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Latex Agglutination
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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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