prac test! (10%) Flashcards
what are the two methods for blood typing
slide method and tube method
principal of the ABO slide grouping/meothod
detects the A or B antigens on red blood cells using the principal of agglutination. this procedure is called the direct/forward grouping as it is accomplished by combining unknown RBC will known antiserum and to observe for agglutination. if the antigen present on the cell corressponds to the antibody in the antiserum, the antibody will bind to the antigen and cause clumping of the cells/agglutination. if the antigen is not present on the cells. no agglutination is observed.
what is the procedure for the side blood typing tests.
- using marker/ wax label the left side A, centre B (draw circle) and right side AB.
2.place one drop of anti-A serum in A, one drop of anti-B serum in B, one drop of anti-AB serum in AB (do not allow dropper to touch the slide-> cross rxn-> false results) - add one drop of well-mixed blood to each part of the slide using pipette (drop should not be larger than the drop of antibody-> more blood than antibody-> insufficnet antibody to bind to antigen-> weak/undetechted agglutination false negaitve )
- mix the blood and antiserum A into a smooth round circle about 10c coin suing a clean wooden applicator stick
- repeat for A and B with a new clean applicator
- rock the slide gently for two minutes and look for agglutination using strong light
- record agglutination results as the follwing + means agglutination present, 0, no agglutination
- determine the nlood group and record
what is ABO blood grouping tube method and why is this preferred over slide method
- more sensitve and reliable methd of determining a patients blood group thanslide testing
it consisits of:
direct/florward grouping which identifies the antigen on the cell
cofirmitory/ reverse grouping which identfies the blood group antibodies in the serum
procedure of the ABO blood grouping-tube method
- Wash blood (remove plasma) specimens given and prepare 3-5% suspension of patients’ red cells. (dilute so can see agglutination)
- Label six tubes for each patient as follows: anti-A, anti-B and anti-A,B (for forward grouping), A cells, B cells and O cells (for reverse grouping).
- Place one drop of anti-A, anti-B and anti-A,B serum into the tubes labelled anti- A, anti-B and anti-A,B respectively.
- Place one drop of patient’s serum into the tubes labelled A cells, B cells and O
cells. - Place one drop of 3-5% suspension of patient’s cells into tubes labelled anti-A, anti-B and anti-A,B.
- Place one drop of known A cells, B cells and O cells into the tubes labelled A cells, B cells and O cells. (with patients serum-antibody)
- Mix all tubes well and centrifuge for 15 seconds at 3500 rpm (1000g).
If preferred, the tubes may be allowed to stand at room temperature for 15-60 minutes. - Gently agitate to dislodge the packed button of red cells and examine for agglutination. Check apparent negative results microscopically.
- Record the results from each tube on worksheet: 0= no agglutination, + =
agglutination. - Determine the blood group of the sample and record.
- You must compare results of forward grouping with results of reverse
grouping of the same sample. Reverse grouping should agree with results of
forward grouping
assay principa
- anti e coli monoclonal antibody is absorbed on microtire plate. E coli 0157 (specific strain of e coli bac) present in pre-enriched sample/standard binds to antibody absorbed on the plate
- FITC conjugated anti E coli monoclonal antibody is added and binds to the E coli captured by the first antibody
- following incubation and wash steps, a HRP-conjugated mouse anti FITC antibody is added and binds to the FITC conjugate E coli monoclonal antibody. unbound HRP conjugated mouse snit FITC antibody is removed during was step
- substate sol reactive with HRP is added to wells
- a coloured product is formed in proportion to the amount of E coli 0157 present in the sample. the rxn is terminated by addition of Stop sol and the absorbance is measured at 450 nm.
- a standard curve is prepared from the proved heat killed E coli 0157 standard and the sample E coli 0157 amount is then determined.
