study matter for pharm-d 1st year

1.lipid profile tests

                                                       Lipoprotein structure (chylomicron)
                        ApoA, ApoB, ApoC, ApoE (apolipoproteins); T(triacylglycerol); C (cholesterol); green (phospholipids)
A lipoprotein is a biochemical assembly that contains both proteins and lipids, bound to the proteins, which allow fats to move through the water inside and outside cells. The proteins serve to emulsify the lipid (otherwise called fat) molecules. Many enzymes,transporters, structural proteins, antigens, adhesins, and toxins are lipoproteins. Examples include the plasma lipoprotein particles classified under high-density(HDL) and low-density (LDL) lipoproteins, which enable fats to be carried in the bloodstream, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins.

functions

The handling of lipoprotein particles in the body is referred to as lipoprotein particle metabolism. It is divided into two pathways, exogenous and endogenous, depending in large part on whether the lipoprotein particles in question are composed chiefly of dietary (exogenous) lipids or whether they originated in the liver (endogenous), through de novo synthesis of triacylglycerols.

The hepatocytes are the main platform for the handling of TGs and cholesterol; the liver can also store certain amounts of glycogen and triacylglycerols. Intriguingly, adipocytes, though being the main storage cells for triacylglycerols, do not produce any kind of lipoprotein particle.

http://www.srmuniv.ac.in/downloads/Lipid_Profile_Tests.pdf

2.nucleic acid metabolism

3. 4. 5. 6. 7. 8. 9. 10. 11.

12.digestive system ppt. 

13.respiratory system ppt.

14.urinary system ppt.

15.sports and physiology
http://www.dikseo.teimes.gr/spoudastirio/E-NOTES/P/Performance_Enhancing_Drugs_Viewpoints.pdf

16.RADIO IMMUNO ASSAYRadio Immuno m

DEFINITION:a sensitive immunological assay, making use of antibodies and radioactive labelling, for the detection and quantification of biologically important substances, such as hormone concentrations in the blood.

The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,

• blood banking
• diagnosis of allergies
• endocrinology

The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay.

The Technique

• A mixture is prepared of
  • radioactive antigen
    • Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes ¹²⁵I or ¹³¹I are often used.
  • antibodies ("First" antibody) against that antigen.
• Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.
• At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules.
• The antibody-bound antigen is separated (see below) from the free antigen in the supernatant fluid, and
• the radioactivity of each is measured.
• From these data, a standard binding curve, like this one shown in red, can be drawn.

• The samples to be assayed (the unknowns) are run in parallel.
• After determining the ratio of bound to free antigen ("cpm Bound/cpm Free") in each unknown, the antigen concentrations can be read directly from the standard curve (as shown above).

• Separating Bound from Free Antigen

  There are several ways of doing this.
  
  • Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first. For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by adding an antirabbit-IgG antiserum (e.g., raised by immunizing a goat with rabbit IgG). This is the method shown in the diagram above.
  • The antigen-specific antibodies can be coupled to the inner walls of a test tube [View another example]. After incubation,
    • the contents ("free") are removed;
    • the tube is washed ("bound"), and
    • the radioactive of both is measured.
  • The antigen-specific antibodies can be coupled to particles, like Sephadex. Centrifugation of the reaction mixture separates
    • the bound counts (in the pellet) from
    • the free counts in the supernatant fluid.
  Radioimmunoassay is widely-used because of its great sensitivity. Using antibodies of high affinity (K₀ = 10⁸–10¹¹ M⁻¹), it is possible to detect a few picograms (10⁻¹² g) of antigen in the tube.
• The greater the specificity of the antiserum, the greater the specificity of the assay.
• The main drawbacks to radioimmunoassay are the expense and hazards of preparing and handling the radioactive antigen.
  
  • Both ¹²⁵I or ¹³¹I emit gamma radiation that requires special counting equipment;
  • The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T₄).
  • 
    
  • Despite these drawbacks, RIA has become a major tool in the clinical laboratory where it is used to assay
    • plasma levels of:
      • most of our hormones;
      • digitoxin or digoxin in patients receiving these drugs;
      • certain abused drugs

for the presence of hepatitis B surface antigen (HBsAg) in donated blood;anti-DNA antibodies in systemic lupus erythematosus (SLE).

17.ELISA(enzyme-linked immunosorbent assay)

Definition:A sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. It is often used as a diagnostic test to determine exposure to a particular infectious agent, such as the AIDS virus, by identifying antibodies present in a blood sample.

                                                        A 96-well microtiter plate used in elisa

ELISAs are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with a protein to which will bind the antibody you want to measure. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies (called primary antibodies). The serum is incubated in a well, and each well contains a different serum (see figure below). A positive control serum and a negative control serum would be included among the 96 samples being tested.

Main ELISA types

*Direct ELISA

- direct detection (primary antibody is conjugated with the enzyme/tag) 

*Indirect ELISA – signal amplification

- indirect detection (labelling the secondary antibody - produced

against the primary antibody type; e.g. anti-mouse IgG, a rabbit Ab)

*Sandwich ELISA – more efficient signal amplification

- indirect antigen immobilization (by capture antibody)

- indirect detection (labelled secondary antibody)

*Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen (sample).
2. These bound antibody/antigen complexes are then added to an antigen-coated well.
3. The plate is washed, so unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition".)
4. The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
6. The reaction is stopped to prevent eventual saturation of the signal.

A "sandwich" ELISA, is used to detect sample antigen. The steps are:

1. A surface is prepared to which a known quantity of capture antibody is bound.
2. Any nonspecific binding sites on the surface are blocked.
3. The antigen-containing sample is applied to the plate.
4. The plate is washed to remove unbound antigen.
5. A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is stuck between two antibodies)
6. Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the antibody's Fc region (nonspecific).
7. The plate is washed to remove the unbound antibody-enzyme conjugates.
8. A chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical signal.
9. The absorbency or fluorescence or electrochemical signal (e.g., current) of the plate wells is measured to determine the presence and quantity of antigen.

For additional info please check the link:http://www3.szote.u-szeged.hu/hurodocs/downloads/biochemistry/lectures/ELISA_lecture_HURO_Keresztes_final_small%20.pdf

18.Active Transport vs Passive Transport
Active and passive transport are biological processes that move oxygen, water and nutrients into cells and remove waste products. Active transport requires chemical energy because it is the movement of biochemicals from areas of lower concentration to areas of higher concentration. On the other hand, passive trasport moves biochemicals from areas of high concentration to areas of low concentration; so it does not require energy.