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Blotting & Elisa Techniques Bio-Technology

The document provides an overview of various blotting techniques including immunoblotting, western blotting, southern blotting, and northern blotting, each used for detecting specific biomolecules like proteins and nucleic acids. It details their procedures, applications in diagnostics, and highlights the differences between them, such as the types of molecules they detect. Additionally, the document discusses the ELISA technique for antigen detection, its types, applications, advantages, and challenges.

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Biotechnology unit-4
DEFINITION OF BLOTTING • Visualization of specific DNA , RNA & protein among many thousands of contaminating molecules requires the convergence of number of techniques which are collectively termed BLOT transfer.
IMMUNOBLOTTING • Viral antigens are detected with a polyclonal or an MAb onto nitrocellulose paper. • After incubation, the protein bands (immune complexes) are visualized with peroxides-conjugated protein and a color reagent. • A color develops in the bands where the antibody binds to the antigen. • Immunoblotting assay mixture of these two technique. WESTERN BLOTTING •Western blotting is based on the principles of immunochromatography, where proteins are separated into polyacrylamide gel according to the isoelectric point and molecular weight.
•A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. •Immunoblotting is performed chiefly in diagnostic laboratories to identify the desirable protein antigens in complex mixtures. • An improved immunoblot method Zestern analysis, is able to address this issue without the electrophoresis step, thus significantly improving the efficiency of protein analysis. • Other related techniques include dot blot analysis, zestern analysis, immunohistochemistry where antibodies are used to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA)
ELISA Enzyme-linked immunosorbent assay (ELISA): ELISA stands for "enzyme- linked immunosorbent assay." This is a rapid immunochemical test that involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). ELISA tests are utilized to detect substances that have antigenic properties, primarily proteins (as opposed to small molecules and ions such as glucose and potassium). Some of these include hormones, bacterial antigens and antibodies. TYPES OF ELISA 1- Direct ELISA 2- Indirect ELISA 3- Sandwich ELISA 4- Competitive ELISA
Diseases That Can Be Diagnosed Using ELISA ELISA can be used to detect some of these conditions: •Ebola •Pernicious anaemia •AIDS •Rotavirus •Lyme disease •Syphilis •Toxoplasmosis •Zika virus •Carcinoma of the epithelial cells
Applications of ELISA •The applications of ELISA are discussed below: •The presence of antibodies and antigens in a sample can be determined. •It is used in the food industry to detect any food allergens present. •To determine the concentration of serum antibody in a virus test. •During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample.
The technique was first described by Harry Towbin in 1979 and named by W. Neal Burnette in 1981. The procedure of western blotting can be understood by the following points: Gel Electrophoresis: The protein samples are separated using gel electrophoresis. SDS-PAGE is commonly used for this step. Transfer: The separated proteins are transferred on a solid support as nitrocellulose or polyvinylidene difluoride membrane. Total Protein Staining: The proteins transferred on the membrane are visualised by staining the membrane using dyes such as Ponceau S, Coomasie R-350 or amido black.
Blocking: This step is done to block the interaction between the membrane and the antibody that will be applied in the next step. Since the target molecule and antibodies are both proteins, there are chances that the membrane might interact with the antibody. Incubation: An antibody with a reporter enzyme is probed on the membrane. A substrate that is specific to the enzyme is applied on the surface. If binding happens, a colourimetric reaction is produced. Detection and Visualization: The unbound probes are washed away and the gel is visualised for the bound proteins.
Applications •Detection of particular protein from a mixture of proteins. •Size and amount estimate of proteins in the mixture. •Verification following a high sensitivity ELISA test for diagnosis of Lyme, HIV infection, BSE, HBV and so on. •Detect condensed isoforms of proteins as well as tagged proteins. Advantage •Effective early diagnostic tool. •Detect minimal immunogenic response form virus or bacteria. •Requires fewer antibodies for testing. •Detect specific protein from a large mixture of different proteins. (Even more than 300,000).
