ELISA

A path breaking technology which has made it possible for us to detect HIV. ELISA is an immunological assay nowadays even used to detect food proteins & is the science behind pregnancy color test. This presentation unlocks the working of this assay.

1. ELISA Moksha CHIB 13FET1003

2. ELISA E nzyme L inked I mmuno S orbent A ssay It is a lab technique, used to measure the concentration of antibodies or antigens (‘ analyte’) in solution by color change . This technique is used in immunology and even in the food industry and was developed in 1970.

3. BRIEF REVIEW ELISA involves coating (binding) an antigen or a protein to a solid support as a membrane or a 96-well micro plate or ELISA plate . Antigens from the sample are attached to a surface. Further, an antibody, an enzyme and then a substrate is added to the adsorbed antigen. This reaction produces a detectable color change . 8×12 cm plate Well- 1cm deep and 0.7 in diameter

4. PRINCIPLE It combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme. It involves the usage of an enzyme to detect the binding of an Antigen (Ag)and an Antibody ( Ab ). The enzyme converts a colorless substrate ( chromogen ) to a colored product, indicating the presence of Ag : Ab binding. Therefore, ELISA can be used to detect either the presence of antigens or antibodies in a sample depending upon how the test is designed.

5. STEPS INVOLVED in ELISA Antigen Binding The sample antigen is binded or immobilized to the micro plate via adsorption to the surface. Binding is achieved by incubating the wells with a solution containing the antigen for 2hrs at RT or overnight at 4°C. The protein adheres due to hydrophobic interactions between the protein & the plastic. Coating is done using carbonate/bicarbonate buffer at a pH 9.6.

6. STEPS INVOLVED in ELISA Blocking  All unbound sites on the solid support are blocked to prevent nonspecific binding of the antibodies. Blocking buffers like BSA , Non fat dry milk powder (casein) in PBS ( Phosphate buffered saline) or TBS ( Tris buffered saline) at a pH 7.4 with minute percentage of Tween 20 are used to block the free sites. Protein in the blocking solution will attach to membranes in places where the target proteins have not attached. Excess blocking agent is removed by washing the plate membrane with washing buffer.

7. STEPS INVOLVED in ELISA Primary Antibody The 1° antibody is added and will be bound only if there is a recognized epitope within sample antigen. Secondary Antibody An enzyme-linked 2° antibody is added with suitable dilutions which will bind to any available 1° antibody (i.e. it is bound to the antigen). 2° antibodies are linked to the enzyme through biconjugation . Enzymes generally used are Horse Radish Peroxidase (HRP) & Alkaline Phosphatase (AP). Plate is washed with buffer or a mild detergent to remove any unbound antibodies or proteins.

8. STEPS INVOLVED in ELISA Detection/ Developing After the final wash step, the plate is developed by adding an enzymatic chromogenic substrate  to give color. The entire plate is placed into a plate reader and the OD is determined for each well. Intensity of color reflects the amount of specific 2° antibody bound to the target. A spectrophotometer is designed to read each well in the 96 well plate. It is interfaced with a computer for data management. Substrates used are pNPP ( p- nitro phenyl phosphate), BCIP (5-bromo,4-chloro,3-indoyl phosphate), NBT (nitro blue tetrazolium), TMB (3,3´,5,5´- tetra methyl benzidine), DAB (3,3-diamino benzidine).

9. ELISA Assay to determine antigen concentration

10. TYPES OF ELISA 3 types of ELISA : 1. Direct ELISA : Only 1° antibody is used. 2. Indirect ELISA : 1° antibody used is then detected by labeled 2° antibody. 3. Sandwich ELISA : A capture antibody is used and bound to the solid surface. A solution containing the antigen is added followed by washing. 

11. ADVANTAGES OF SANDWICH OVER INDIRECT ELISA A major disadvantage of the indirect ELISA is the method of antigen immobilization is not specific. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.  Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement. Simplifies the assay. Increases the specificity and the sensitivity of the assay.

