Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM

GeNei Latex Agglutination Teaching Kit Manual

Cat No. KT53 KT53A

Bangalore Genei, 2007 Bangalore Genei, 2007

New Cat No. 106206 106207

Revision No.: 00200605

Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM
CONTENTS
Page No.

Objective Principle Kit Description Materials Provided Procedure Observation & Interpretation Ordering Information

3 3 4 5 6 8 9

Bangalore Genei, 2007

Bangalore Genei, 2007

Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM

Objective:
To learn the technique of agglutination.

Principle:
All methods of detecting or quantitating antigen or antibody take advantage of the fact that they react to form a complex. At the optimum antigen-antibody concentration, this complex precipitates out. However, if the antigen is particulate in nature, agglutination of antigen-antibody complex is observed. In the latex agglutination method, antigen is coated onto an inert matrix i.e., latex, which behaves as particulate antigen. Hence, when antigen coated latex is mixed with an antibody, agglutination occurs which can be visualized by the naked eye. However, if the antibody is incubated with antigen prior to mixing with latex, agglutination is inhibited; this is because free antibodies are not available for agglutination.

Bangalore Genei, 2007

Bangalore Genei, 2007

Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM

Kit Description:
In this kit, latex beads, antigen and test antiserum are provided. The experiment involves coating of the latex beads with the antigen followed by blocking of the unreacted sites. The coated latex beads are reacted with the test antiserum and if the antiserum is specific for the antigen coated, agglutination is observed. The specificity of the agglutination is further checked by agglutination inhibition reaction. KT53 : The kit is designed to carry out 10 latex agglutination experiments. The kit is designed to carry out 20 latex agglutination experiments.

Materials Provided:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.
Quantity Materials Latex Beads Glycine-Saline buffer Blocking buffer Antigen for coating Test Antiserum Glass Plates KT53
(10 expts.)

KT53A
(20 expts.)

Store 4C 4C 4C 4C 4C 4C

0.2 ml 5 ml 20 ml 1.25 ml 50 l 5 Nos.

0.4 ml 10 ml 40 ml 2 x 1. 25 ml 0.1 ml 5 Nos.

KT53A :

Duration of experiment: Experiment is carried out over a span of two days, approximate time taken on each day is indicated below: Day 1: 2 hours 30 minutes (Coating of latex) Day 2: 1 hour (Agglutination test and Interpretation)

Materials Required:
Equipment : Microcentrifuge. Reagent : Distilled water. Other Requirements : Micropipette, Tips, 1.5 ml vials, Tooth picks.

Bangalore Genei, 2007

Bangalore Genei, 2007

Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM

Note:
Read the entire procedure before starting the experiment. Reconstitute the antigen for coating with 1.25 ml of glycine-saline buffer. Mix well. Store at 4C and use within 3 months. In case of KT53A, reconstitute one vial at a time. KT53: Reconstitute the test antiserum with 0.05 ml of glycine-saline buffer. Store at 4C and use within 3 months. KT53A: Reconstitute the test antiserum with 0.1 ml of glycine-saline buffer. Store at 4C and use within 3 months.

Day 2: Agglutination Test 7. Dilute the test antiserum 50 times with glycine-saline buffer i.e. to 200 l of glycine-saline buffer add 4 l of test antisera. Note: Use this diluted antiserum on the same day. 8. Add 50 l of antigen to 50 l of diluted antiserum in a 1.5 ml vial, mix well and incubate at room temperature for 10 minutes. This will be used for testing the agglutination inhibition reaction. 9. Pipette 10 l of coated latex onto a glass plates, as shown below: a b c

Procedure:
Day1: Coating of Latex 1. Add 40 l of glycine-saline buffer to 20 l of latex beads taken in a 1.5 ml vial. 2. Add 60 l of antigen to the latex. 3. Incubate at 37C for 2 hours. 4. After 2 hours, spin down at 5000 rpm for 10 minutes and carefully aspirate the supernatant. 5. Resuspend the pellet in 1 ml of blocking buffer and spin down at 5000 rpm for 10 minutes. Repeat the washing once more. 6. Add 90 l of blocking buffer to the pellet, mix well and incubate at 4C, overnight. 10. Add 10 l of diluted test antiserum to a. 11. Add 10 l of antiserum mixed with antigen (from step 8) to b. 12. Add 10 l of glycine-saline buffer to c. 13. Mix each of them with a separate tooth pick and gently swirl the plate by hand, observing for any signs of agglutination. Agglutination is observed within 2 minutes as clumping of latex particles leaving a clear liquid phase (curdling). Absence of agglutination is seen as a homogenous suspension (milky).

Bangalore Genei, 2007

Bangalore Genei, 2007

Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM

Observation:
Note down your observations as follows:
Case a b c Agglutination

Ordering Information
Product Size Cat # KT53

GeNeiTM Latex Agglutination 1 Pack Teaching Kit (Consumables for 10 experiments) 1 Pack GeNeiTM Latex Agglutination Teaching Kit (Consumables for 20 experiments)

KT53A

Denote + ve: Presence of agglutination - ve: Absence of agglutination.

Interpretation:
Agglutination is observed in the first case (a) in which coated latex is mixed with antiserum, indicating that the antiserum has antibodies against the antigen used for coating. In the second case (b) no agglutination is observed because having pre-incubated antibody with the antigen, antigen-binding sites on the antibodies are already occupied by the antigen. Since free antibodies are not available for agglutination, inhibition is observed. This also confirms that the agglutination seen in case (a) is specific. In the third case (c) no agglutination is observed as antibody was not added and only buffer was used. Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com

Bangalore Genei, 2007

Bangalore Genei, 2007

Latex Agglutination

GeNeiTM

Latex Agglutination

GeNeiTM

Notes:

Bangalore Genei, 2007

10

Bangalore Genei, 2007