Lab material

StudyBlue printing of Lab material html, body, div, span, applet, object, iframe, h1, h2, h3, h4, h5, h6, p, blockquote, pre, a, abbr, acronym, address, big, cite, code, del, dfn, em, font, img, ins, kbd, q, s, samp, small, strike, strong, sub, sup, tt, var, b, u, i, center, fieldset, form, label, legend, table, caption, tbody, tfoot, thead, tr, th, td { margin: 0; padding: 0; border: 0; outline: 0; font-size: 100%; background: transparent; } body { line-height: 1; } blockquote, q { quotes: none; } blockquote:before, blockquote:after, q:before, q:after { content: ''; content: none; } /* remember to define focus styles! */ :focus { outline: 0; } /* remember to highlight inserts somehow! */ ins { text-decoration: none; } del { text-decoration: line-through; } /* tables still need 'cellspacing="0"' in the markup */ table { border-collapse: collapse; border-spacing: 0; } /* end RESET */ .header { min-width:800px; } .logo { padding:6px 20px 2px 20px; margin:0; font-size:25px; font-weight:bold; color:#808285; position:relative; border-bottom: 1px solid #c5c5c5; } .logo-blue { color:#70adc4; } .logo-desc { font-weight:normal; font-size:19px; color:#cccccc; margin-top:50px; position:absolute; display: none; } .back-button { position:absolute; top:20px; right:20px; font-size:13px; line-height:25px; color:rgb(0,175,225); font-weight:normal; } .back-button a { color:rgb(0,175,225); } .instructions { padding:0; margin:0; width:100%; position:relative; color:rgb(100,100,100); } .step-holder { border-left:1px solid #ededed; margin-left:20px; } .steps { padding:15px 0; float:left; width:24%; border-right:1px solid #ededed; text-align:center; } .steps-01 { } .steps-02 { } .steps-03 { } .steps-04 { } .label { padding:5px 10px; } .print-button { } .print-button a { background-color:rgb(0,175,225); color:white; line-height: 19px; padding:9px 8px 5px 30px; font-size:14px; text-decoration:none; background-image: url(images/printer.png); background-repeat: no-repeat; background-position: 7px 50%; -moz-border-radius: 5px; -webkit-border-radius: 5px; } .print-button a:hover { background-color:black; } .theNote .content { width: 8.0in !important; margin: 5px auto; padding:20px; background-color:white; } .theNote .header { border-bottom: 1px dashed #C8C8C8; font-size: 17px; padding: 0 0 10px; line-height: 19px; color: #00ADE1; min-width:500px; } .theNote .body { font-size: 14px; line-height: 19px; padding: 10px 0; } .theNote{ padding:6px 0; clear:both; background-color: rgb(200,200,200); } .theNote h3{ color: rgb(100,100,100); } .theNote h1, .theNote h3{ background-color:white; padding:2px 20px; width:8.0in !important; margin: 0 auto; font-size: 15px; } .theNote h1{ padding-top: 10px; font-size: 15px; } .theNote h1:first-child{ font-size: 20px; } .theNote h3 { font-size: 14px; font-weight: normal; } #options { border: 3px double #ccc; padding: 5px 12px; margin: 10px 50px 10px 20px; float: left; } #info { border-top: 1px solid #ccc; padding-top: 5px; font-style: italic; } li { margin: 5px 10px 5px 25px; } ul li { list-style: disc; } ol li { list-style: decimal; } img { border: 0; } table { clear: both; width: 100%; border: 1px solid #c5c5c5; border-width: 1px 0; margin: 0; page-break-after: always; } table#page { page-break-after: auto; } td { text-align: center; font-size: 12px; border-bottom: 1px dashed #c5c5c5; height: 1.75in; width: 50%; padding-left: 15px; } .leftside { border-right: 1px solid #cccccc; padding: 0 15px 0 0; } .bottom td { border-bottom: none; } .clearfix { clear:both; line-height:1px; height:1px; } img { max-width:80%; max-height:150px; margin:20px; } @media print {.header { display: none; } .content .header{ display:inherit; } table { border: 1px dashed #bbb; border-width: 1px 0; } .theNote{ background-color:white; } } How are Abs are used in lab solid phase assays: assay in which one reactant (either Ag or Ab) is immobilized on a solid phase/support -culture dish with wells or nitrocellulose membrane ELISA Enzyme Linked Immunosorbant Assay -use to test if Ab is present and can quantitate Ab -96 well plates -STEPS: Ex. HIV testing 1) add antigen to wells of plate : Ag is core and envelope proteins from HIV virus 2) add antibody specific to Ag : Ab is from patient's serum -wash 3) add enzyme-conjugated secondary antibody : anti-human IgG -wash 4) Add substrate -choose such that the substrate is changed by enzyme --> colored product -color is positive; no color is negative -intensity of color - estimate amount of Ab ---If Negative result: human IgG for Ag won't be there, all is washed away Western Blot -confirmatory test during HIV testing and many other uses as well -more sensitive than ELISA -Steps: 1) Add mixture of proteins into wells at top of polyacrylamide gel -if HIV test: proteins from HIV virus -separation based on SIZE 2) Take SDS-PAGE gel and blot: -gel --> electrophoretic transfer --> nitrocellulose membrane 3) Add antibodies and allow to interact with proteins: specific for 1 or more of the proteins -primary Ab: Ab for HIV positive person -secondary Ab: anti-human IgG -enzyme conjugated just like previous -secondary binds to primary 4) add substrate --> colored product -colored band: positive -no colored band: negative -Better than ELISA because proteins are separated, so we know which ones are reacting -false positive: 1/250K -false negative: in US .003% -not yet producing Abs -T helpers depleted which help make Ab Techniques to detect Ab on intact cells/tissues Why? -learn about characteristics of the cell/tissue (ex: cancer cells) -distinguish different types of cells/tissues Ex: males age 50: PSA antigen (prostate specific antigen) Ex: females: breast cancer, upregulation of p185HER2 -3 types of T cells: -each have different functions -Tc has CD8- -Th has CD4- -E. coli has surface antigen O: 157, H:7 --> highly dangerous strain 1) Immunofluorescence 2) Immunoelectron microscopy 3) FACS Immunofluorescence 1) obtain Ab specific for Ag you wish to detect 2) couple Ab to fluorescent tag (stain) -many choices for tags -variety of colors available -common stains: fluorescein: yellow/green, rhodamine: red 3) apply Ab to intact cells/tissues 4) View under fluorescent microscope (or confocal) Immunoelectron Microscopy 1) obtain Ab specific for Ag you wish to detect 2) couple to microscopic gold bead 3) apply Ab to intact cells/tissues 4) view under electron microscope (black dots = gold beads) FACS analysis Fluorescent activated cell sorting ; aka flow cytometry Advantage: cells stay viable and unharmed 1) obtain Ab specific for CD4- (Th) 2) add fluorescent tag (anti-CD4) 3) add antibody to mixture of 2 cells -Th positive: stained -Tc negative: not stained 4) add cells to reservoir of instrument 5) cells exit single file 6) analyzed by laser (positive: Th) 7) fluorescence --> detector -positive for stain will be marked with a negative charge 8) negative cells attracted toward positive plate -common to sort based on size --> when laser shines, marks cells above/below certain size