12. DIRECT ELISA INDIRECT ELISA SANDWICH ELISA Only primary antibody is used. Both primary and secondary antibodies are used. A capture antibody is used which is bound to the solid surface before the antigen is added. Antigen immobilisation is not specific. Antigen immobilisation is not specific. Antigen immobilisation is specific. Any protein in the sample may attach to the plate. Any protein in the sample may attach to the plate. A specific will be attached as a ‘capture’ antibody is used. This increases the specific antigen immobilisation. There is a competition between the serum protein and the other proteins. As any protein can bind to the surface, there is a competition between the serum and the other proteins. No competition as only the specific serum proteins will be attached due to the inclusion of a ‘capture’ antibody. It requires purification of the sample to be tested. It also requires the purification of the sample to be tested. There is no need of sample purification as only the specific antigen will bind. The selectivity is questionable. The selectivity and specificity is a major disadvantage. Thus it simplifies the assay and increases the selectivity. Less sensitive but a faster method Highly sensitive Highly sensitive

13. COMPETITIVE ELISA Plate is precoated with an antigen to which Ag:Ab complex is added. The more antigens in the sample, the fewer antibodies will be able to bind to the antigen in the well, hence “competition”. After washing, the unbound Ag:Ab complex is removed. The 2° antibody that is specific to the primary antibody and conjugated with an enzyme is added followed by addition of a substrate. For Competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. The major advantage of this ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

14. WHAT CAN BE TESTED USING ELISA Protein with multiple epitope can be detected using Sandwich ELISA because it requires two antibodies which can react with different epitopes . Pure form of an antigen can be detected using Competitive ELISA as the purified analyte is immobilized and the other antigen competes. Small target molecules which do not adsorb by themselves can be attached to a larger protein which provides the means to attach to a desired epitope for detection. Membrane proteins from the cells can be adsorbed and thus detected by reducing the concentration of the detergents in which these cells are generally maintained.

15. APPLICATIONS OF ELISA Detection of HIV antibodies Detection of microorganisms and the toxins produced by them. For example: Salmonella, Listeria , Bacillus cereus enterotoxin . Food Allergens - It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs. Serological blood test for  coeliac disease .  ELISA can also be used in t oxicology as a rapid presumptive screen for certain classes of drugs. ELISA can be used as a detection method for detection of Mycobacterium antibodies in tuberculosis, detection of rotavirus in faeces , detection of hepatitis B markers in serum, detection of enterotoxin of  E. coli  in faeces , detection of HIV antibodies in blood samples .

16. COMPONENETS IN ELISA KIT Immunosorbent : Solid supporter which has been coated with antigens & antibodies, can be stored in low temperature of about 2-8°C. Polystyrene is generally used in ELISA Conjugate: Conjugate is the antibody (antigen) linked with the enzyme, and is the key substance for ELISA. A good conjugate possess not only catalytic activity of enzyme but also immunological competence of antibody. It should be stable and must be least contaminated. Substrate : the chemical activity of the substrate will be compromised if it is exposed to light or comes into contact with the metal. Controls: Need to be diluted and then added to the plate. Sample Diluents & Wash Solution Stop solution

17. ELISA DEVICE ELISA pipettes Can be single or multi channeled.  Adjustable -volume (1–20 μL, 10–100 μL, 20–200 μL, etc.) Washer systems Manual or semi/fully automated systems that wash one row or column at a time ELISA plate readers Manual or semi/fully automated readers that read one row or well at a time

18. ELISA DETECTION STRATERGIES Chromogenic Assay - The substrate forms a colored product under the catalysis of the enzyme catalysed, which is directly related to the amount of test substance. The Optical Density is proportional to the concentration of the analyte to be measured. Chemiflourescent Assay- An enzyme converts a substrate to a reaction product that fluoresces when excited by light of a particular wavelength. Chemiluminescent Assay- The chemiluminescent substance is excited by the oxidation and catalysis forming intermediates. When the intermediates return back to their stable ground state, a photon is released, which is detected by the luminescent signal instrument

