such a way that all of the complement proteins but none of the antibodies within it are destroyed.
  • A known amount of standard complement proteins are added to the serum.
  • The antigen of interest is added to the serum.
  • If the patient’s serum contains antibodies against the antigen of interest, then antigen-antibody complex will be formed that will fix the complement (figure-7). The complement proteins will react with these complexes and be depleted.
  • Sheep red blood cells (sRBCs), which have been pre-bound to anti-sRBC antibodies are added to the serum. When complement fixation occurs, there will be no complement left in the serum to react with the sRBC-antibody complexes. However, if the patient’s serum contains no antibodies against the antigen of interest, the complement will not be depleted and it will react with the sRBC-antibody complexes, lysing the sRBCs and spilling their contents into the solution, thereby turning the solution pink. The solution when turns to pink, it confirms the test negative.   
  • It is equally possible to detect antigen in a patient’s serum. In this case, the patient's serum is supplemented with specific antibody to induce formation of complexes; addition of complement and indicator sRBC is performed as the above mentioned antibody detection procedure. 

 

Figure 7: Complement fixation
Figure 7: Complement fixation

 

 

Enzyme-linked Immunosorbent Assay (ELISA)

The ELISA is a fundamental tool of clinical immunology, which has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries. ELISA, or Enzyme-linked immunosorbent assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, or anibody in a liquid sample or wet sample. It depends on an enzyme-substrate reaction that generates a colored reaction product. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. A number of enzymes i.e., alkaline phosphatase, horseradish peroxidase, and β-galactosidase, have been employed for ELISA.

       A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody. Four types of ELISA methods are there, but generally three methods are employed for detection of either antigen or antibody. Direct ELISA is the simplest type of ELISA. Antibody can be determined with an indirect ELISA whereas antigen can be determined with a sandwitch ELISA or competitive ELISA. Each assay can be used qualitatively or quantitatively by comparison with standard curves prepared with known concentrations of antibody or antigen.

Direct ELISA

The direct ELISA is a method for detecting and measuring antigen concentration in a sample. It is the simplest type of ELISA among the four types. In direct ELISA, the presence of a particular antigen in a sample, is detected by using a capture monoclonal antibody.

The procedure for direct ELISA includes:

  • The wells of a microtiter plate are coated with a sample containing the target antigen. The antigen is fixed to the surface to render it immobile.
  • The plate wells are then coated with a blocking buffer.
  • In a separate reaction, an enzyme is linked to an antibody.
  • The enzyme-antibody conjugate is added to the wells to adsorb to the antigen.
  • The plate is washed to remove any excess enzyme-antibody conjugate.
  • Then a substrate is applied for the enzyme, and which is converted by the enzyme to elicit a chromogenic or fluorescent signal. So the substrate detects the presence of the  enzyme and the antigen.  
  • The amount of colored product is measured by a specialized spectrophotometric plate readers.

The advantage of direct ELISA is that it is relatively quick because of the use of only one antibody but direct ELISA requires the labeling of every primary antibody, which can be time-consuming and more expensive than in indirect methods. Certain antibodies may be unsuitable for direct labeling. Direct methods do not allow for signal amplification in contrast to methods that use a secondary antibody. Direct ELISA can be used to test specific antibody-to-antigen reactions, and helps to eliminate cross-reactivity between other antibodies.

Indirect ELISA

Indirect ELISA helps detecting antibody. It is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

The procedure of indirect ELISA includes:

  • A buffered solution of the antigen to be tested for is added to each well of a microtiter plate. Coating is achieved through passive adsorption of the antigen to the assay microplate. This process occurs though hydrophobic interactions between the micro titer plate and non-polar protein residues. After incubation any excess antigen is removed by washing steps by flooding and emptying the wells with neutral phosphate buffered saline ( PBS ) or deionized water. A solution of nonreacting protein, such as bovine serum albumin or casein, is added to block any plastic surface in the well that remains uncoated by the antigen.  
  • Serum or some other sample containing primary antibody is added to the antigen-coated microtiter well. The antibody specific to the test antigen, binds the coated antigen on incubation.
  • Excess antibody or any unbound antibodies are removed by washing and is followed by addition of blocking solution.  
  • The presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary anti-isotype antibody, which binds to the primary antibody (figure-8).
  • Any free secondary antibody is washed away, and a substrate for the enzyme is then added. Often, this substrate changes color upon reaction with the enzyme, which shows the secondary antibody has bound to primary antibody. The higher the concentration of the primary antibody present in the serum, the stronger the color change. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers.
  • Serum antibodies to HIV can be detected by indirected ELISA within six weeks of infection. In this assay, recombinant envelope and core proteins of HIV are adsorbed to solid-phase antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins.

