Practical Immuno 1 PDF
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Uploaded by PolishedVeena6642
CEU Cardenal Herrera Universidad
Prof Dr Slaven Erceg
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Summary
These notes detail various immunological techniques, such as antibody titers, antigen detection, and immunodiffusion. They cover topics like monoclonal and polyclonal antibodies, and include visual aids and diagrams of the immunoprecipitation process, emphasizing solid-phase and liquid-phase techniques. The notes use scientific terminology and are likely part of a microbiology or immunology course for undergraduate students.
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PRÁCTICAL COURSE 1 ANTIBODY TITER Identification of the interactiones antígen-antibody using de precipitation methods Immunology Prof Dr Slaven Erceg COVID-19 ANTIGEN DETECTION RT-PCR ANTIB...
PRÁCTICAL COURSE 1 ANTIBODY TITER Identification of the interactiones antígen-antibody using de precipitation methods Immunology Prof Dr Slaven Erceg COVID-19 ANTIGEN DETECTION RT-PCR ANTIBODY DETECTION POLICLONAL Y MONOCLONAL ANTIBODIES Epitopes antigen Policlonal Recognition of Unique epitope monoclonal antibodies various apitopes policlonal monoclonal FIRST PHASE: Ag-Ab reaction SECOND PHASE: VISUALIZATION OF Ag-Ab REACTION Immunoprecipitation When soluble antibody binds to soluble antigen (sensitization) there will come a point where lattice formation will occur resulting in precipitation occurring resulting in a visible reaction These immune complexes have fallen out of solution. The Ab at the bottom in the illustration at right is still in the soluble phase. Immunoprecipitation:conditions High concentration of Antigen and antibody Polyclonal antibody Polyvalent antigen (multiple epitopes) Equimolar concentrations of both Measurement of Precipitation by Light Scattering Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. Passive Immunodiffusion Reactions of antigens and antibodies in agar gel. Migrate towards each other and where they meet in optimal proportions form a precipitate. “Passive” because they are allowed to react to completion with no enhancements such as an electrical charge applied. Ouchterlony Gel Diffusion Holes punched in agar. Known antibody or antigen added to center well. Known sample added to outer well. Wait for bands to form. This is a QUALITATIVE technique, simply determines the presence NOT the concentration. Ouchterlony Immunodiffusion INMUNOPRECIPITATION IN SOLID PHASE: agar PATIENT´S SERUM 1/1 1/107 1/10 antigen 1/106 1/102 1/105 1/103 1/104 http://www.youtube.com/watch?v=h-KptLVJpU0 INMUNOPRECIPITATION EN LIQUID PHASE: Conjugated polymers 5 min mix 5 min mix Plate mixer Plate mixer Add 20 ml of the Add 20 ml of rabbit serum immunocomplex beads to the wells o 20 ml pure rabbit IgG that contain 100 ml of blocking Measure the antibodies against buffer to all wells absorbance at 405nm rabbit IgG 20microL 62.5 125 250 500 X1 X2 ng/ml de rabbit IgG + 20microL Anti-IgG con sepharose + blocking buffer reading Plate reader 405microM Abs 62.5 125 250 500 ng/ml de rabbit serum D C B A
