Immunoassays

Immunology - Applications

Bacterial infections usually result in an immune response in the host. The
products of the immune response react specifically with the bacterial
immunogens that stimulated their production. Assays have been developed that
use bacterial antigens to detect the antibodies produced by the immune system
and antibodies to detect antigens. The immune response of the host to some
bacterial antigens may result in specific immunity to, or recovery from, a
particular infection. Antibodies to other bacterial antigens do not effect
the course of the disease nor do they provide immunity. They are important
for use in immunoassay procedures. This program will deal with the uses
immunology has been put to in the bacteriology laboratory under three major
headings: 1. Detection of Bacterial Antigens; 2. Response to Bacterial
Antigens and; 3. Principles of Immunoassays. There are advantages and
disadvantages to the use of antigens and antibodies as reagents. Advantages:
The specificity of antibodies for antigens in reactions means little
interference from other substances. A wide range of choices exists for
procedures depending on the sensitivity required. Manual manipulation is
usually simple, but most methods have the ability to be semi- and fully
automated with easily obtained and inexpensive equipment. Reagents are
inexpensive with a long shelf life and are chemically non-toxic. A variety
of labels may allow multiple, simultaneous assays. Some assays are quicker
than culture and do not rely on viable organisms. Disadvantages; A
biological hazard may exist depending on source of reagent (usually sera; eg.
Hepatitis B). The stability of the antigen/antibody reaction may require
critical control of some variable conditions like temperature or timing. The
purity of antibody or antigen, cross-reactivity (sometimes advantageous), or
presence of haptens (incomplete antigens) must be established. Expertise is
required in the manufacture and labeling procedures to ensure the quality of
the reagents. A number of control procedures are usually required to check
reactivity and monitor interference from non-specific or non-immunologic
reactions. Some procedures are slow, but frequently another assay technique
can be employed. The procedures should not be relied upon to replace culture
techniques.

Detection of Bacterial Antigens

Antigenic determinants are found on the microbial cell wall, capsule, and
flagella or they are released into the cells environment. Assays for
microbial antigens can therefore involve intact cells or fluid from the
microbial environment. The antigens can be obtained from single colonies on
plated media or broth cultures and also from sample sites. Detection of
bacterial antigens has become important in the laboratory diagnosis of
infectious diseases. It is very useful in body fluid specimens where rapid
results can direct antimicrobial therapy. In patients previously treated
with antibiotics, antigen detection may be the only way to establish the
cause of infection. There is also a correlation between the quantity of
antigen in the fluid and the prognosis of the infection. Antigens detected
in broth cultures can give a rapid presumptive identification of a bacterial
species. Direct agglutination reactions can also aid in bacterial
identification and serogrouping is important for epidemiology. Antigens can
be proteins, polysaccharides, or lipopolysaccharides in nature. Physiological
and biochemical activities of these substances are important for selecting an
assay method. Proteins are usually toxins, enzymes, or structural elements
like flagella, fimbriae, or cell wall proteins. Polysaccharides are found in
the capsule, on the cell wall, or are released. Lipoprotein antigens are
usually cell wall bound. Assays useful for antigen detection include
precipitation reactions (especially counterimmunoelectrophoresis),
agglutination reactions, enzyme-immunoassays, and fluorescent-immunoassays.
For more information on the assays, see Principles of Immunoassays. Antigen
detection methods can be used for any bacteria. Some methods have become
associated very closely with specific organisms. Antigen detection is
extremely useful for the following genera/species; Staphylococci,
Streptococci, Neisseria sp., H. influenzae, Enterobacteracae, Bordetella sp.,
Legionella sp., Corynebacterium sp., Clostridia, Chlamydia, and Rickettsia. ;

Counterimmunoelectrophoresis

Counterimmunoelectrophoresis (CIE) is useful for the detection of bacterial
antigens. It is faster and more sensitive than other immunodiffusion
procedures, but has been replaced in clinical laboratories by the even more
rapid latex agglutination procedure. CIE can be used for antigen detection in
any body fluid, suspension or extract of organisms, or broth culture. CIE is
based on the principle of immunodiffusion enhanced by electrophoresis to
drive the antigen and antibody toward each other. Test specimens are put in
a cathodic well, and the antiserum in an anodic well in an alkaline buffered
agarose layer. Most bacterial antigens detected by CIE are polysaccharides
and will migrate toward the anode. Antibodies are less negatively charged
and will move toward the cathode with the buffer ions. A precipitin line is
formed in the agarose where the reacting antigen and antibody are at optimal
proportions. Examination of the precipitin can be enhanced by staining the
CIE plate. Cautions: Extremely high titres of antigen or antibody will
prevent the formation of the precipitate. Possible cross- reactions and
migrational characteristics of the antigen must also be considered. Bacterial
antigens detected: Streptococci groups A-F, S. pneumoniae, Neisseria
meningitidis, H. influenzae, some Enterobacteracae, Pseudomonas aeruginosa.
Antigen concentration in a body fluid may be determined by testing serial
dilutions of the fluid.

