BIOL 640 1st Edition Lecture 12 Outline of Last Lecture I. Double immunodiffusion assay Outline of Current Lecture II.ImmunoelectrophoresisIII. Hemagglutination based assayIV. Hemagglutination inhibition assayCurrent LectureImmunology lecture 13- Hemagglutination based assays: When human IG immunizes a rabbit, polyclonal Ab with heavy chains and light chains form. The precipitation line has a spur indicating H chain epitopes. Since diffusion depends on size, H larger than L, H migrates slower. 2 precipitation lines are seen: H line is lower than L line.Ouchterlong : Only simple ones can be used as precipitation lines are readable. If more complex substances are studied, hazy and complicated prepitation lines are seen. This system is slow.Immunoelectrophoresis:It is faster as radio labeling is done.Hemagglutination based assay:RBC is used for blood typing to observe visual clumping of cells. For Rh typing, Ab is divalent and can not form complex lattice. Therefore, low density of Rh antigen is added on cells. Sandwiching is the process where Ab binding to Ab facilitate read off results.Conjugate Ag to RBC: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.Precipitate is formed. This test for Ab is called passive hemagglutination assay.Conjugate large Ag to RBC:Test for Ag called reverse passive agglutination.Test for different phases: Used for Hepatitis B typing:Test for both phases- 1. virus particles- viremic phase and denoted by reverse passive hemagglutination phase. 2. Ab detected in blood called as sero- converted phase and by passive agglutination assay.Hemagglutination Inhibition assay:Test for HCG (human chrionic gonadotrophin)- small peptide formed during pregnancy. Urine is tested. Reagent used: 1. HCG conjugate. 2. Alpha HCG.Run test:If sample contains free HCG it competes for Ab. Therfore, more alpha HCG more agglutination.To test a population of cells when only subpopulation has Ag, use immunofluorescence.Direct immunofluorescence:Ab labeled directly to fluorochrome. Then identify cells that carry component by fluorescence. This is expensive.Indirect fluorescence:Primary Ab recognizes Ag and secondary Ab recognizes primary Ab and labeled with fluorochrome. This is called signal amplification and saves money. If all primary Ab are from same species, we only need to buy one secondary Ab that is fluor-labeled.Assay Tag Example ApplicationImmunofluor- Dye Fluorescent Tissues sections, cellsescence Rhodamine VisualizationRadio immuno Radioactive I129 Small soluble molecules AssayELISA, Enzyme Horse raddish Small soluble moleculesWestern peroxidase,Blots Alkaline PhosphataseRadio immuno assay:It is extremely sensitive i.e. sensitivity refers to the lowest amount we can detect in pico grams, pg. It usually employs solid support. It has highly quantitative cpm (counts per minute) radiation picked up. It usually employs sandwiching. It is easier to clean up.Problems:1. Radioactive so needs protective gear and needs waste management.2. It also needs large and expensive detector. Example: PCB is a highly toxic small molecule and this method is used to detect PCB level in