immunology

S.K Barolia

1. The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum. ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured in a single experiment. These plates need to be special absorbent plates (e.g. NUNC Immuno plates) to ensure the antibody or antigen sticks to the surface. Each ELISA measures a specific antigen, and kits for a variety of antigens are widely available. ELISA stands for enzyme-linked immunoassay . It is a commonly used laboratory test to detect antibodies in the blood. An antibody is a protein produced by the body’s immune system when it detects harmful substances, called antigens. ELISA is an effective and widely used technique in microbiology and virology—in particular, for investigating infectious pathogens.

2. The ELISA pictured in Figure 1 is what is known as a sandwich ELISA, here two sets of antibodies are used to detect secreted products, e.g. cytokines. The method is stepwise in the order shown. The 1st step is to coat the ELISA plate with capture antibody , any excess, unbound antibody is then washed from the plate. The capture antibody is an antibody raised against the antigen of interest.

3. Next the sample (e.g. urine, serum, or cell supernatant) is added. Any antigen found in the sample will bind to the capture antibody already coating the plate. Samples are usually added in duplicate or triplicate (to allow for statistical analysis), and in varying concentrations to guarantee it falls within the levels of detection of the assay. Again any excess sample is washed from the plate. In step 3, detection antibody is added. This antibody is labelled with an enzyme, usually horse radish peroxidase or alkaline phosphatase. Detection antibody binds to any target antigen already bound to the plate. Finally, a substrate is added to the plate. ELISA assays are usually chromogenic using a reaction that converts the substrate (e.g. TMB or ABTS) into a coloured product which can be measured using a plate reader.

4. Determination of antigen concentration in a sample requires production of a standard curve using antigens of a known concentration (shown in Figure 2 ). The concentration of antigen in a sample can then be calculated using the optical density (OD). Figure 1 . ELISA method. Described above is a sandwich ELISA, showing the steps in the assay, numbered in order 1-4.

5. ELISA stands for enzyme-linked immunoassay . It is a commonly used laboratory test to detect antibodies in the blood . An antibody is a protein produced by the body’s immune system when it detects harmful substances, called antigens . ELISA is an effective and widely used technique in microbiology and virology—in particular, for investigating infectious pathogens .

6. ELISA Principle What is the principle of enzyme linked immunosorbent assay? ELISA is an abbreviation for “Enzyme Linked Immunosorbent Assay” and it is a common laboratory technique. Toget the task of measuring the analyte’s (antibodies or antigens most likely) concentration in solutions done, we use the ELISA technique. As an indicator to the amount of analytes exist in the original sample, a colored end product is the final result of the ELISA steps. ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins . It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. The principle of ELISA depends on having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from unbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

7. General ELISA Procedure How does the ELISA test work? Unless you are using a kit with a plate that is pre-coated with antibody, an ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate. This is followed by a blocking step (Blocking ELISA principle: all unbound sites are coated with a blocking agent.)Following a series of washes, the plate is incubated with enzyme-conjugated antibody. Another series of was hes removes all unbound antibody. A substrate is then added, producing a calorimetric signal. Finally, the plate is read. Because the assay uses surface binding for separation, several washes are repeated in each ELISA step to remove unbound material. During this process, it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next assay step. To ensure uniformity, specialized plate washers are often used . ELISAs can be quite complex and include multiple intervening steps, especially when measuring protein concentration in heterogeneous samples such as blood. The most complex and varying step in the overall process is detection, where multiple layers of antibodies can be used to amplify signals.

8. There are four basic ELISA formats, allowing for a certain amount of flexibility which can be adjusted based on theantibodies available, the results required, or the complexity of the samples: 1.Direct ELISA 2.Indirect ELISA 3.Sandwich ELISA 4.Competition or Inhibition ELISA

9. Advantages Quick because only one antibody and fewer steps are used. Cross-reactivity of secondary antibody is eliminated. Disadvantages Immuno reactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. Labeling primary antibodies for each specific ELISA system is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Minimal signal amplification.

10. Indirect ELISA For indirect detection, the antigen coated to a multi-well plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody.  The secondary antibody is usually an anti-species antibody and is often polyclonal. The indirect assay, the most popular format for ELISA, has the advantages and disadvantages:  Advantages A wide variety of labeled secondary antibodies are available commercially. Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection Maximum immuno reactivity of the primary antibody is retained because it is not labeled. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Disadvantages Cross-reactivity might occur with the secondary antibody, resulting in a nonspecific signal. An extra incubation step is required in the procedure.

