Serological Identification of Patients Antibodies

The serology laboratory tests patients sera to detect specific antibodies. The results of such tests may
provide a serological diagnosis of an infectious disease in which antibody was produced in specific response to
the microbial antigens of the infecting microorganism. Demonstrating increasing quantities of antibody in the
serum during the course of disease from its onset (when there may be little or no antibody) and acute stages
through convalescence (when large amounts of antibody have been produced) constitutes evidence of current
active infection and indicates the nature of the etiologic agent.
The interaction of a patients antibody with a specific antigen may be demonstrated in one of several
ways. Descriptive terms for antibodies refer to the type of visible reaction produced.
Agglutinins are antibodies that produce agglutination, a reaction that occurs when bacterial cells or
other particles are visibly clumped by antibody combined with antigens on the cell surfaces.
Precipitins are antibodies that produce precipitation of soluble antigens (free in solution and
unassociated with cells). When antibodies combine with such antigens, the large complexes that result simply
precipitate out of solution in visible aggregates.
Antitoxins are antibodies produced in response to antigenic toxins. Since toxins are soluble antigens, in
vitro interactions with antitoxins are seen as precipitation.
Opsonins are antibodies that coat the surfaces of microorganisms by combining with their surface
antigens. This coating on the bacterial cell makes them highly susceptible to phagocytosis by white blood cells.
(The word opsonin has a Greek root that means to relish food.)
Serological tests may be performed in vitro (in the test tube) or in vivo (in the body of an animal or
human). In in vitro tests, quantitative methods are often employed. An in vivo test may employ experimental
animals or cell cultures to demonstrate neutralization of an antigen by its antibody.
When antibody combines with antigen in the test tube, the antigen is neutralized or inactivated. If it is a
virus or other microorganism, it can no longer infect tissues or cell cultures. If it is a toxin combined with
antitoxin, it is no longer toxic for tissues. If the antigen-antibody complex is injected into an animal, it will not
cause disease or damage, whereas a non-immune control animal injected with the antigen alone will display
characteristic symptoms of disease, according to the nature of the antigen. Certain harmful antigens will also be
ineffective when injected into animals if specific antibodies have been administered to the animal a short time
before (passive immunity).
Skin tests constitute another type of in vivo serological test. Depending on the nature of the antigen
injected intradermally, a humoral or cellular (delayed hypersensitivity) immune response may be demonstrated.
Patients immune to diphtheria experience no reaction at the site of injected diphtheria toxin (Schick test)
because their circulating antitoxin neutralizes this antigen. Conversely, when persons who are (or have been)
infected by tubercle bacilli are injected with a purified protein derivative of this microorganism, the response is a
reddened area of induration at the injection site. This reaction results from vasodilation and infiltration of
lymphocytes.
To quantitate antibody in serum, serial dilutions of the serum are made by setting up a row of test
tubes, each containing the same measured volume of saline diluent. A measured quantity of serum is added to
the first tube and mixed well. The dilution in this tube is noted (1:2, 1:4, 1:10, or whatever). A measured aliquot
of this first dilution is then removed and placed in the second tube, containing measured saline. Material in the
second tube is mixed, and an aliquot is removed and placed in the third tube. The procedure is repeated down
the line of tubes, so that a graded series of serum dilutions is obtained. The antigen is then added in a constant
volume per tube. After allowing time (at the right temperature) for antigen-antibody combination to occur, the
tubes are examined for visible evidence of such combination. The reciprocal of the last (highest) dilution of
serum that produces a visible reaction is reported as the titer of the serum because it indicates the relative
quantity of antibody present. If two sera are compared for reactivity with the same antigen, the one that can be
diluted furthest and still show reactivity is said to have the higher titer, that is, the most antibody.
In serological diagnosis of infectious disease, it is almost always necessary to test two samples of the
patients serum: one drawn soon after the onset of symptoms during the acute stage, and another taken 10 to
14 days later. The reason for this is that antibody production takes time to begin and to build up to detectable
concentrations during the course of active infection. The first sample may show no antibody, or a low titer that
could reflect either past infection or previous vaccination with the microbial antigen in question. If the second
sample shows at least a fourfold or greater increase in titer as compared with the first, it is evident that current
active infection has induced a rising production of antibody. Such laboratory information is of great value both
in diagnosis and in evaluation of the immunologic status of the patient with respect to any antigen tested.

Laboratory Exercise 4
Serial Dilution
Purpose:

To know how to prepare a ten-fold and two-fold dilution and,

To recognize the importance of serial dilution in interpreting titers of serological tests

Materials:

5ml test tubes (15 pc), 1ml and 5 ml pipettes, liquid w/ dye, diluent (tap water), test tube rack

Procedures:
A. Two-fold Dilution
1. Arrange test tubes in a rack.
2. Dispense 0.5 ml diluents in all tubes.
3. Use the same pipette to measure 0.5ml of dye and add on the first tube.
4. From tube 1, transfer 0.5 ml of suspension to tube 2.
5. Repeat step 4 for all remaining tubes.
6. Discard 0.5ml from tube 10.
7. Resulting dilutions in the tubes are:
Tube #
1
2
3
4
5

Dilution
1: 2
1: 4
1: 8
1: 16
1: 32

Tube #
6
7
8
9
10

Dilution
1: 64
1: 128
1: 256
1: 512
1: 1024

8. Determine the titer (the last tube in which color is visible).

B. Ten-fold Dilution
1. Arrange 5 tubes in a rack.
2. Dispense 0.9 ml of diluents in all tubes.
3. Use the same pipette to measure 0.1 ml of dye and add to the first tube. Mix by pipetting in and
out, then transfer 0.1ml to test tube 2.
4. Repeat step 3 for the remaining tubes.
5. Discard 0.1 ml from tube 5.
6. The resulting dilutions in the tubes are:
Tube #

1
1:10

2
1:100

3
1:1000

4
1:10,000

5
1:100,000

7. Determine the titer (the last tube in which color is visible).

8. Compare the titer with the result in two-fold dilution.

Study Guide:
1. Define serum titer.
2. What is the significance of determining antibody titer? What is the importance of dilution in
serological tests?
3. What are acute and convalescent sera? Why must they both be tested in making a serological
diagnosis of infectious disease?
4. Give three examples of diagnostic tests in which antibody titers are determined.
5. Define toxin, antitoxin, and toxoid.
6. Define natural and acquired immunity.
7. How do immunological tests for detecting microorganisms or their antigens in patient specimens
differ from serological tests to detect antibodies in patient sera?
8. Why is immunity to tuberculosis detected by a skin test rather than by a test for patients serum
antibodies?
9. What is the RPR test?
10. What is the importance of testing for rubella antibodies in women?