writing lab report about antigen-antibody interaction

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I have to write a lab report 6 pages minimum about antigen- antibody interaction: radial immunodiffusion and quantitative ELISA. The lab report should cover 5 different sections. Those sections, the instruction paper in the attachment, are objective, background, results, interpretation of results and discussion, and answers to the given questions. On the results section, we need to check the plate and measure diameter of precipitin rings, and Calculate unknown antigen concentration. In addition, we need to make scatter graph, leaner one, measuring ELISA result.

As I mentioned, I am attaching the instruction paper as well as the ppt and all the results I got.

writing lab report about antigen-antibody interaction
writing lab report about antigen-antibody interaction
writing lab report about antigen-antibody interaction
writing lab report about antigen-antibody interaction
BCHB 507/508 Laboratory Applications of Biotechnology Session 14 – Lab 273 and 278 Antigen – Antibody Interaction Radial Immunodiffusion, Quantitative ELISA & Simulation of HIV Definitions  Antigens = A substance that when introduced into the body stimulates the production of an antibody   Epitope = antigenic determinant Antibodies Immunoglobulins (Ig)  gamma globulin proteins that are found in blood  used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses   Immunoassay = A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample Epitopes = Antigenic Determinants http://highered.mheducation.com/sites/9834092339/student_view0/chapter51/antigenic_determinants__epitopes_.html What are Antibodies?    Serum Protein Globulins Made up of a light and heavy polypeptide chain In response to “non-self” antigenic stimulants Monoclonal vs. Polyclonal Antibodies MONOCLONAL ANTIBODY     Produced from a single B-cell  identical antibodies Will only recognize the same epitope of a specific antigen Limited lifespan B-cells + myeloma = immortalized hybridoma  constant supply of highly specific monoclonal antibody May only recognize a particular protein form (phosphorylation) POLYCLONAL ANTIBODY     Produced from multiple B cell clones  Mixed population of antibodies Each antibody recognizes a different epitope of a specific antigen Variability in polyclonal antibody production between batch preparations Tolerant of small changes in protein structure Antibody – Antigen Binding    Antigen – antibody reactions are performed to determine the presence of either the antigen or antibody. ( serological tests ). One of the two components has to be known. e.g. with a known antigen, such as influenza virus , a test can determine whether antibody to the virus is present or not . http://www.ilbe.com/2828854492 Precipitation of Ag & Ab    Soluble Ag & Ab interact and form a lattice that develops into a visible precipitate Antibodies that aggregate soluble antigens are called precipitins Occurs best when antigen and antibody are present in optimal proportions (Equivalence) http://www.microbiologybook.org/mayer/ab-ag-rx.htm Precipitation curve    1. Zone of antibody excess precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant 2. Zone equivalence maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant 3. Zone of antigen excess precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant http://www.life.umd.edu/classroom/bsci423/song/Lab5.html Single Radial Immunodiffusion 1. 2. 3. Antibody is incorporated into molten agarose Plates are prepared (punch holes) Antigen is introduced into the wells. Single Radial Immunodiffusion (cont’d) 4. 5. As antigen diffuses into agar, precipitation rings form depending on the concentration of the antigen Radial Immunodiffusion is used to measure IgG, IgM and complement components. Radial Immunodiffusion Procedure Experiment 1. 2. 3. 4. 5. 6. 7. Label and cut wells into agarose plates Prepare serial dilution of antigen samples Load center well with Unknown sample Load outer wells with serially diluted antigen samples Incubate plates in chamber Check plate, measure diameter of precipitin rings and diagram/photo results Calculate unknown antigen concentration undiluted 1:2 1:1 6 Unknown 1:8 1:4 Determination of antigen concentration A plot of precipitation ring diameters versus concentrations is made for the samples with known antigen concentrations ELISA - understanding the acronym  EL Enzyme-linked  IS Immunosorbent  A    Assay Antibody  Allows for specific detection of antigen/antibody Solid phase (sorbent)  Allows one to wash away all the material that is not specifically captured Enzymatic amplification  Allows one to quantify color change/intensity using an absorbance plate reader www.edvotek.com/278.pdf Types of ELISA 14  Qualitative ELISA   Positive or Negative results Quantitative ELISA   Optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.   Sandwich ELISA = Direct ELISA Indirect ELISA Competitive ELISA Sandwich ELISA • Purpose: Used to detect specific antigen • Procedure: • Coat plate with Antibody. Block any non specific binding sites on the surface. • Apply the antigen-containing sample to the plate. Wash plate. • Apply enzyme linked primary antibodies as detection antibodies. Wash plate. • Apply a chemical which is converted by the enzyme into a coloured product. • Measure the absorbency of the plate wells to determine the presence and quantity of antigen http://imgarcade.com/1/indirect-sandwich-elisa/ Indirect ELISA • Purpose: Used to detect specific antibody • Procedure: • Coat plate with antigen Block any non specific binding sites on the surface. • Add mixture of the serum antibodies of unknown concentration to wells. • Add enzyme-conjugated secondary antibody. • Add substrate for this enzyme • Observe color change  The higher the concentration of the primary, the stronger the color change. • Use spectrometer to measure color strength http://imgarcade.com/1/indirect-sandwich-elisa/ Competitive ELISA • Purpose: • Procedure: • Incubate unlabeled antibody with its antigen (sample) • Add these bound antibody/antigen complexes to an antigen-coated well. Wash plate • Add enzyme-conjugated secondary antibody • Add substrate • The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The less antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal. http://imgarcade.com/1/indirect-sandwich-elisa/ Applications of ELISA     Detect hormonal changes (pregnancy) Detect viruses (hepatitis, HIV, venereal diseases) Measure antibody levels (allergies, vaccines) Detect circulatory inflammatory markers (cytokines) 18 Traditional ELISA    Requires 2 antibodies: primary and secondary antibody Enzyme covalently bound to secondary antibody Colorless substrate enzymatically converted to colored product www.edvotek.com/271.pdf Quantitative ELISA Experiment            Label plate. Prepare primary antibody dilutions with PBS (store on ice until use). Load antigens into wells, incubate and remove excess. Wash plate with PBS. Add blocking agent to the wells, incubate and remove excess. Wash plate with PBS. Add primary antibody dilutions to the wells, incubate and remove excess antibodies. Wash plate with PBS. Add secondary antibody samples to the wells, incubate and remove excess antibody from the wells. Wash plate with PBS. Add substrate to each of the wells, incubate and read plate. Lab Math – Normality  Normality is the gram molecular weight of a compound divided by the number of hydrogen ions present in the solution. 1 liter of 1M HCl (36.46 MW) contains 1 M of hydrogen ions so 1M = 1N. H2SO4 (98.0 MW) has 2M of hydrogen per mole H2SO4 so to make a 1N solution you would use 49 g per liter (a 0.5 M solution). Resources  Performing Radial Immunodiffusion assay  https://www.youtube.com/watch?v=Fnx5CkGRBEM  Performing ELISA  https://youtu.be/zR_xlV5v_f4

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School: Purdue University


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Awesome! Exactly what I wanted.

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