agglutination (2)

8/13/2019 Agglutination (2)

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AGGLUTINATION

Agglutination:

Is the clumping of antibody antigen complex to form insoluble andvisible aggregates.

The word agglutination comes from the Latin agglutinare, meaning "to glue to."

Antigen:

Antigen is any molecule that binds specifically to an antibody .

Antibody:Antibodies (immunoglobulins) are proteins secreted by the immune system

to identify and neutralize foreign objects (antigens), such as bacteria and

viruses .

Factors affecting the agglutination reaction in vitro:

1) Antigen to antibody ratio: he ratio bet!een antigen and antibody influences the detection of

antigen"antibody complexes.

Antigen or antibody excess ma#e invisible reaction.

Prozone phenomenon (antibody excess)

here are too many antibodies.

Antibodies saturating all antigen sites

$o antibodies forming cross"lin#ages bet!een cells or particles

so, no agglutination appears ( false"negative reactions).
http://en.wikipedia.org/wiki/Latin_languagehttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Immune_systemhttp://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Latin_languagehttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Immune_systemhttp://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Virus


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!one of e ui#alence

Antibodies and antigens are present in an optimum ratio.

his leads to cross"lin#ages bet!een acells or particles, so

agglutination appers (positive reaction).

Post$zone phenomenon (%ntigen excess)

here are too many antigens

Any agglutination is hidden by masses of unagglutinated

antigens that gives false"negative reactions.


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These false$negati#e reactions can be detected by repeating the

test at a higher dilution of sample, which reduces the antigen or

antibody concentration into the range that produces #isible

agglutination.

Nu ber of Antigen !ites : he more antigen sites on a cell result in more cross"lin#ages bet!een

cells.

hese cross"lin#ages result in more visible agglutination.

!i"e and !tructure of the Antibody : %arger antibody causes more cross"lin#ages bet!een different cells. he larger antibodies (Ig&) can reach bet!een more antigen sites on

different cells and therefore causing stronger agglutination reactions.

Ig& antibodies also have more binding sites to react !ith antigens and

potentially causing cross"lin#ages bet!een ' different cells.

#istance bet$een cells: he small Ig antibodies usually cannot reach bet!een t!o cells as

the distance bet!een its t!o arms is narro!.

he larger antibodies, Ig&, can reach bet!een cells that are further

apart and cause agglutination.

entrifugation of the cells attempts to bring the cells closer together,

so enhance agglutination.

%) &ell surfce electric charge '(eta otential): *eta potential is an electric charge surrounding the red blood cells

ma#es the cells maintain a certain distance from each other.


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It ma#es repulsion bet!een the red blood cells !hen suspended in

saline.

It is cause by sialic acid groups on the red blood cell membrane !hich

gives the cells a negative charge. he positive ions in saline attracted to the negatively charged red

blood cells.

he net positive charge surrounding cells in saline #eeps them far

apart due to repulsion from electric charges.

+maller antibodies (Ig ) cannot cause agglutination !hen zeta

potential exists o overcome zeta potential techni ues add albumin to test mixture as

- " groups of albumin neutralize positive charge.

&lassification of agglutination reactions:

/. 0irect agglutination reactions1

23 (Ag) 4 specific Ab 5 agglutination


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&xamples

A3- blood group typing

2h (0) Ag

6. 7assive (indirect) agglutination

Patient serum, %b ('g ) soluble %g adsorbed particle *

agglutination

7assive Agglutination is a very sensitive method for antibody

detection.

23 , bacterial cells or inert particles such latex can be used asa carrier for soluble antigens.

he soluble antigen is absorbed onto the carrier cell or particle,

then reacted !ith the specific antibody.

his ma#es the reaction more visible than precipitation.

&xample

o +heumatoid factor detection

2heumatoid factor is Ig&"anti Ig !hich agglutinates

latex beads coated !ith Ig .

o $+eacti#e Protein detection ( +P)

27 is an immunologic reaction bet!een serum

containing 27 (antigen) and the corresponding antibody

coated on latex particles.

Test procedure

Add 1 dro &*+ late, to 1 dro of atient seru ' ay be diluted)


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then stir - i,.

-rades of the positi#e reaction

84 %arge clumping !ith clear fluid in the bac#ground94 &oderate clumping !ith fairly clear fluid in the

bac#ground

64 +mall clumping !ith a slightly opa ue fluid in the

bac#ground

/4 :ery small clumping !ith fluid definitely opa ue

+ractical $or/ sheetGive an account on:


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Factors Affecting agglutination tests in vitro:

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Ty es of agglutination test and $rite an e,a le for each:

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+rocedure of &*+ detection:

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#irect Antiglobulin Test '#AT)

0#irect &oo bs test

Ai :

o detect the coated red blood cells !ith incomplete immunoglobulin (Ig ) invivo.

+rinci le 1

Ig 4 23 5 give complex !ith no agglutination (in vivo).

Add anti human globulin ;Ig&< to the complex 5 agglutination (invitro)

!a le:

3lood dra!n into & T% is preferred but oxalated or citrated blood may

be used.

he blood sample should be tested as soon as possible after collection

and should not be stored.

*eagents2 34ui ent and !u lies:

7olyspecific A ( oombs)


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Isotonic saline

oombs ontrol ells

7ipettes

/6 x =' mm tubes entrifuge

&icroscope

+rocedure:

/. 7ut /"6 drops of blood in >0 A tube.

6. ?ash this tube three times !ith isotonic saline.9. 7repare a '"/@ suspension from the !ashed cells.

8. Add one drop of the !ashed '"/@ suspension to a tube.

'. hen, add one drop 7olyspecific Antihuman lobulin and sha#e to mix.

B. entrifuge the tube for 9@ seconds.

=. Immediately resuspend gently and examine for agglutination.

Inter retation:

/. $o agglutination indicates a negative 0A " nothing is coating the

patientCs cells in vivo.

6. Agglutination !ith 7olyspecific A indicates a positive 0A , due to

either Ig antibodies or complement coating the patientCs cells.

+reccautions:

/. he !ashing step must proceed uninterrupted. A delay in !ashing may

cause antibodies to be eluted off cells and !ashed a!ay.

6. A must be added to the cells immediately follo!ing !ashing.


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Antibodies may elute from the cells if they are allo!ed to sit in saline

!ithout the addition of A .

9. he oombs ontrol ells must be positive. her reasons for false

negative test include1

Inade uate !ashing of the cells. 2esidual serum neutralizes the A

so that it cannot react !ith the antibody"coated oombs ontrol

ells at the end of the procedure.

3e sure the cells are thoroughly resuspended bet!een each !ash.

A delay in adding A . Antibodies that !ere attached to the red

cells may elute off into the saline, and neutralize the A !hen it is

added.

Add the A immediately after the final !ash.

A !as omitted ;inactive< or !as diluted out by too much residual

saline in the tube.

0rain the last !ash !ell and blot before adding A .


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+ractical $or/ sheetGive an account on:The ai of direct &oo bs test:

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+rocedure of direct antiglobin test:

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agglutination (2)

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