KT53

Latex Agglutination Latex Agglutination

© © © © © Bangalore Genei, 2007 © © © © © Bangalore Genei, 2007

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GeNei™™™™™ Latex AgglutinationTeaching Kit

Manual

Cat No. New Cat No.

KT53 106206

KT53A 106207Revision No.: 00200605



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CONTENTS

Page No.

Objective 3

Principle 3

Kit Description 4

Materials Provided 5

Procedure 6

Observation & Interpretation 8

Ordering Information 9

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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 acomplex. At the optimum antigen-antibody concentration,this complex precipitates out. However, if the antigen isparticulate in nature, agglutination of antigen-antibodycomplex is observed.

In the latex agglutination method, antigen is coated ontoan 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 mixingwith latex, agglutination is inhibited; this is because freeantibodies are not available for agglutination.

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Kit Description:In this kit, latex beads, antigen and test antiserum are

provided. The experiment involves coating of the latex beadswith the antigen followed by blocking of the unreacted sites.The coated latex beads are reacted with the test antiserumand if the antiserum is specific for the antigen coated,agglutination is observed. The specificity of the agglutinationis further checked by agglutination inhibition reaction.

KT53 : The kit is designed to carry out 10 latexagglutination experiments.

KT53A : The kit is designed to carry out 20 latexagglutination experiments.

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

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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.

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

Tooth picks.

Quantity

Materials KT53 KT53A Store(10 expts.) (20 expts.)

Latex Beads 0.2 ml 0.4 ml 4°C

Glycine-Saline buffer 5 ml 10 ml 4°C

Blocking buffer 20 ml 40 ml 4°C

Antigen for coating 1.25 ml 2 x 1. 25 ml 4°C

Test Antiserum 50 µl 0.1 ml 4°C

Glass Plates 5 Nos. 5 Nos. 4°C



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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 4°C and usewithin 3 months. In case of KT53A, reconstitute onevial at a time.

• KT53: Reconstitute the test antiserum with 0.05 ml ofglycine-saline buffer. Store at 4°C and use within3 months.

• KT53A: Reconstitute the test antiserum with 0.1 ml ofglycine-saline buffer. Store at 4°C and use within3 months.

Procedure:Day1: Coating of Latex

1. Add 40 µl of glycine-saline buffer to 20 µl of latex beadstaken in a 1.5 ml vial.

2. Add 60 µl of antigen to the latex.3. Incubate at 37°C 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 washingonce more.

6. Add 90 µl of blocking buffer to the pellet, mix well andincubate at 4°C, overnight.

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Day 2: Agglutination Test7. Dilute the test antiserum 50 times with glycine-saline

buffer i.e. to 200 µl of glycine-saline buffer add 4 µl oftest antisera.Note: Use this diluted antiserum on the same day.

8. Add 50 µl of antigen to 50 µl of diluted antiserum in a1.5 ml vial, mix well and incubate at room temperaturefor 10 minutes. This will be used for testing theagglutination inhibition reaction.

9. Pipette 10 µl of coated latex onto a glass plates, asshown below:

a b c

10. Add 10 µl of diluted test antiserum to ‘a’.11. Add 10 µl of antiserum mixed with antigen

(from step 8) to ‘b’.12. Add 10 µl 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 ofagglutination. Agglutination is observed within 2 minutesas clumping of latex particles leaving a clear liquid phase(curdling). Absence of agglutination is seen as ahomogenous suspension (milky).

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Observation:Note down your observations as follows:

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Ordering Information

Product Size Cat #

GeNeiTM Latex Agglutination 1 Pack KT53Teaching Kit(Consumables for 10 experiments)

GeNeiTM Latex Agglutination 1 Pack KT53ATeaching Kit(Consumables for 20 experiments)

Email:Sales:[email protected]

Customer Support:[email protected]

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 thatthe antiserum has antibodies against the antigen usedfor coating.

• In the second case (b) no agglutination is observedbecause having pre-incubated antibody with the antigen,antigen-binding sites on the antibodies are alreadyoccupied by the antigen. Since free antibodies are notavailable for agglutination, inhibition is observed. This alsoconfirms that the agglutination seen in case (a) isspecific.

• In the third case (c) no agglutination is observed asantibody was not added and only buffer was used.

Case Agglutination

a

b

c



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Notes:

KT53

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