 

Figure 8: Indirect ELISA
Figure 8: Indirect ELISA

      

       Indirect ELISA has sensitivity, since more than one labeled antibody is bound per primary antibody. It has flexibility too, since different primary detection antibodies can be used with a single labeled secondary antibody. Apart from that, it is cost effective, since fewer labeled antibodies are required.

       A major disadvantage of the indirect ELISA is the method of antigen immobilization is not specific; when serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich or direct ELISA provides a solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.

Sandwitch ELISA

Sandwitch ELISA helps detecting presence of antigen in a sample and to use it as a diagnostic tool for medicine. The Sandwich ELISA measures the amount of antigen between two layers of antibodies. The two layers of antibody consist of capture and detection antibody.  Here, either monoclonal antibodies or polyclonal antibodies can be used as capture and detection antibodies. The antigen to be measured must contain at least two antigenic sites capable of binding to antibody, since at least two antibodies act in the sandwich.

The procedure for sandwitch ELISA includes:

  • In sandwitch ELISA, the antibody (capture antibody) rather than the antigen is coated on the surface of a microtiter well.
  • Any nonspecific binding sites on the surface are blocked with the help of blocking solution.
  • The sample containing antigen is added and allowed to react with the immobilized antibody on the microtiter well.
  • The microtiter plate is washed to remove any unbound or excess antigen.
  • After washing, a second enzyme-linked antibody (detection antibody) specific for a different epitope on the antigen is added and is allowed to react with the bound antigen.
  • The plate is washed to remove the unbound antibody-enzyme conjugates.
  • After any free second antibody is removed by washing, substrate is added and the colored reaction product is measured (figure-9). A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. TMB (3, 3’, 5, 5’-tetramethyl benzidine) is the most commonly used substrate for the enzyme horseradish peroxidase (HRP).
  • The absorbency of the plate wells is measured to determine the presence and quantity of antigen. Specially designed spectrophotometers are available which reads through the microtiter wells either singly or in rows. Most ELISA readers can be set to measure the absorbance of the colors produced by the action of antibody- conjugated enzymes on their respective substrates.

 

Figure 9: Sandwitch ELISA
Figure 9: Sandwitch ELISA

      

       The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive than indirect ELISA or competitive ELISA. Sandwitch ELISA has high specificity, since the two antibodies help in capturing and detecting the antigen. The process both has flexibility and sensitivity.
 

Competitive ELISA

Competitive ELISA is another variation for measuring amounts of antigen. The procedures of competitive ELISA is different from that of sandwitch ELISA and indirect ELISA.

The procedure for competitive ELISA includes:

  • Unlabeled primary antibody is first incubated in solution with a sample containing antigen.
  • The antigen-antibody mixture is then added to an antigen coated microtiter well (figure-10).
  • The more antigen present in the sample, the less free antibody will be available to the antigen-coated well. Therefore competition arises.
  • The plate is washed to remove any unbound antibody.
  • The enzyme-conjugated secondary antibody, specific for the isotype of the primary antibody is added to determine the amount of primary antibody bound to the well.
  • A substrate is added, and color change is measured.
  • In competitive ELISA, the concentration of antigen in the original sample is inversely proportional to the color produced.

 

Figure 10: Competitive ELISA
Figure 10: Competitive ELISA

      

       Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal. So, here the microtiter well is coated with an antibody.

       In competitive ELISA, the advantage is that the antigen does not require purification prior to measurement. Competitive ELISA has also high specificity compared to indirect ELISA.

 

The ELISPOT assay

The enzyme-linked immunospot (Elispot) assay is a modification of the ELISA assay, allows the quantitative determination of the number of cells in a population that are specific for a given antigen or an antigen for which one has a specific antibody. Based on the sandwich enzyme-linked immunosorbent assay (ELISA), the ELISPOT assay derives its specificity and sensitivity by employing high affinity capture and detection antibodies and enzyme-amplification.

The procedure for the ELISPOT assay (figure-11) includes:

  • The wells of the microtiter plate are coated with the antigen (capture antigen) recognized by the antibody of interest or with the antibody (capture antibody) specific for the antigen whose producion is being assayed.
  • This assay is commonly used to detect cytokine secreted from different cells.
  • A suspension of cell population thought to contain some members secreting cytokine are added to the wells coated with relevant antibodies (capture antibodies).  It is allowed to be incubated.
  • After the incubation period, the wells are washed and enzyme-labeled anti-cytokine antibodies (detection antibodies) are added.
  • Then again the wells are washed to remove any unbound antibody. After washing the wells, a chromogenic substrate that forms an insoluble colored product is added.
  • The colored product precipitates and forms a spot only on the areas of the wells, where cytokine-secreting cells had been deposited.
  • The number of cytokine-secreting cells present in the added cell suspension were identified by counting the number of colored spots.