19. FOOD ALLERGIES Food allergies are reaction to food proteins. They may be categorized as Immunoglobulin E ( IgE )–mediated (immediate) reactions- Cause flushing, itchy skin, wheezing, vomiting, throat swelling, and even anaphylaxis immediately after exposure. Non– IgE -mediated (delayed) hypersensitivity reactions- Cause localized (e.g., contact dermatitis) or generalized reactions, which are usually gastrointestinal or dermatological in nature. Some allergic disorders are both IgE and non- IgE or mixed reactions .

20. ELISA FOR FOOD INDUSTRY Certain allergens are responsible for 90% food allergen cases. ELISA can detect potential allergic reactions in the body. Traditional food allergen detection uses ELISA to find protein.   Because the ELISA is protein-based, it is proficient at determining the exact source of an allergen, such as dairy or wheat.

21. EXAMPLES IN FOOD INDUSTRY CASEIN: Casein is a source of food allergies and must be excluded from the diet of susceptible individuals. Because it is present in high levels in milk, casein is a useful indicator of the presence of milk residue. This has applications in Non Dairy Products which can be tested for any presence of milk protein. Takes 45 minutes. HAZELNUTS : These nuts contain several allergenic proteins that can pose a serious threat to allergic consumers.  Hazelnut proteins in the presence or absence of soluble wheat proteins are modified with glucose via the Maillard reaction. Changes in hazelnut proteins , such as the formation of protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe aggregation dramatically affects the hazelnut protein detection by the ELISA kits. The observed impact was highly dependent on the type of ELISA kit used.

22. EXAMPLES IN FOOD INDUSTRY GLUTEN: Gluten contains proteins like gliadin and glutenin found in seeds of wheat, barley and rye. Around 1% of the population of North America & Europe is sensitive to gluten . Therefore, adoption of gluten free diet is mandatory avoiding foods containing wheat, rye, barley and other related cereal grains.  There are possibilities that traces of these seeds are present in the food. Thus, ELISA kits are used to determine the traces of gluten. ELISA Technologies also offers a rapid, lateral flow gluten test —  EZ Gluten .

23. HOW GLUTEN TEST WORKS ?? Food sample is ground to a fine consistency. Then added to gluten extraction solution and then mixed. Few drops of sample extract are placed in a test tube. EZ Gluten test strip is placed in a test tube to absorb the sample extract. After 10 mins , strip can be read.

24. ADVANTAGES Highly sensitive due to enzyme ‘linkage’. Small amount of enzyme can induce large amount of catalytic reactions. Strongly specific due to the selectivity of the antibody or the antigen. Detects the actual allergen protein molecule. Flexible; more than one systems can be used to measure the same analyte.

25. DISADVANTAGES Some matrices can interfere with the ELISA method, such as chocolate. Can cause cross reactivity as seen between different types of nuts. This method is also not the most suitable for cooked or heated products because the protein molecules are denatured or broken down and the allergen is no longer detectable.

26. RECENT INVENTION Scientists at Imperial College London in 2012 developed PLASMONIC ELISA technology for detecting a variety of pathogens that needs nothing but a eye to read the results.  Prostate specific antigen (PSA ) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10 −18  g ml −1 .   When blood serum is introduced into a small plastic bottle containing a special mix that includes gold nanoparticles , they combine in specific ways depending on whether an analyte being investigated is present or not.  If it is, the solution turns a shade of blue; if not, it turns red .

27. REFERENCES Research paper : DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF PECAN RESIDUES IN PROCESSED FOODS by Denise Tran ELISA Encyclopedia by Sino Biological Inc. ELISA Technologies, Laboratory Testing services and Diagnostic Kits website. Wikipedia website