ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA is a biochemical technique used mainly in immunology to detect the presence of an antigen or antibody in a sample.

• Used to detect both antigen and antibody.
• Detection based on → Enzyme catalysed reaction or fluorescent probe.
• Uses an immune reaction like RIA (Radioimmunoassay)  

Advantages of ELISA ›  

•   Antigens and antibodies detectable at leavels of about 10^-10 g/ml or 1 part in 10 billion. (Sensitive at nanogram levels or lower.
• Less expensive 
•  Safer (No radiation hazards) 
•  Reliable and accurate as (RIA) 
•  High speed
• Minimal reagents
• Reproducible
• Quantitative e.g. Thereyptic Drug monitoring (TDDM)
• Qualitative e.g. HIV testing
•  Greater scope › wells can be coated with antibodies or antigens

Principle ›

The principle of ELISA is based on these two observations:

1. Antibodies and antigens can attach to polystyrene plastic plates (or other solid-phase supports) and still maintain their full immunologic capabilities
2. Antibodies and antigens can be bounded to enzymes, and the resulting complexes are still fully functional both immunologically and enzymatically.

It is enzyme activity which is measure of quantity of antigen or antibody present in test sample.

Components of Test ›

  1. Enzymes ► Antiglobulin linked with enzyme β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase. Horseradish peroxidase (HRP) glycoprotein with four lysine residues. The enzyme acts as a catalyst to oxidize substrate in presence of Hydrogen peroxide to produce a blue colour. Reaction stopped with dilute acid to cause complex to turn yellow. 

  2. Antibody ►IgG fraction of serum (Purified by affinity chromatography). Reagent proteins include antibodies such as goat-anti-human-IgG. Or, bacterial proteins that bind to antibodies such as Staphylococcal protein A or Streptococcal protein G. 

Biotin and Avidin also used. Avidin has several high-affinity binding sites for biotin thus it can be used to bridge molecules that have been “tagged” with biotin. 

   3. Substrate ►TMB (3,3’,5,5’-tetramethyl benzidine), P-nitrophenyl phosphate. 

   4. Solid phase (to which proteins can sticks) ►Plastic (Polystyrene) microtiter plate.

Types of ELISA ›

1.    . Sandwitch ELISA
    2. Indirect ELISA 
    3.Competitive ELISA 
   
   
   Sandwitch ELISA ►
   
   
                                                                           

                                             Antibody (antiserum) adsorbs to well surface  

                                                                             

 

Test antigen is added and binds to antibody (antiserum) if it is homologous. Plate washed to remove unbound antigen.

Enzyme-labeled specific antibody (Detection antibody) is added and binds to test antigen forming an antibody-antigen-antibody “Sandwitch”. Plate washed to remove unbound antibody-enzyme conjugates.

Enzyme substrate is added. Degradation of substrate molecules (Hydrolysis) is accompanied by a colour change (By visually or by colorimetry)

 

• The rate of enzyme action is directly proportional to quantity of enzyme-labeled antibody present and that, in turn, is proportional to amount of test antigen.

☺USES› Assay Hepatitis B antigen with great success.

 

Indirect ELISA ►

 

Antigen adsorbs to the well surface. A solution of nonreacting protion, such as bovine serum albumin or casein, is added to well (usually 96 well plates) any plastic surface in well that remains uncoated by antigen.

 

Test antiserum is added and specific antibody will bind to antigen (Primary antibody could also be in the serum of donor to be tested for reactivity towards antigen).

Enzyme-labeled antihuman antibody is added and will link to specific antibody previously bound.

Enzyme substrate is added. Degradation of the substrate molecules is accompanied by a colour change.

• Higher the concentration of primary antibody present in serum→stronger colour change.

• Enzyme acts as an amplifier of signals.

Disadvantage › Antigen immobilization is not specific; when serum is used as source of test antigen, all proteins in sample may stick to microtiter plate well, so small concentration of analyte in serum must compete with other serum proteins when binding to well surface.
Uses ›
  1. Immunodiagnosis of many infectious pathogens such as viruses, parasites and fungi. 

  2. Antiserum sample from pregnant woman can be screened simultaneously for infections of    

       Rubella virus (agent for german measles, which causes congenital malformations and fetal death) 

       Type 2 Herpes virus (causes severe congenital nervous system malformations and small heads).  

Applications ›

1. Determining serum antibody concentration (such as with HIV test or West Nile virus) 
2. In food industry in detecting potential food allergens, such as milk, peanuts, walnuts,   almonds, eggs.Measuring toxins in contaminated food.  
3. Used in toxicology as a rapid presumptive screen for certain classes of drugs. 
4. Dr Dennis E Bidwell and Alister Voller created the ELISA test to detect various kind of diseases such as Malaria, Chagas disease, Johne’s disease.  
5.  In vitro diagnostics in medical laboratories in medical laboratories. 
6. Detection of Mycobacterium Antibodies in tuberculosis. 
7. Detection of rota virus in feces. 
8.  Detection of Hepatitis B markers in serum
9. Detection of enter toxin of E.Coli in feces
10. Detection of HIV antibodies in blood samples. 
11. Detection of Rota virus and Hepatitis A virus (stool samples) which are not grown in cell culture.   
12. Assay of 

         a). Measuring levels of Hormones→ Oestrogen, Progesterone,        

                                                                     T₃, T₄, TSH (for thyroid function)

                                                                      LH (determining the time of ovulation)

                                                                      HCG (as a test for pregnancy).

        b). Oncoprotein (α_2-Hepatoglobulin)

        c). Snake poisoning

        d). Virological infections

            →Hepatities A Antigen

            → Hepatities B Antigen

            →Herpes simplex

            →Rota

            →Rubella

            →E.B.

            →CMV

            →Antibodies to Influenza and Measle viruses

     e). In bacteriological conditions

            →Enteric infections,

            →E.Coli antibodies in UTI in urine specimen

            →Mycobactrium tuberculosis

            →Leprosy

    f).Other infections→ Syphilis, Chlamydia, Toxoplasma gondii
 

13.  Fungal Diseases→ Candidiasis, Aspergillosis    
14. Measuring rheumatoid factors
15. Therapeutic drug monitoring→Barbiturates, Morphine, Digoxin.
16. Analysis of hormones, metabolites, vitamins, diagnostic markers→ Eg. ACTH, T3, T4, FSH,   
     Glucagon, Insulin, Testosterone, Vitamin B12, Prostaglandins, Glucocorticoids.