11. The following figure shows the difference between direct and indirect ELISA

12. Sandwich ELISA  Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture antibody) is coated to the wells.  The sample solution is then added to the well. A second antibody (known as detection antibody) follows this step in order to measure the concentration of the sample . Advantages High specificity: the antigen/ analyte is specifically captured and detected Suitable for complex (or crude/impure) samples: the antigen does not require purification prior to measurement Flexibility and sensitivity: both direct or indirect detection methods can be used

13. Competitive ELISA  The key event of competitive ELISA (also known as inhibition ELISA) is the process of competitive reaction between the sample antigen and antigen bound to the wells of a microtiter plate with the primary antibody. What is the principle of competitive ELISA technique?  First, the primary antibody is incubated with the sample antigen and the resulting antibody–antigen complexes are added to wells that have been coated with the same antigen. After an incubation period, any unbound antibody is washed off. The more antigens in the sample, the more primary antibodies will be bound to the sample antigen.  Therefore, there will be a smaller amount of primary antibody available to bind to the antigen coated on the well, resulting in a signal reduction. The main advantage of this type of ELISA arises from its high sensitivity to compositional differences in complex antigen mixtures, even when the specific detecting antibody is present in relatively small amounts.  

14. Immunology Dr.S.K.Barolia

15. 1- Prepare a surface to which a known quantity of capture antibody is bound. 2- Block any nonspecific binding sites on the surface. 3- Add an antigen-containing sample to the plate. 4- Wash the plate, so that unbound antigen is removed. 5- A specific antibody is added, and binds to antigen (hence the ‘sandwich, the Ag is stuck between two antibodies) 6- Add enzyme-linked secondary antibodies as detection antibodies that also bind specifically to the antibody’s Fc region (non-specific). 7- Wash the plate, so that the unbound antibody-enzyme conjugates are removed. 8- Add a substrate that is converted by the enzyme into a color or fluorescent or electrochemical signal. 9- Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

16. Sandwich ELISA Principle  Sandwich ELISA is used to detect the presence of an antigen. Sandwich ELISA typically requires the use of matched antibody pairs, where each antibody is specific for different, non-overlapping parts of the antigen molecule. The fi­rst antibody termed the capture antibody is coated to the plate. Next, the analyte or sample solution is added to the well.  A second antibody layer, the detection antibody, follows this step in order to measure the concentration of the analyte . If the detection antibody is conjugated to an enzyme, then the assay is called a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection antibody will be needed resulting in an indirect sandwich ELISA .

17. There are several  applications of ELISA test, for ex., food industry, vaccine development, immunology, diagnosis, toxicology, drug monitoring, pharmaceutical industry, and transplantation.  Food Industry ELISA plays a major role in the food industry. It is the main platform for identifying food allergens such as those present in milk, peanuts, walnuts, almonds, and eggs. ELISA can also be employed to corroborate the authenticity of the food products. This technique is of great help to avoid possible economic losses caused by fraudulent substitution. Vaccine Development  Vaccine development is a very important application of ELISA which serves as a great candidate for the process of serves as a great candidate for . The sera sample from an immunized animal or human model can be tested to detect the presence of antibodies against certain types of antigens, which were intentionally injected to the host. 

18. Immunology The defender of the body, the immune system, can operate in cellular or  humoral (innate or adaptive)  modes. Measuring and monitoring the changes of the immune response underlay the foundation for understanding immune disease. Various studies have demonstrated ELISA as the gold standard method that is rapid and cost-effective for such measurements and monitoring. Humoral Immunity ELISA has shown great potential in studying the humoral response of the immune system towards different classes of infections as well. Humoral immunity response involves the substances (antibodies and other components) that exist in the body fluids. Monitoring and measurement of these components are of great importance.

19. Pregnancy Test A number of different biomolecular entities including human chorionic gonadotropin ( hCG ), luteinizing hormone (LH), follicle stimulating hormone (FSH),  estriol (E3) , and thyrotropin -stimulating hormone (TSH) can be expressed due to the pregnancy. ELISA can detect some of these proteins from the maternal blood, saliva, or urine at the early stages of the pregnancy . HCG is one of the common hormones that can be detected by ELISA during the first month after fertilization. Another biomolecule associated with pregnancy is estriol (E3) that can be detected with ELISA in the saliva at the 6th week of pregnancy. Cancer Detection Highly sensitive detection of cancer provides with the early stage diagnostic, which is crucial for patient survival. Cancer biomarkers, however, are some of the most challenging biomolecular entities as target analytes . Advancements of ELISA technique have promised its application in detection of cancer biomarkers.

20. Diagnosis In the area of diagnosis, ELISA has proven to be a capable platform applied worldwide for detecting a variety of disease types in humans and animals. A number of different commercial ELISA kits are available in the market for detection of HIV, Influenza, Dengue fever, Ebola,  Chagas disease , Leishmaniasis , Lyme disease,  West Nile virus , among others. Even in plants pathology, ELISA technique is attracting increasing attention. ELISA has successfully overcome the drawbacks of the previous serological analyses performed in phyto -diagnosis