PSB ELISA/Hemo

Re: the Cell Counting lab, you should be able to: • given dimensions of a field(s) (that is, the “squares”) to be counted, calculate the volume in mm3 and convert to mLFirst volume of square is DxWxH then 1 cubic meter = 0.001ml • take an accurate cell count from a haemocytometer  • from the count, determine cells/mL based on the volume of the field (i.e. the haemocytometer squares) considered in the count  1cm3 = 0.001ml pretty much 10^4 Average viable cell count per square x Dilution Factor x 104/ • from a determination of cells/mL in the field, correct if necessary for any initia  dilution of the suspension made prior to loading the haemocytometer times back by diloution factor  • given a cell/mL count in a “concentrated” suspension, determine the dilution necessary to plate out cells at a more “dilute” density in a given volume of culture medium?? • determine the volume of the drug to apply and calculate from its stock concentration the concentration in the cell culture Remember that apart from the volume calculation, everything else you have done already, although in different contexts (i.e. determining amounts and concentrations; making dilutions; etc.).

Re: the ELISA lab, you should be able to: • Show that you understand the general order of events in a sandwich ELISA: primary, block, antigen, secondary, revealing agent, enzyme reaction; also knowing when washes are performed and why Washed are done using a buffer and a type id detergent to get rid of non bound regents decress the background  and increase the signal - no interferance. to much washing can cause lessen sensitivity as abs or antigen are eroded from the wells Antibody catching Protien Capture Dectition antibody streptian enzyme conjugate addition of substrate  analisis • Describe one or more controls that one should perform to test the reliability of an ELISA and explain what the controls are for (e.g. what does a particular control tell you has or has not happened in the assay?)

Different types of ELISA diffrences in how the antigen is captured or immolobies to the well. Then how captured antigen is then detected direct - 1ab with enzyme indirect primary ab -> secondary ab w. enzyme.  sandwich - capture AB -> antefin ->2 more abs 1 with enzyme  directab  with reporter attached goes strait on  indirect use of seccondary abswider varritety so more accurate 

Polu

Polyclonal antiserum has had antigens injected for the purpose of makig antibodies compsition  varry a lot moncolnal antibodies slpleen of immunised mouse?fuse with emortal cell line 

HEAMO

ELISA