Materials Required

1. 1.5 ml Vials.
2. Microcentrifuge.
3. Pipette.
4. Microtips.
5. Laboratory refrigerator.
6. Glycine saline buffer.
7. Blocking buffer.
8. Antigen for coating.
9. Latex beads.
10. Test antiserum.
11. Glass slides.
12. Beaker.
13. Tooth pick.

Procedure

Coating of Latex

1. To 20 μl of latex beads taken in a 1.5 ml vial add 40 μl of glycine-saline buffer.
2. Add 60 μl of antigen to the latex and incubate at 37oC for 2 hours.
3. Spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant.
4. Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10 minutes.
5. Repeat the washing once more.
6. Add 90 μl of blocking buffer to the pellet, mix well.
7. Incubate at 4oC, overnight.

Agglutination Test

1. To 200 μl of glycine-saline buffer taken in a vial,add 4 μl of test antisera. ( 50 times diluted ).
2. 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.
3. Pipette 10 μl of coated latex onto a glass slides.
4. Add 10 μl of diluted test antiserum to slide A.
5. Add 10 μl of antiserum mixed with antigen (from step 8) to B.
6. Add 10 μl of glycine-saline buffer to C.
7. 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 ).
8. 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.