IMMUNOLOGICAL TECHNIQUES IN BIOTECHNOLOGY

FINALS QUESTIONS & ANSWERS

Section A
1. Differentiate between plasma and serum Plasma contains fibrinogen which assists in clotting therefore when it is separated from blood it does not lose the ability to clot. Serum, on the other hand is the part of blood that remains when fibrinogen is separated from blood. 2. Differentiate between primary and secondary antibody Primary antibody is the initial antibody used to bind with specific antigen in a antigen coated well in ELISA technique. Secondary antibody is antibody which will binds on the primary antibodies that binds to the antigen. 3. Differentiate between ELISA and Western Blotting ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample while Western Blotting is a analytical technique used to detect specific proteins in the given sample. It uses gel electrophoresis to separate native proteins. 4. Differentiate between Northern Blotting and Southern Blotting Northern Blotting refers to the identification of detailed sequences of RNA in which the RNA fragments are separated by electrophoresis. Southern Blotting refers to the identification of detailed sequences of DNA in which the DNA fragments are separated by electrophoresis. 5. Differentiate between monoclonal and polyclonal antibody Monoclonal antibodies represent a single B lymphocyte generating antibodies to one specific epitope while polyclonal antibody ontains a mixture of antibodies (mainly IgG), often recognizing multiple epitopes on the antigen. 6. Write the principle of ELISA and Spectrophotometer Enzyme-linked Immunosorbent Assays (ELISA) combine the specificity of antibodies with the sensitivity of a simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISA can provide a useful measurement of antigen or antibody concentration A spectrophotometer is used to measure the amount of light a absorbs. It works in Beer Lamberts Law. The amount of light the chemical on the particles absorbed is correlated to the amount of substance present in the sample.

7. Expansion of PEG, APS, SDS, TEMED, and MEM PEG = Polyethylene glycol APS = Ammonium persulphate SDS = Sodium deodecyl sulphate TEMED = Tetramethylethylenediamine MEM = Minimum Essential Media 8. 3 applications of monoclonal antibody Monoclonal antibodies can be used to treat viral diseases, traditionally considered "untreatable". Monoclonal antibodies can be used to classify strains of a single pathogen, Monoclonal antibodies used in several diagnostic tests such as pregnancy test to detect the presence of HCG (hormones). 9. What are the types of bond present in antibody structure ? Hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. 10. Explain the different between molecules which are antigenic and immunogenic. Antigenic molecules have the ability to combine specifically with the final products of the immune response while immunogenic is a molecules such as an antigen or epitope, that have the ability to provoke an immune response 11. Define immunoelectrophoresis and their types Immunoelectrophoresis is a technique used to measure proteins called immunoglobulins (IgG, IgA, IgM) present in the blood sample. Therere 2 types of immunoelectrophoresis, Rocket immunoelectrophoresis and counter-current immunoelectrophoresis. 12. Differentiate between Avidity and Affinity Affinity is the strength of binding of one molecule to a ligand. For example, the strength of binding of an antibody to an antigen however avidity is the sum of total strength of binding of more than one molecules to ligands. 13. Explain the role of secondary antibody in ELISA The secondary antibody, which is conjugated to an enzyme, recognizes and binds to primary antibodies of antigen-antibody complexes.

14. Write down the principle of Double Immunodiffusion with diagram Both antigen and antibody are allowed to diffuse into the gel depending on the similarity between the antibody, different geometrical patterns are produced.

Section B
Question 1 a) Explain the agglutination reaction Agglutination reactions are used to assess the presence of antibodies in a specimen by mixing it with particulate antigens. There are 2 types of agglutination reaction, direct agglutination and indirect agglutination. Direct agglutionation consist of two methods. First method is known as Slide agglutination. Slide agglutination is used to identify the blood group and Rh factors in the given blood sample. It uses a direct method carried out on the slides by mixing the Anti-A, Anti-B and Anti-D to the blood sample and observe the agglutination. Second method is Tube agglutination reaction or also known as Widal Test. Widal test is used to study the interaction of Ag-Ab complex. It is a semi-quantitative test by mixing the known antigen directly with serial dilution of the serum sample. Agglutination is detected when RBC sedimented at the bottom of the test tube. Indirect agglutination is usally used in pregnancy test. This test is used to determine the presence of HCG in the given urine sample. The HCG present in the urine sample will react with the monoclonal antibody anti-HCG coated on the cord. b) Brief the detection of chemokines ( 5 points) - Neuronal cell lines U373 and U87 were obtained from the ATCC (American Tissue Culture Collection. - Culture maintained in MEM medium with non-essential amino acids, sodium pyruvate, 10% fetal bovine serum, 90% Balanced Salt Solution (BSS). - Add 10-7 to 10-15 M concentration of morphine and incubate for 24 hours to 98 hours at 37 oC in a CO2 incubator. - Centrifuge and the supernatant stored at -70 oC - Chemokines quantified by ELISA, RNA extracted from the cells for RT-PCR and northern blotting c) Write the principle of ELISA Enzyme-linked Immunosorbent Assays (ELISA) combine the specificity of antibodies with the sensitivity of a simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISA can provide a useful measurement of antigen or antibody concentration

Question 2 a) Pregnancy test used monoclonal antibodies. What molecules do they detect in this kit? Molecules detected in this kit is HCG (Human chorionic gonadotropin). HCG is a marker present in blood and urine sample which are used to detect pregnancy. b) What is mean by Fc fragment in the antibody ? Fc fragment is present in IgG structure. The function of Fc fragment is act as complement fixation. Fc denotes the ability of Antibody to crystallise. c) Which cell types releases cytokines All nucleated cell such as macrophage, endothelial cell, and epithelial cell. Most abundant number of cytokines is secreted by TH (T Helper) cell. d) Where would you find IgD in significant quantity? Most quantity of IgD is found in blood serum and its structure exhibit 2 heavy and light chain. It is also expressed in B cell along with IgM e) What is the role of aminoptherin in HAT medium? Aminopterin block the De novo systhesis but hypoxanthine and thymidine will help the cell to proceed the Salvage pathway during the production of Antibody in hybridoma technology.