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1588745070antisera Titration and Avidity

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236 views9 pages

1588745070antisera Titration and Avidity

Uploaded by

Tanupreet
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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MLS 306

ANTISERA POTENCY, TITRATION


AND AVIDITY

AKINBO D.B. Lecture Series


Antisera potency and specificity
• An antiserum must be specific for the antigen to be detected
under the test conditions.

• The specificity of an antiserum can be established by testing


equal volumes of serum against 5% red cells suspension.

• An agglutination reaction would only occur when reacted


with the corresponding cells.

• The serum to be employed for grouping must have sufficient


titre against the corresponding cells in use as this determines
the potency of the antisera.
Antisera Titration Avidity
• Avidity refers to the ability of a serum to react very quickly and
strongly with its corresponding antigen.

• Avidity is based on affinity, specificity and strength of the serum;


affinity refers to strength of binding of single epitope to single
antigen binding site.

• A suitable antisera should readily produce complete


agglutination in 30 seconds when mixed with its corresponding
cell suspension.

• The avidity of the anti-A is of clinical relevance in the detection


of weak subgroups of A.
Test for avidity
 If anti-A is reacted against A cells, agglutination occurs in
10secs.

 If anti-B is reacted against B cells, agglutination occurs in


10secs.

 If anti-A+B is reacted against A cells, agglutination occurs in


10secs.

 If anti-A+B is reacted against B cells, agglutination occurs in


20secs.

NB. “A” cells react quicker and stronger with AB serum than “B”
cells because the “A” component of the antiserum A+B is
stronger than the “B” component.
Antisera titration

• The stronger the antibody, the greater the ability of


the serum to pick the weakest antigen.

Tube No. 1 2 3 4 5 6 7 8 9 10 11

Dilution (antisera) N ½ ¼ 1/8 1/1 1/32 1/64 1/128 1/256 1/512 1/1024
6
Anti-A vs 2-5% A C C C C # # # # ++ + +/-
cells
Anti-AB vs 2-5% A C C C C # # # # ++ + +/-
cells
Anti-A+B vs 2-5% B C C C # # # # ++ + +/- -
cells
Human Sources of Antisera
• For human donors, selection is carried out by picking out the
choice of antiserum from the specific blood group.

 Collection of Group A blood yields anti-B


 Collection of Group B blood yields anti-A
 Collection of Group O blood yields anti-A+B

• A potent anti-A serum for use as a good diagnostic reagent should


have a titre of 512 when titrated against A cells.

• Anti B should have a titre of 256 with B cells.

• Anti-A+B should also give titre of 512 and 256 when titrated with
A and B cells respectively.
Absorption of Antisera
• Adsorption: Unwanted, cold and atypical agglutinins are
adsorbed by mixing a volume of the serum with 1 volume
of 10-20% pooled O “Rh” positive cell suspension.

• This is then kept at 4 ̊C overnight for the removal of


unwanted, non-specific cold reacting antibodies.

• The sera are centrifuged and separated on the following


day with the separated serum kept at 56ͦC to remove
complements and lysins.
Standardization of antisera
• The prepared antisera would be titrated against A2, B and O
cells.

• The result would then appear in this pattern:

Antisera A cell B cell O cell

A2 C - -

B - C -

A+B C V -
Antisera Preparation..
• Dispense the prepared antisera into aliquots of about 5-
10mls.

• Preservation: Add a drop of sodium azide.

• Coloration:
*Add methylene blue to the prepared anti-A to give a blue
color.
*Add Eosin to the prepared anti-B to give a yellow color

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