Immuno Final Q&A

7/28/2019 Immuno Final Q&A

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IMMUNOLOGICAL TECHNIQUES IN BIOTECHNOLOGY FINALS QUESTIONS & ANSWERS

Section A1. Differentiate between plasma and serum

Plasma contains fibrinogen which assists in clotting therefore when it is separatedfrom 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 antibodyPrimary antibody is the initial antibody used to bind with specific antigen in a antigencoated well in ELISA technique. Secondary antibody is antibody which will binds onthe primary antibodies that binds to the antigen.

3. Differentiate between ELISA and Western BlottingELISA 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 techniqueused to detect specific proteins in the given sample. It uses gel electrophoresis toseparate native proteins.

4. Differentiate between Northern Blotting and Southern BlottingNorthern Blotting refers to the identification of detailed sequences of RNA in whichthe RNA fragments are separated by electrophoresis. Southern Blotting refers to theidentification of detailed sequences of DNA in which the DNA fragments are separatedby electrophoresis.

5. Differentiate between monoclonal and polyclonal antibodyMonoclonal antibodies represent a single B lymphocyte generating antibodies to onespecific epitope while polyclonal antibody ontains a mixture of antibodies (mainlyIgG), often recognizing multiple epitopes on the antigen.

6. Write the principle of ELISA and SpectrophotometerEnzyme-linked Immunosorbent Assays (ELISA) combine the specificity of antibodieswith the sensitivity of a simple enzyme assays, by using antibodies or antigens coupledto 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 inBeer Lamberts Law. The amount of light the chemical on the particles absorbed iscorrelated to the amount of substance present in the sample.


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7. Expansion of PEG, APS, SDS, TEMED, and MEMPEG = Polyethylene glycolAPS = Ammonium persulphateSDS = Sodium deodecyl sulphateTEMED = Tetramethylethylenediamine

MEM = Minimum Essential Media

8. 3 applications of monoclonal antibodyMonoclonal 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 todetect 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 orepitope, that have the ability to provoke an immune response

11. Define immunoelectrophoresis and their typesImmunoelectrophoresis 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-currentimmunoelectrophoresis.

12. Differentiate between Avidity and AffinityAffinity is the strength of binding of one molecule to a ligand. For example, thestrength of binding of an antibody to an antigen however avidity is the sum of totalstrength 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 toprimary antibodies of antigen-antibody complexes.


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


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Section BQuestion 1

a) Explain the agglutination reactionAgglutination 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, directagglutination and indirect agglutination.

Direct agglutionation consist of two methods. First method is known as Slideagglutination. Slide agglutination is used to identify the blood group and Rh factors inthe given blood sample. It uses a direct method carried out on the slides by mixing theAnti-A, Anti-B and Anti-D to the blood sample and observe the agglutination. Secondmethod is Tube agglutination reaction or also known as Widal Test. Widal test is usedto study the interaction of Ag-Ab complex. It is a semi-quantitative test by mixing theknown antigen directly with serial dilution of the serum sample. Agglutination isdetected when RBC sedimented at the bottom of the test tube.

Indirect agglutination is usally used in pregnancy test. This test is used to determinethe presence of HCG in the given urine sample. The HCG present in the urine samplewill 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 TissueCulture Collection.- Culture maintained in MEM medium with non-essential amino acids, sodiumpyruvate, 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 hoursat 37 oC in a CO 2 incubator.- Centrifuge and the supernatant stored at -70 oC- Chemokines quantified by ELISA, RNA extracted from the cells for RT-PCR andnorthern blotting

c) Write the principle of ELISAEnzyme-linked Immunosorbent Assays (ELISA) combine the specificity of antibodieswith the sensitivity of a simple enzyme assays, by using antibodies or antigens coupledto an easily-assayed enzyme. ELISA can provide a useful measurement of antigen orantibody concentration


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Question 2a) 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 amarker 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 ascomplement fixation. Fc denotes the ability of Antibody to crystallise.

c) Which cell types releases cytokinesAll nucleated cell such as macrophage, endothelial cell, and epithelial cell. Mostabundant number of cytokines is secreted by T H (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 andlight 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 helpthe cell to proceed the Salvage pathway during the production of Antibody inhybridoma technology.

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Immuno Final Q&A