 

Figure 11: The ELISPOT assay
Figure 11: The ELISPOT assay

      

       There are several advantages of the ELISPOT assay. Elispot assay has high sensitivity. Frozen or thawed biological samples can be applicable for the assay. This assay requires minimun biological samples. It is also compatible with other assays.

 

Radioimmuoassay (RIA)

Radioimmunoassay is a very sensitive in vitro assay technique used for separation of a protein from a mixture using the specificity of antibody-antigen binding and quantitation using radioactivity. The technique was first developed  in 1960 by two endocrinologists, S.A. Berson and Rosalyn Yalow, to determine levels of insulin-anti-insulin complexes in diabetics. Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. 

       The principle of RIA involves competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody. The method of RIA starts with making a known quantity of antigen radioactive by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. The labeled antigen is then mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. Then test samples of unlabeled antigen of unknown concentration are added in progressively larger amounts. The test sample may be a complex mixture, such as serum or body fluids, that contains the unlabeled antigen. This causes the unlabeled antigen to compete with the radio-labeled antigen for antibody binding sites (figure-12).

 

Figure 12: The radioimmunoassay (RIA)
Figure 12: The radioimmunoassay (RIA)

      

       As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. Then the bound antigens are separated by the unbound ones by precipitating the antigen-antibody complex with a secondary anti-isotype antiserum. For example, if the antigen-antibody complex contains rabbit IgG antibody, then goat anti-rabbit IgG will bind to the rabbit IgG and precipitate the complex. After separation, the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a binding curve can then be generated which allows the amount of antigen in the test sample to be derived. A standard curve is obtained by adding increasing concentrations of unlabeled antigen to a fixed quantity of radio-labeled antigen and specific antibody (figure-13).

 

Figure 13: A standard curve of RIA
Figure 13: A standard curve of RIA

 

       From the plot of the percentage of labeled antigen bound versus the concentration of unlabeled antigen, the concentration of antigen in unknown serum samples can be determined by using the linear part of the curve.

 

Immunoflorescence

Immunofluorescence is an antigen-antibody reaction where the antibodies are tagged (labelled) with a fluorescent dye and the antigen-antibody complex is visualized using ultra-violet (fluorescent) microscope. Fluorescent molecules absorb light of one wavelength and emit light of another wavelength. If antibody molecules are tagged with a fluorescent dye, or flurochrome, immune complexes containing these fluorescently labeled antibodies can be detected by colored light emission when excited by light of appropriate wavelength. In immunoflorescence, fluorescent compounds such as fluorescein and rhodamine are commonly used. Phycoerythrin, an intensely colored and highly fluorescent pigment obtained from the algae, is also routinely used. It can be categorised into direct and indirect immunoflorescence, given briefly below.

Direct immunoflorescence

This technique is used to detect antigen in clinical specimens using specific fluorochrome labeled antibody. In direct immunoflorescence, the specific antibody is directly conjugated with a fluorescent dye. The procedure begins with fixation of cells with membrane antigens (mAg) to a slide (figure-14a). Then the cells are stained with anti-mAg antibodies that are labeled with flurochromes. After a period of incubation, the slide is washed to remove any unbound excess labeled antibody. After washing, the slide is viewed under fluorescent microscope. When viewed under fluorescent microscope, the field is dark and areas with bound antibody fluoresce green.
This technique can be used to detect viral, parasitic, tumor antigens from patient specimens or monolayer of cells.

 

Indirect immunoflorescence

Indirect immunofluorescence is employed to detect antibodies in a test sample, for example in a patient's serum. Here, primary antibodies which are unlabeled, allowed to react with cells having membrane antigens. After a period of incubation, the slide is washed to remove any unbound antibodies. After washing, the cells are stained with flurochrome-labeled secondary antibodies (fluorescein-labeled goat anti-mouse antibodies). This antibody binds to Fc portion of first antibody and persists despite washing. The presence of secondary antibodies is detected by observing under fluorescent microscope (figure-14b).

 

Figure 14: Direct method and indirect method of immunoflorescence
Figure 14: Direct method and indirect method of immunoflorescence

      

       Indirect immunoflorescence staining has two advantages over direct staining. First, the supply of primary antibodies is often a limiting factor and loss of primary antibody occurs during conjugation reaction. Indirect methods avoid the loss of Primary antibody which is not conjugated  with flourochrome. Secondly, indirect methods increase the sensitivity of staining because multiple molecules of the flurochrome reagent bind to each primary antibody molecule, increasing the amount of light emitted at the location of each primary antibody molecule.

       Immunoflorescence has been applied to identify the CD4+ and CD8+ T-cell populations. Immunoflorescence is suitable for detecting antigen-antibody complexes in autoimmune disease, detecting complement components in tissues and the major application of it is localizing antigens in tissue sections or in sub-cellular compartments.