Precipitation

When a soluble antigen and its antibody are mixed, antigen antibody complexes
form. Precipitation is the result of specific cross links between antigens
and antibodies and the complexes of ag/ab until they become so large that
they become insoluble. At ag/ab proportions that are closest to an optimal
ratio, the complexes precipitate most rapidly. The antigen/antibody reaction
is detected usually as a band of insoluble precipitate at the interface
between antigen and antibody where the optimal ag/ab proportion exists. The
precipitate is more stable if agar is incorporated in the test procedure.
These procedures are known as immunodiffusion assays and can be used
qualitatively and/or quantitatively. The technique first described by
Ouchterlony has produced more advances in the knowledge of immunology than
any other assay, but has been replaced clinically by more rapid and/or
sensitive methods.

If electrophoresis is applied to immunodiffusion, the antigen or antibody is
separated into component fractions and the resolving power of immunodiffusion
is greatly increased. Also see counterimmunoelecrophoresis. Examples of
precipitation reactions for bacterial antigens include many classic
bacteriology procedures like capillary tube precipitation (Lancefield's
grouping for Streptococci) and immunodiffusion in agar (Elek test for C.
diphtheria toxin). The Quellung reaction of capsular swelling in S.
pneumoniae is a form of preciptation reaction where the refractive index of
the capsules is changed because of the ag/ab reaction. Cautions: The type of
antigen and antibody under investigation can affect the type of assay chosen.
The optimal ag/ab ratio is affected by temperature, pH and salt concentration
of the solutions and by the way in which the antigen is added to the
antibody. The precipitate formed may be unstable, so observation timing may
be important.

Agglutination

Agglutination differs from precipitation by the form of the antigen involved.
Instead of soluble molecules, agglutination is the aggregation of insoluble
particles into larger clumps. Much less particulate antigen is needed for an
ag/ab reaction to be visible. A suspension of bacteria will agglutinate when
specific antisera is added. Microscopically small inert particles, such as
poly-styrene latex, can be coated with various soluble antigens or
antibodies. Latex agglutination has replaced many of the older immunoassay
procedures. Agglutination procedures are rapid and can be used to quickly
identify or confirm a bacterial species. They are usually performed on a
glass slide by mixing antigen and antibody and rocking the slide back and
forth for a few minutes. Agglutination of bacteria can be used to detect
antibodies in a patient's serum. The Widal test for typhoid and paratyphoid
is still used. Other agglutination tests for the presence of antibodies in
bacterial diseases include Brucella abortus and Franciella tularensis. The
Weil-Felix test is also useful for the diagnosis of rickettsial infections
such as typhus and spotted fever. This test employs antigens from Proteus
vulgaris strains Ox-2, Ox-19, and Ox-k to detect cross-reacting antibodies
produced during a rickettsial infection. Many latex kits for bacterial
surface antigens, toxins and antibodies are available commercially.
Antibodies detected include Streptococcal antistreptolysin O (ASO) and anti-
deoxyribonuclease (ADN). Kits are available for bacterial surface antigens
like Streptococci (groups A-F), Streptococcus pneumoniae, Campylobacter sp.,
E. coli 0157:H7 that use colonies from plated media. Some of these methods
require an extraction procedure to remove the antigens from the cell wall or
to remove interfering capsular material. Soluble antigens can be detected
in CSF or other body fluids for N. meningitidis, H. influenzae type b, S.
pneumoniae and E. coli giving an extremely rapid and non-culture dependant
diagnosis of meningitis. The toxin of C. difficle is detectable by latex
agglutination. Cautions: The reliability of the reagents in agglutination
procedures must be confirmed during each performance of the test by use of
appropriate control organisms or antisera.

Immunofluorescent (IF) Assays

Immunoglobulins and many antigens can be coupled to fluorescent dyes. The
reactions of these antigens or antibodies can be made visible in histological
or cytological preparation with a fluorescence microscope. Fluorescent dyes
or fluorochromes are a group of substances that achieve elevated but unstable
energy levels by absorbing light at a certain wavelength and immediately
emitting light at another wavelength. The most widely used fluorochromes in
IF studies are fluorescein and rhodamine. They are conjugated to a specific
antibody (direct method) or to an anti-species antibody (indirect method).
Sensitivity of IF is dependent on the immune properties of the reagents and
also on the optical system employed. IF methods of studying ag/ab reactions
have three advantages. First, these methods make it possible to locate
antigens in histological or cytological preparations. Second, because whole
cells or tissue sections may be examined, most or all of their antigenic
components are available as reaction sites. Third, a variety of test
procedures exists for the detection of antigen, antibodies, or complement. In
bacteriology, IF is usually used to detect bacterial cells in specimens or to
confirm the identification of an isolate. These assays are called
fluorescent antibody (FA) stains and are usually done by the direct method.
Direct FA conjugates are available for B. pertussis, some Enterobacteracae,
Legionella sp., N. gonorrhoea and N. meningitidis, Chlamydia sp., and
Treponema pallidum. Antibodies to treponema (syphilis) are detected by an
indirect FA test. Cautions: The purity of the antibody/conjugate is the most
important consideration in IF procedures. Reagents are available
commercially for most tests, but not for some specialized applications. IF
assays are less sensitive than radioimmunoassays or enzymeimmunoassays, but
is more sensitive than precipitation. Over-washing of stained preparations
can move some soluble antigens to other tissue sites. Background staining or
autofluorescence can interfere.

