Immu Practical Flashcards
(43 cards)
how are antibodies prepared for viewing
attached to flourescent dye or enzyme that converts a colourless substrate to colored. Can be detected on microscope (immunohistochemistry) or spectorphotometer (ELISA/EIA)
immunoflouresence
antibodies labelled with flourescent dye. Stained tissues are examined with microscope that exposes to ultraviolet light. Emits light at a specific wavelength that shows as color. This can be used to idenfiy where antibody was bound and location.
direct immunoflourecense
antibody bound molecules detected with flourescent dye directly attached to molecules.
indirect immunoflourescense
bound antibody is detected using flourescent anti-immunoglobulin. Then bound antibody binds to antigen while unbound leftover is washed off. Flourochrome-labelled second antibody specific for the Fc region of first antibody is used to bind to and reveal first or primary antibody (revealing presence of antigen in tissue)
immunohistochemistry
enzyme labelled antibodies used to reveal presence of antigens on cells in tissue. Antibody bound cells revealed when it is added to the section of the slide. Where anzyme labelled antibody is bound, the enzyme converts into coloured product and you can see location of antigen using standard microscope
immunological function of flourescnet cells showing in figures
antigen presenting
pros and cons of direct immunohistology
pros: faster and less prone to errors. Cons: less specific, not as sensitive.
pros and cons of indirect immunohistology
more specific and better for high abundances. Cons: slower and more room for errors
pros and cons for immunohistochemistry
pros: more specific, flourescent microscope not needed. Cons: takes longer to process. Not as helpful to visualize multiple labels.
pros and cons of immunoflourescnece
pros: helpful for research and diagnosis, quicker, good for visualizing multiple lables. Cons: less specific and more complex (needs flourescent microscope)
mean error calculation
(set volume-average volume)/set volvume x 100
haemocytometer
a way to count cells. Cells on top and left are counted. Bottom and right are not.
concentration of cells formula
(nx10^4xdilution factor)/number of 1mm squares counted
why is it important to count 50-100 cells/1mm square instead of 1000/1mm square?
There is more room for error if we could 1000 cells/1mm. The counting could be less accurate or take more time.
when do people develop antibodies
when exposed to pathogens (infection or immunisation)
agglutination
a way to observe antigen antibody interactions. Often used to type blood cells
antibody excess
too many antibodies relative to antigens, antibodies will bind to antigen particles individually and frevent formation of cross linked networks, causing agglutination
antigen excess
too many antigens, antibodies will bind only to a few antigen particles, preventing formation of cross linked networks and causing agglutination
rising titre
rising or increasing antibodies in a sample
Why do we use a ‘V’ bottom microplate instead of a ‘flat’ bottom microplate?
easier to pipette from
how is rising titre used diagnositcally
shows that soemone in producing more antibodies which could indicate infection
glycolipids
the antigens on red cells
what is different between the blood types
they are different terminal sugars on each molecule
why do blood types have antibodies against terminal sugars of others without previous encounter
bacteria have complex surface carbohydrate motifs. A few mimic blood types. Thus immune system has recognized some antigens are foreign and some as self.
