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ELISA ( Enzyme Linked Immunosorbent Assay ) : It is a common type of enzyme immunoassays, which depends on a enzyme . In this type , an enzyme linked with an antibody reacts with colorless substrate to generate a colored product .i.e. chromogenic substrate . Enzymes : Enzymes that can be used for ERead more
ELISA ( Enzyme Linked Immunosorbent Assay ) : It is a common type of enzyme immunoassays, which depends on a enzyme . In this type , an enzyme linked with an antibody reacts with colorless substrate to generate a colored product .i.e. chromogenic substrate .
Enzymes : Enzymes that can be used for ELISA are alkaline phosphatase , horseradish peroxidase , urease and beta galactosidase.
Types of ELISA : It can be divided into 2 types : Qualitative and Quantitative.
A) Qualitative ELISA: it provides simple results for a sample i.e. positive or negative result .
B ) Quantitative ELISA: it provides results in form of standard curve i.e. concentration of the target molecule in a sample via standard curve.
There are main 4 categories of ELISA :-
1) Direct ELISA :- it’s one of the simplest ELISA technique.
Process : a) addition of antigen- The antigen present in the sample is first immobilized to the wall of the wells of microtiter plate , then wells are washed thoroughly leaving behind only the adsorbed antigen.
b) addition of enzyme linked antibody – an enzyme linked antibody ( complementary to the antigen of interest) is added to the wells where it would bind to the antigen . Then well is again washed and it would leave bound antigen-antibody complex on the surface of well .
c) addition of substrate- A substrate is added, that will be converted by the enzyme-linked with antibodies into a detectable product.
d) detection – it can be done on the basis of color, fluorescence, or luminescence.
2) Indirect ELISA :- in this method , 2 different type of antibodies are used , in which secondary antibody would bind to the enzyme and primary antibody would only act as a bridge between the antigen and secondary antibody .
This method can be used to detect the presence of antibodies against HIV. In this test ,
A) antigens are added to the bottom of the well.
B) primary antibodies from patient are added to the coated well and allowed to bind to the antigen.
C) secondary enzyme linked antibodies to the human antibodies are allowed to react in the well. Then unbound antibodies are removed by washing.
D) enzyme substrate is added and amount of colored product that forms is measured.
3) Sandwich ELISA :- in this method , an antigen can be detected.
process – a) addition of antibody -the antibody is immobilized to the microtiter well. These antibodies would bind to the specific antigen present in the sample with the high affinity .
b) addition of secondary antibody – Then well is washed and a second enzyme linked antibody specific to different epitope on the antigen is added and allowed to bind to the antigen . Then free secondary antibodies are washed away.
c) addition of substrate- The addition of substrate is followed by for the enzyme and the amount of colored product that form is measured.
4) Competitive ELISA :- it’s one of the complex techniques of the ELISA. It involve use of inhibitor antigen , that’s why also known as inhibition ELISA. in this method , 2 antigens compete with each other in order to binding to antibodies.
process- a) addition of primary antibody- The unlabeled primary antibody is first incubated with sample having the antigen of interest. It would lead to formation of antigen-antibody complex.
b) binding with inhibitor antigen- Antigen- antibody mixture is added to the plate coated with inhibitor antigen. The free primary antibody in mixture binds to the inhibitor antigen on plate. Then plate is washed off .
c) addition of secondary antibody- the enzyme labeled secondary antibody is added to plate and binds to primary antibody bound to inhibitor antigen .
d) addition of substrate and detection- Substrate is added to react with enzyme and emit a visible signal for detection.
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PCR (Polymerase Chain Reaction) amplification is a fundamental technique used in molecular biology to exponentially replicate specific segments of DNA. It revolutionized genetic research by enabling the rapid production of billions of copies of a targeted DNA sequence from a minute starting materialRead more
PCR (Polymerase Chain Reaction) amplification is a fundamental technique used in molecular biology to exponentially replicate specific segments of DNA. It revolutionized genetic research by enabling the rapid production of billions of copies of a targeted DNA sequence from a minute starting material.
The process involves several key steps:
1. **Denaturation**: The DNA sample, containing the target sequence, is heated to around 95°C, causing the double-stranded DNA to separate into two single strands.
2. **Annealing**: The reaction is cooled to a temperature typically between 50-65°C. Short DNA sequences called primers, which are designed to bind to specific regions flanking the target sequence, anneal or bind to their complementary sequences on the single-stranded DNA.
3. **Extension**: The temperature is raised to about 72°C, which is optimal for DNA polymerase activity. DNA polymerase, an enzyme, extends the primers by synthesizing new DNA strands complementary to the template DNA.
These three steps are repeated in cycles (usually 20-40 cycles), each cycle doubling the amount of DNA, resulting in an exponential amplification of the target DNA sequence. The final product is a large amount of DNA identical to the original target sequence, which can then be used for various applications in research, diagnostics, forensic analysis, and more.
PCR amplification is highly precise, sensitive, and has numerous applications, including genetic testing, sequencing, cloning, and detecting genetic mutations or pathogens.
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