BIOL 640 1st Edition Lecture 13 Outline of Last Lecture I. Hemagglutination Inhibition assay Outline of Current Lecture II.Radio immuno assayIII. ELISAIV. Competitive ELISACurrent LectureImmunology Lecture 14- RIA and ELISA.RIA- Is there Ab against HIV?If there sis a virus:1. viremic stage is short.2. Lag phase where the virus hides is about 10 years.3. Virus replication is where the cells that are needed for Ab production are killed.Radio immuno assay is extremely sensitive but radio active materials are dangerous.ELISA: Enzyme linked immunosorbant assay:Enzymes: horse raddish peroxidase, alkaline phosphatase and others.Ab is conjugated with enzyme. The color is detected using a simplified spectrophotometer aka ELISA plate reader. The enzyme and substrate are standard and read only 2 or 3 colors.Indirect ELISA:These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Secondary Ab recognizes constant region for detection. Only ½ the plate is standard solution. Then a known amount of Ab is added. Standard curve is used for analysis. Enzyme conjugated Ab has to bind to Ag binding to Ab and the Ab has to be large.Limitations:It can not be used for measuring small molecules such as DNP molecule. Also, the enzyme conjugated Abhas to see parts of the molecule of Ab. Enzyme conjugated Ab needs to be purchased.Competitive ELISA:1. The objective is to measure excess Ab that is not binding to sample Ag.2. Less amount of sample gives high levels of sensitivity.3. Sample has to be pipetted fast enough so that same time for Ab.Ag formation and dissociate to free Ab is avoided by maintaining same temperature and
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