       Fluorescent antibody techniques are important qualitative tools but they do not give quantitative data. Flow cytometer, automate the analysis and separation of cells stained with fluorescent antibody. The flow cytometer uses a laser beam and light detector to count single intact cells in suspension. Every time a cell passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded. Those cells having a fluorescently labeled antibody bound to their cell surface antigens are excited by the laser and emit light that is detected by a second detector system located at a right angle to the laser beam.

       The flow cytometer has multiple applications to clinical and research problems. Flow cytometry can also analyze cell population that have been labeled with two or three different fluorescent antibodies.

 


Immunoprecipitation

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. It also provides a sensitive assay for the presence of a particular antigen in a given cell or tissue type. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure. There are two general methods for immunoprecipitation, i.e., the direct capture method and the indirect capture method.

Direct method

An antibody (monoclonal or polyclonal) against a specific protein is pre-immobilized onto an insoluble support, such as agarose or magnetic beads, and then incubated with a cell lysate containing the target protein. During the incubation period, the lysate is gently agitated so that the proteins that are targeted by the antibodies are captured onto the beads via the antibodies, in other words, they become immunoprecipitated.  The immobilized antigen-antibody complexes are collected from the lysate, and then eluted from the support and analyzed.
 

Indirect method

In indirect method, the antibody is not pre-immobilized onto an insoluble support rather, the antibody that is specific for a particular protein antigen, added directly to a cell lysate containing the target protein. Free, nonbound antibodies are allowed to form immune complexes in the lysate. After some time, secondary antibodies, specific for the primary antibodies, are attached to synthetic beads and added to the antigen-antibody complexes in the lysate. At this point, the antibodies, which are now bound to their targets, will stick to the beads.  The antigen-antibody complexes are then eluted from the support and analyzed (figure-14).

 

Figure 15: Immunoprecipitation
Figure 15: Immunoprecipitation

      

       Both the direct method and the indirect method gives the same end-result with the protein or protein complexes bound to the antibodies which themselves are immobilized onto the beads. The direct method is a preferred choice, but the indirect approach is sometimes preferred when the concentration of the protein target is low or when the specific affinity of the antibody for the protein is weak.

Types of immnoprecipitation

There are generally four types of immunoprecipitation (IP) techniques, i.e., individual protein immunoprecipitation, protein complex immunoprecipitation (Co-IP), chromatin immunoprecipitation (Ch-IP) and RNA immunoprecipitation (RNA-IP).
 

Individual protein immunoprecipitation

It Involves using an antibody that is specific for a known protein to isolate that particular protein out of a solution containing many different proteins. These solutions will often be in the form of a crude lysate of a plant or animal tissue.
 

Protein complex immunoprecipitation (Co-IP)

Immunoprecipitation of intact protein complexes (i.e. antigen along with any proteins or ligands that are bound to it) is known as co-immunoprecipitation (Co-IP). Co-IP is a powerful technique that is used regularly by molecular biologists to analyze protein–protein interactions.

       Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this known member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify unknown members of the complex. This works when the proteins involved in the complex bind to each other tightly, making it possible to pull multiple members of the complex out of solution by latching onto one member with an antibody. Immunoprecipitated proteins and their binding partners are commonly detected by SDS-PAGE and Western blot analysis.
 

Chromatin immunoprecipitation (Ch-IP)

Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. This technique gives a picture of the protein–DNA interactions that occur inside the nucleus of living cells or tissues.

       DNA-binding proteins (including transcription factors and histones) in living cells can be cross-linked to the DNA that they are binding. By using an antibody that is specific to a putative DNA binding protein, one can immunoprecipitate the protein–DNA complex out of cellular lysates. The purified protein–DNA complexes are then heated, allowing the separation of DNA from the proteins. The DNA is then identified by PCR, sequenced and applied to microarrays or analyzed according to the requirement.
 

RNA immunoprecipitation

The principle of RNA immunoprecipitation is similar to Ch-IP, except that here RNA-binding proteins are immunoprecipitated instead of DNA-binding proteins. RNA immunoprecipitation is also an in vivo method in that live cells are lysed and the immunoprecipitation is performed with an antibody that targets the protein of interest. By isolating the protein, the RNA will also be isolated as it is bound to the protein. By performing an RNA extraction, the purified RNA-protein complexes can be separated and immunoprecipitated RNAs can then be identified by RT-PCR and cDNA sequencing.

 

References

1. Kuby, Janis etal.(2003). Immunology, 5th Edn. W.H. Freeman & Company Publishers.

2. Lichtman, Andrew H., Abbas, Abul K., Basic Immunology, 3rd Edn. Saunders Elsevier Inc.

3. Roitt, Ivan M. etal. Essential Immunology, 12th Edn. Willey-Blackwell Publishers.

4. www.wikipedia.org

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