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Education Marks Proper Humanity


Education Marks Proper Humanity


Education Marks Proper Humanity


Education Marks Proper Humanity


Education Marks Proper Humanity

Showing posts with label CSIR-NET-LIFE-SCIENCE. Show all posts
Showing posts with label CSIR-NET-LIFE-SCIENCE. Show all posts

Sunday, November 29, 2020

Monoclonal antibodies & Hybridoma technology- CSIR-NET/ICMR/DBT (Life Sciences)

Monoclonal antibodies & Hybridoma technology

Monoclonal antibodies (mAb)

  • Antibodies produced by infection or immunization are polyclonal because natural antigens have multiple epitopes, each o which generates clones of lymphocytes.
  • This results in antisera containing antibodies from different clones of lymphocytes with specificities against different epitopes of antigens.
  • Monoclonal antibodies (mAb) are monospecific antibodies. These antibodies are produced from a clone of single lymphocytes directed against a single antigenic determinant or epitope.

Hybridoma technology: 

  • It is a method of forming hybrid cell lines (called hybridomas) by fusing a specific Ab-producing B-cell with myeloma (cancerous B-cell).
  • The production of mAb was invented by Cesar Milstein and Georges J.F Kohler in 1975, and they shared the Nobel Prize in 1984 for Medicine & Physiology.
  • A hybridoma is somatic cell hybrids produced by fusing antibodies forming spleen cells with myeloma cells.
  • Ab- producing B-cells normally die after several weeks in cell culture, in vitro. Therefore, Ab- producing B-cells are fused with B-cell tumors called myelomas. These myelomas are capable of dividing indefinitely and are called immortal cell lines.
  • The immortal cell lines that result from the B cell-myeloma fusion are hybrid cell lines are called hybridomas.
  • The hybridoma cell lines share the properties of both fusion partners. They grow in vitro and produce antibodies.
  • Hybridomas are selected by the use of a selective medium in which the myeloma cells die, but hybridomas survive. The most widely used selective medium involves the inclusion of the antibiotic aminopterin in the growth medium.

Aminopterin (Selective medium):

  • It is a synthetic derivative of pterin. 
  • This folate analog acts as a competitive inhibitor for the enzyme dihydrofolate reductase which catalyzes the reduction of dihydrofolate into tetrahydrofolate.
  • Normal animal cell synthesizes purine nucleotides and thymidylate for DNA synthesis by a de novo pathway requiring tetrahydrofolate. Thus, the addition of aminopterin inhibits the de novo nucleotide synthesis pathway.
  • However, normal cells survive in this medium as they are able to use the salvage pathway for nucleotide acid synthesis.
  • But, if the cells are unable to produce the enzyme Hypoxanthine-Guanine Phospho Ribosyl Transferase (HGPRT), they are unable to utilize the salvage pathway, therefore, die in the aminopterin-containing medium.

Production of monoclonal antibodies (mAb) step-to-step:

  • During the next several weeks, antigen-specific B-cells proliferate and begin producing antibodies in the mouse.
  • Spleen tissue, rich in B-cells, is then removed from the mouse, and the B-cells are fused with myeloma cells.
  • Myeloma cells are engineered to be deficient in the enzyme Hypoxanthine-Guanine Phospho Ribosyl Transferase (HGPRT -ve).
  • After fusion of lymphocytes with HGPRT negative myeloma cells, aminopterin-containing medium, supplemented with hypoxanthine and thymidine to ensure an adequate supply of substrate for the salvage pathway (HAT medium) is added, which kills myeloma cells but allows hybridomas to survive as they inherit HGPRT from the lymphocyte present.
  • Unfused lymphocytes die after a short period of culture, which results in a pure preparation of hybridomas.

Saturday, November 28, 2020

Culture Technique - CSIR-NET/ICMR/DBT (Life Sciences)

 Culture Technique- Microbiology

I. Culture Techniques

  • Microbiology as a science is very dependent on several specialized laboratory techniques. Laboratory steps routinely employed in microbiology are- Inoculation, Incubation, Isolation, Inspection, and Identification.
  • To prepare a culture (visible growth specimen), a medium (nutrient substrate) is inoculated (implanted, seeded) with a sample.
  • Methods of Isolation: Cells are spread over a large area (streak plate and spread plate) or are diluted in a large volume (pour plate) so that individuals cells are completely separated and can grow into colonies.
  • The cultures are incubated, subcultured, observed microscopically, and identified by the morphological, physiological, genetic methods.


II. Artificial Media: 

Artificial nutrient media vary according to their physical form, chemical characteristics, and purpose.

A. Physical Subgroups:

  • They can be liquid (broth, milk), semisolid, or solid, depending upon the absence or the quantity of solidifying agent (usually agar and gelatin).

B. Chemical Subgroups:

  • A synthetic medium is any preparation that is chemically defined, but a medium containing a poorly identified component is nonsynthetic.

