Flash content link: https://vlab.amrita.edu/repo/BIOTECH/IMM/Latex_agglutination/index.swf
Materials Required
- 1.5 ml Vials.
- Microcentrifuge.
- Pipette.
- Microtips.
- Laboratory refrigerator.
- Glycine saline buffer.
- Blocking buffer.
- Antigen for coating.
- Latex beads.
- Test antiserum.
- Glass slides.
- Beaker.
- Tooth pick.
Procedure
Coating of Latex
- To 20 μl of latex beads taken in a 1.5 ml vial add 40 μl of glycine-saline buffer.
- Add 60 μl of antigen to the latex and incubate at 37oC for 2 hours.
- Spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant.
- Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10 minutes.
- Repeat the washing once more.
- Add 90 μl of blocking buffer to the pellet, mix well.
- Incubate at 4oC, overnight.
Agglutination Test
- To 200 μl of glycine-saline buffer taken in a vial,add 4 μl of test antisera. ( 50 times diluted ).
- Add 50 μl of antigen to 50 μl of diluted antiserum in a 1.5 ml vial, mix well and incubate at room temperature for 10 minutes.
- Pipette 10 μl of coated latex onto a glass slides.
- Add 10 μl of diluted test antiserum to slide A.
- Add 10 μl of antiserum mixed with antigen (from step 8) to B.
- Add 10 μl of glycine-saline buffer to C.
- Take a tooth pick and mix the content in each slide. Discard the tooth pick after using in one slide (take a new one for the next slide ).
- After mixing, wait for 2 minutes to observe the result.
Result
- Presence of White clumps indicates a positive result i.e suspected particle is present.
- Absence of white clumps indicates a negative result i.e Suspected particle is not present.
Reagent preparation
Preparation of Glycine saline buffer ( 0.5 M) stock solution
Dissolve components Glycine ( 14 g), Sodium hydroxide, solid (0.7 g), Sodium chloride ( 17 g), Sodium azide ( 1g ) in 500 ml of distilled water and adjust to pH 8.6 with alkali as required. Make upto 1000 ml with distilled water.
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