The McGill Physiology Virtual Lab

Immunology Laboratory


Antibody can be detected in the serum of animals. If red blood cells are used as a source of antigen, the assay is called hemagglutination. An animal receiving Sheep red blood cells (SRBC) will develop antibodies against SRBC.  In the experiment, SRBC are added to serially diluted serum from this immunized animal. The  antibodies in the serum will agglutinate to SRBC added in a test tube, resulting in a specific looking pattern at the bottom of the tube. Hemagglutination

  • measures the relative concentration of antibody in serum

  • is expressed as Titer

  • an indirect assay can also be devised: the antigen is linked to RBC

You are given two sera, from a "normal" mouse and one that was immunized with SRBC.

Using micro titer plates place 0.1 ml of balanced salt solution into each well of rows A and B. Add 0.1 ml of serum #1 to the first well, mix and transfer 0.1 ml to the next well in row A and so on until you reach the end.

Repeat the procedure for serum 2 in row B. Add 0.1 ml of a 1% suspension of SRBC. Incubate for 30 minutes at 37C and then place in the refrigerator. Best results are obtained if you examine the plates the next morning; however you should have results by the end of the laboratory session. Explain your results and report the titer of the two antisera.

 Serial dilution (doubling dilution): this technique is referred to as serial dilution, where you dilute a reagent or compound in series or in sequence. Since you take the same quantity of reagent from one well to the other, and the first well has the same volume as you put in: you are doubling your dilutions.

Antibody may be detected and measured by hemagglutination at lower concentrations than those detectable by other techniques. This relies on the ability of antibody to cross-link red blood cells by interacting with the antigens on their surface.
The agglutination of an antigen, as a result of crosslinking by antibodies, is dependent on the correct proportion of antigen to antibody.
Reading the 96-well plate:

If sufficient antibody is present to agglutinate and form cross-linking with the antigen, the antibody-antigen complex forms a mat at the bottom of the well. If insufficient antibody is present, the cells roll down the sloping sides of the well to form a red pellet or "button" at the bottom of the well.
Hemagglutination is expressed as titer: it is the inverse of the last dilution that is positive; e.g: 1/1000.


In the zone of equivalence, the correct proportion of antibody to antigen occurs, resulting in a visible mat formed by Ag-Ab complex crosslinking.

At high concentration of antibodies: Ag-Ab complex crosslinking is prevented to occur; every epitope on one antigen particle may bind to a single antibody molecule.
At higher dilution of serum, agglutination may occur: crosslinking is possible.

Testing serum at only one concentration may give misleading conclusions.
What might the absence of agglutination reflect?

    Depending on the starting dilution, the figure above could be derived.
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