TSO preperation
Homogenize (mix) 25 g of samples (ground beef, milk, lettuce, etc) in 225 mL of Modified Tryptone Soya Broth. (used to grow bac)
2. Incubate 18-24 hrs at 37ºC to 41.5ºC. (optimum temp for e coli growth)
3. After the enrichment incubation, transfer 1 mL of the culture to a microcentrifuge tube and inactivate the bacteria by heating the sample at 90-100ºC for 30 min. (eliminate risk of infection during testing, safety hazard)
4. Cool down to room temperature before ELISA steps. (it can denature/ affect results)
preperation and carrying out of the E coli 0157 ELISA kit
1.Add 100 μL of heat inactivated E.coli O157 sample or standard to Anti- E.coli O157 Antibody Coated Plate (antibody alr absorbed onto plate)
2. Cover with a Plate Cover and incubate at 37ºC for 2 hours (to facilitate atigen-antibody binding)
3. Remove plate cover and empty wells.
4. Wash microwell strips 5 times with 250 μL 1X Wash Buffer per well with thorough aspiration (in, out) between each wash. (remove unbound molecules)
5. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer. (interview with results)
6. Add 100 μL of the diluted FITC-Conjugated Anti-E.coli O157 Monoclonal Antibody to the allocated well. (bind to atigen alr primarily binded)
7. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker. (for even distribution and binding of antibody)
AFTER 1 HOUR
8. Remove plate cover and empty wells. Wash the strip wells 5 times according to step 4 above.
9. Add 100 μL of the diluted HRP-Conjugated Anti-FITC Monoclonal Antibody to allocated wells.
10. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
AFTER 1 HOUR
11. Remove plate cover and empty wells. Wash microwell strips 5 times according to step 5 above. Proceed immediately to the next step.
12. Warm Substrate Solution to room temperature. (for optimal enzymatic activity when HRP interacts with substrate)
13. Add 100 uL of Substrate Solution to allocated wells, including the blank wells.
14. Incubate at room temperature on an orbital shaker. Actual incubation
time may vary from 2-30 minutes.
Note: Watch plate carefully; if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
15. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. (preserve colour change) Results should be read immediately
(color will fade over time-> difficult to obtain accurate absorbance reading).
16. Read absorbance of each microwell on a spectrophotometer using 450
nm as the primary wave length
purpose of washing step (100ul of Wash Buffer sol)
-remove unbound molecules by washing the sold phase, ensuring that only molecules of interest remains attached
excess antibodies/ unbindied molecules can cause background noise -bind to other components-> false positvive)
purpose of substrate addition
binds with enxyme (HRP) -> catalyse rxn-> production of detectable sigh (colour)
what are common substrates
- chemogenic (colour producing) or flurogenic (forescence-producing, glowing) compound
purpose of creating the standard curve
establishes a relationship between known concentrations of the antigen (from the standard solutions) and the measured absorbance at a specific wavelength (usually 450 nm).. see the light amt (what we insert) and we can find the concentration of the E coli
different types of antigens that can be found on the surface of bacterial cells,
H antigen: found on All motile bacteria and most of these antigens are flagellin proteins. (Found on the flagellin)
Both gram-positive and gram-negative bacteria can be characterized by their H antigen. (if they have flaggelin)
o antigen: found on LPS of gram neg bac
K antigen: found on capsule of antigen
principal of agglutination test to identify bacteria
Agglutination occurs when the antibody binds to epitopes (sections of antigen) on two different objects, linking them together.
assay can be direct or passive
direct assay: interaction of antibody with the cellular antigen (direct binding to the antigen on the bac)
passive assay: coated latex particle (eg antigen) (to antibody)
what needs to happen for agglutination occue, when it doesnt happen what does it mean
In order for agglutination between antibody and antigen to occur, the
antibody and antigen epitope must be combined in the proper proportions
called the zone of equivalence
When this happens, the antibody molecules bind to epitopes on two or more
different antigens, forming a cross-linked network. If enough antigens and
antibodies are present, the mass of agglutination becomes visible to the naked
eye as clumping.
antibody molecules to high in conc-> no cross linked network formed even if they recognise the antigen-> called the prozone effect
if antigen is in acess-> no formation of cross linked network-> postzone effect