Enzymeimmuno Assays (EIA, ELISA)

Immunoglobulins and some antigens can be coupled to enzymes so that the
antigen/antibody reactions can be visualized by colourmetric or histochemical
examination. Histochemical staining with enzyme conjugated antibodies
enables the resulting tissue to be examined with routine light microscopes.
Changing the enzyme/ substrate system allows the tissue to be examined by
electron- microscopy. EIA is also known as enzyme linked immunosorbant
assay (ELISA) because either the antigen or antibody can be fixed to a solid-
phase surface. The enzyme is co-valently bound to the antibody. The reaction
between the antigen and antibody stays bound to the surface and is made
visible by the enzyme/substrate reaction. EIA is an extremely sensitive assay
technique because of the amplification of the ag/ab reaction by the enzyme. A
small number of enzyme molecules bound can convert a large number of
substrate molecules to a measurable level. ELISA can be used to detect
bacterial antigens in body fluids, but has not been highly utilized for this
type of assay as latex agglutination is more rapid and as sensitive. ELISA is
widely used for the detection and quantitation of antibodies to bacterial
antigens and has largely replaced complement fixations procedures. Cautions:
All unreacted sites on the solid-phase medium must be blocked or non-specific
reactions will occur. Washing between reagent additions is critical. The
reactivity of the reagents must be assured. EIA is affected by the problems
in immunoassays and also by enzyme kinetics, but careful design and
monitoring of the test system will ensure accurate results.

Other Immuno- and Non-immunoassays

Neutralization of a specific biological activity of an antigen by its
antibody may be used as assay technique. Neutralization is the principle of
the TPI (Treponema pallidum immobilization) test for syphilis, the Nagler
reaction for C.perfringens toxin, and the Anti-Streptolysin O Titer for
Strept. pyogenes. Non-specific immunoassays C-reactive protein (CRP) is an
abnormal alpha globulin that appears rapidly in the serum of patients who
have an inflammatory condition because of infection or other non-infectious
conditions. CRP is detected by mixing patient's serum with anti-CRP in a
latex agglutination procedure. The test is useful for diagnosis and
monitoring of patients with pelvic inflammatory disease (PID). The most
commonly used test for screening for syphilis is a not specific for syphilis.
The antigen used is a lipid fraction isolated from beef heart called
cardiolipin. The antibody which reacts with this antigen is called reagin
and is detected by a rapid slide precipitation (flocculation) test.
Non-immuno assays A rapid slide latex test has been developed for Staph.
aureus. The test is not an immune reaction but immunoglobulin is used. S.
aureus produces two biologically active proteins called coagulase and protein
A. The latex beads are coated with fibrinogen and the Fc portion of IgG.
Coagulase bound to the cell wall will cause clumping of the latex because of
the reaction on fibrinogen (converted to fibrin). Protein A binds to the Fc
portion of IgG and will also clump the latex. The method is extremely rapid
(20 seconds), and strong. Sensitivity and specificity are both greater than
99.5%. Pure isolates are required for testing because some other bacteria
may possess plasma binding factors.

Response to Bacterial Antigens

Serological testing can be an important addition to the diagnosis of
bacterial diseases. Care must be exercised in the interpretation of
serological results because antibodies may be present in the absence of
obvious disease. Serological tests vary in their sensitivity and
specificity, antigens vary in their ability to be immunogenic, and patients
vary in their response. Timing is important in serum collection, and it is
necessary to collect two samples. The first sample should be collected soon
after the onset of symptoms (acute stage) and the second should be collected
one or more weeks later (convalescent stage). Titration is performed on both
samples. When the second sample is run, the first sample should be repeated
to eliminate day-to-day, and batch-to-batch variation in assay. An increase
in titre of two dilutions (four-fold increase) or greater is considered
significant and suggestive of the cause of infection. Bacterial which produce
diseases with significant serological responses are S. pyogenes, Yersinia
entercolitica and pestis, Mycoplasma sp., Legionella sp., Treponema pallidum,
Leptospira, Rickettsia sp. and N gonorrhoeae. Immunoassay procedures used to
detect antibody responses include neutralization, agglutination of bacteria,
red blood cells, and latex, immunofluorescence, enzymeimmunoassay.

Immunoassays: References

Much of the information in the immunoassay section was obtained from the
following references, and the student is encouraged to use reference sources
for more details than are given here.

Lennette, E. H. et al. Manual of Clinical Microbiology, 4th ed. American
Society for Microiology, Washington, D. C. 1985.

Rose, N. R. and Friedman, H. et al. Manual of Clinical Immunology, 2nd ed.
American Society for Microbiology, Washington, D. C. 1980.

Rose, N. R. et al. Principles of Immunology, 2nd ed. MacMillan Publishing
Co. Inc., New York 1979