Disadvantages •Requires specific primary antibodies to perform test on desired protein of interest. •Challenging and hence requires well trained staffs. •Poorer results as antibodies may revel off-target bindings. •Detecting and imaging the results can be expensive as equipment cost is high.
Southern blotting Southern blotting is a molecular biology technique used for DNA detection, characterization, and quantification. An example of RFLP(restriction fragment length polymorphism), southern blotting can be defined as an analytical technique for identifying specific sequences of DNA by separating fragments on a gel and transferring them to a second medium (carrier membrane) on which hybridization testing may be carried out. During southern blotting, the DNA fragments are immobilized as a result, the membrane carries a semi-permanent reproduction of the banding pattern of the gel. The DNA are then exposed to hybridization analysis allowing bands with sequence resemblance to a labeled probe to be identified.
Procedure of Southern Blotting 1. Extract and purify DNA from cells We separate the DNA to be tested from the rest of the cellular material in the nucleus. We then incubate the specimen with detergent to promote cell lysis (frees cellular proteins and DNA). Proteins are removed through organic and non-organic extraction. We then use alcohol precipitation to purify the DNA from the solution. Visible DNA fibers are removed and suspended in buffer. 2. DNA fragmentation We use restriction endonuclease enzyme to break long nucleotide sequence into smaller fragments for purification or identification process.
3. Gel electrophoresis Sorts the complex mixture of DNA fragments according to size. The percentage and size of the gel to be used must be determined. Gels consist of microscopic pores and are solid (agarose/Polyacrylmide) Generally, 0.7 – 2% gel is considered to be adequate for most of the applications. Nucleic acids have negative charge and move from left to right. The large molecules are held up while smaller ones move faster causing separation by size. Gels are stained with ethidium bromide to permit photography under UV light. A single band form is given to intact high quality DNA.
4. Denature DNA DNA obtained are double stranded in nature. Alkalis are used to denature the restriction fragments in the gel that makes double stranded DNA to become single stranded. To avoid re-hybridization, we use NaCl so that DNA is neutralized. 5. Blotting Transfer DNA from the gel to solid support (carrier membrane). We dry the blot (around 80°C) or use UV radiation to make it permanent. 6. Washing Despite using blocking agents, some excess probe binds to the membrane. Wash buffers containing NaCl and detergent washes away the excess probe.
Applications of Southern Blotting •Identify specific DNA from a DNA sample. •Identification of viral and bacterial infections. •Important in the study of gene mutation, deletion and rearrangements. •DNA fingerprinting (maternity and paternity analysis, forensic studies and personal identification). •Diagnosis of neonatal and genetic diseases including cancer. •Discovery of RFLP (restriction fragment length polymorphism) to map crucial genomes.
Northern Blotting •Purpose: Detects specific RNA sequences (and their abundance) within a sample. •Key Difference: Targets RNA, unlike Southern blotting (DNA) or Western blotting (proteins). •Steps: • RNA Isolation: Extract RNA from cells/tissues. • Gel Electrophoresis: Separate RNA fragments by size using an electric field. • Transfer: Blot RNA from gel onto a membrane (usually nylon or nitrocellulose). • Hybridization: Incubate membrane with a labeled probe (DNA or RNA) that's complementary to the target RNA sequence. • Detection: Visualize the probe-RNA complex using: • Radioactive Labeling: Probe emits radiation, detected by X-ray film. • Non-Radioactive Labeling: Probe linked to an enzyme that produces a colored or chemiluminescent signal. Applications: •Gene Expression Studies: Determine if a gene is active (producing RNA) in a sample. •RNA Sizing: Estimate the size of specific RNA transcripts. •RNA Quantification: Compare relative amounts of RNA in different samples. •Medical Research: Study RNA viruses, gene expression changes in diseases. Advantages: •Sensitive: Detects even low levels of RNA. •Specific: Identifies unique RNA sequences. •Quantitative: Provides information on RNA abundance.
Blotting & Elisa Techniques Bio-Technology