C. Functional Subgroups:

1. A large general-purpose medium:  It is used to grow a wide assortment of microbial types. Example: Tryptic soy broth (TSB).

2. Enriched-medium: It is supplemented with blood or tissue infusion to culture fastidious species. Example: Sheep-blood agar, chocolate (heated blood) agar.

3. Selective-medium: It permits preferential growth of certain organisms in a mixture and inhibits others. Highly selective media are used expressly to favor the growth of one organism over another. 

Example: Mueller telluride, Enterococcus faecalis broth, Phenylethanol agar, Tomato juice agar, Salmonella/Shigella (SS) agar, Lowenstein-Jensen. 

  • Sabouraud's dextrose agar = pH of 5.6  discourages bacterial growth. Used to isolate fungi.
  • Brilliant green agar = Green dye selectively inhibits gram-positive bacteria. Used to isolate gram-negative Salmonella.
  • Bismuth sulfite agar = Used to isolate Salmonella typhi. Inhibits the growth of most other bacteria.
  • MacConkey agar = used to distinguish and select for Salmonella.
4. Differential medium: It distinguished among different microbes by bringing out their variations in a particular reaction. 

Example: Blood agar, Eosin methylene blue (EMB) agar, Spirit blue agar, Urea broth, Surfur indole motility (SIM), Triple-sugar iron agar (TSIA), XLD agar.

  • Mannitol salt agar = used to distinguish and select for Staphylococcus aureus.

6. Carbohydrate Fermentation medium: The ability to utilize sugars can be determined with a carbohydrate fermentation medium.

7. Transport medium: To covey fragile microbes, special transport media are needed to stabilize viability.

8. Assay medium: It is used to evaluate the effectiveness of antimicrobial agents.

9. Enumeration medium: Environmental and Industrial surveillance routinely call for enumeration media to determine the number of microbes in food, drinking water, soil, sewage, and other resources.

10. In certain instances, microorganisms have to be grown in animals and bird embryos.


Friday, November 13, 2020

Cell Signaling - CSIR-NET (Life Sciences)

 Cell Signaling

  •  All cells receive and acknowledge signals from their surroundings. 
  • This is competent by a variety of signal molecules that are secreted or expressed on the surface of one cell and bind to receptors expressed by other cells, thereby integrating and coordinating the functions of the many individual cells that make up organisms.
  • Each cell is programmed to acknowledge specific extracellular signal molecules.

Extracellular signaling usually involves the following steps:

  • Synthesis and deliverance of the signaling molecules by the signaling cell;
  • Transport of the signal to the target cell;
  • Binding of the signal by a specific receptor leading to its activation;
  • Initiation of signal-transduction pathways.

Endocrine Signaling:

  • In endocrine signaling, the signaling molecules act on the target cells distantly located from their site of synthesis.
  • It is long-range signaling in which the signal molecule is transported by the bloodstream.
Fig: Endocrine signaling

Paracrine Signaling:

  • In paracrine signaling, the signaling molecules released by a cell affect target cells only nearby. An example of this is the action of neurotransmitters in carrying signals between nerve cells at a synapse.
Fig: Paracrine signaling

Autocrine Signaling:

  • In autocrine signaling, the signaling molecules produce an effect on the same cell that produces it.
  • One important example of such is the response of cells of the vertebrate immune system to foreign antigens.
  • Certain types of T-lymphocytes respond to antigenic stimulation by synthesizing a growth factor that derives their own proliferation, thereby increasing the number of responsive T-lymphocytes and amplifying the immune response.
Fig: Autocrine signaling

Juxtacrine Signaling: Signaling across gap junctions.

  • In juxtacrine signaling, signal molecules do not diffuse from the cell producing it, and cell bearing signal molecules interact with the receptor proteins of adjacent responding cells.
  • Unlike another mode of cell signaling, it requires physical contact between the cells involved.
  • Notch signaling and classical cadherin signaling are examples of juxtacrine signaling.


Wednesday, September 30, 2020

Chromosomal Basis of Inheritance - CSIR NET

Chromosomal Basis of Inheritance

In 1902, Walter S. Sutton and T. Boveri proposed the chromosomal theory of heredity.

  • The theory provides a way to explain how cellular transmission or chromosomes passes genetic determinant (i.e. genes) from parent to offspring.

According to this view:

  • Chromosomes are replicated & passed along generation after generation from parent to offspring.
  • One member of each pair segregates into one daughter nucleus & the other segregates into different daughter nucleus. Therefore, gametes contain one set of chromosomes (i.e. they are haploid).
  • During gamete formation, different types of chromosomes segregate independently of each other.
  • Each parent contributes one set of chromosomes to its offspring. Hence, the chromosome theory of inheritance describes the relationship between Mendel's Law & chromosomal transmission.
Fig: Segregation of homologous chromosome during meiosis explains Mendel's law of segregation


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