Serological techniques and immune assays

Eman abd el-raouf ahmedImmunology department

The content

Serological assayImmune assay

Types of serologic reactions.

A. Agglutination

B-precipitation

A-agglutination

a/Direct agglutinat

ion

b/ Indirect agglutin

ation

c/ Conglutination

g/ Latex agglutination

f/ Indirect (passive)

haemagglutination

e/ Haemagglutina

tion - inhibition test

d/ Direct haemagglutin

ation

A. AgglutinationA corpuscular antigen - agglutinogen - is agglutinated when the specific antibody - agglutinin - is added. Agglutination can be read either visually or in the microscope . Presence or absence of clumping is noted.

a/Direct agglutinationtests the presence of antibodies in the serum. It is used most often as Widal reaction for diagnosing typhus and paratyphus. Direct agglutination is also used for detecting antibodies in tularemia, brucellosis (Wright reaction), listeriosis, rickettsial disease (Weil-Felix reaction).

b/ Indirect agglutinationtests the presence of unknown microbial antigen structure with a diagnostic serum. Slide agglutination is most often used method for the identification of the bacteria by means of specific diagnostic sera, especially in enterobacteria. Specific antisera are prepared by immunization of animals with bacterial strain.

.

c/ ConglutinationSpecific immunoglobulin G (e.g. against Streptococcus spp., Hemophilic influenza, Neisseria meningitidis) is bound with Fc fragment to protein A which is present on the surface of bacterial cell wall of Staphylococcus

aurous, strain Cowan I. After addition of the antigen the clumps are formed on the slide within 1 minute.

This reaction is used for identifying serological types of Streptococcus spp., Neisseria meningitidis and Hemophilic influenza

d/ Direct haemagglutinationis an agglutination of red blood cells. Haemagglutinaytion can be caused by antierythrocytic antibodies, by several viruses (e.g.myxoviruses, paramyxoviruses) and bacteria (Bordetella pertussis) which contain antigen called haemagglutinin. Haemagglutination is caused after their binding on the receptors present on the erythrocyte surface. Direct haemagglutination is used for the diagnosing of infectious mononucleosis (Paul-Bunnel reaction), for the detection of cold agglutinins in atypical pneumonia caused by Mycoplasma pneumonia (agglutination of human group 0 red blood cells at low temperatures). e/ Haemagglutination - inhibition testis used in virology. Many viruses agglutinate red blood cells because of containing haemagglutinin . Since the process is specifically inhibited by antibodies against the virus, haemagglutination inhibition can be used as an test for identifying viruses and measuring antibodies.

.

f/ Indirect (passive) haemagglutinationRed blood cells which are first treated with tannic acid or formalin are the carriers of soluble antigens. Antigen is either adsorbed or bound on the erythrocyte surface. The method is sensitive and is used to detect antibodies against enterobacteria, Clostridium tetani, Treponema pallidum (TPHA test) and against some tissue antigens (e.g. thyroid gland antigens). g/ Latex agglutinationInert latex particles are the carriers of antigens. There are many commercial latex particle tests, e.g. for grouping streptococci, detecting bacterial antigens from cerebrospinal fluid, for detecting rheumatoid factors, for detecting some viruses from obtained samples (rotaviruses and adenoviruses from the stools). Also antibodies against rubella can be detected by latex particle test

Agglutination reactionsa. Agglutination reactions serve to detect ant quantitate agglutinins and to identify cellular antigens such as bacterial cells, white blood cells, and red blood cells .

(1 )When cells interact in vitro with the appropriate antibody, they clump together and eventually form

masses that become large enough to be seen. (When antibody agglutinates bacteria in the body ,opsonization occurs .)

(a ) Agglutination occurs because antibodies are at least bivalent) . ., i e they have at least two combining

sites .)(b ) Two sites on the antibody and multiple sites on the antigen

result in antigen antibody lattice formation that can build up into increasingly larger complexes

(2 )The aggregates may be seen in the test tube or under the microscope. (3) If red blood cells are employed as the agglutinogen, the process is hem agglutination. b. The classic application of the agglutination reaction is the Widal test for the diagnosis of typhoid fever. (1) In this test, the antibody content of the patient's serum is measured by adding a constant amount of antigen (e.g., Salmonella typhi) to serially diluted serum, that is, to progressively more dilute serum samples. (2) After appropriate incubation, the serum samples are examined for visible agglutination. The highest dilution of serum that shows agglutination is referred to as the titer.

A-ring precipitation test

d/ Immunoelectrop

horesis

e/ Countercurrent

immunoelectrophoresis

b/ Slide precipitation

C-gel-diffusion precipitation

c/ Gel - diffusion precipitation

c/ Gel - diffusion precipitation

B. Precipitation

B-precipitationa/ Ring precipitation test

A solution of antigen is layered on the surface of the antibody in a small tube or capillary tube. A narrow ring of precipitate occurs near

the junction of two fluids. The result can be read visually. The concentration of immunoprecipitate is possible to be measured by this

method or by laser nephelometry. This type of test is used for grouping streptococci (according to C

polysaccharide), for determining unknown proteins in forensic medicine.

b/ Slide precipitation

is carried out on a slide and the occurrence of precipitate is detected in the light microscope . This type of precipitation test is used for

diagnosing of lues (quick reagin reaction).

c/ Gel - diffusion precipitationAntigen and antibody meet in an agar medium and a thin line of precipitate is

produced there (antigen - antibody complex).1. Single diffusion

Antigen diffuses in the agar medium (antibody is homogenously spread in the agar). It is carried out either in the tubes - single gel - diffusion by Oudin or on

the slide - single radial immunodiffusion by Mancini. The principle of the reaction: The antigen is placed in a well cut in an agar gel

containing suitable diluted antibody. A ring of precipitate forms where the reactants meet in optimal proportions. The higher is the concentration of the

examined antigen, the greater is the diameter of the ring. According to the diameter of the ring it is possible to count the concentration of the examined

antigen. This type of immunodiffusion is used for quantitative determination of

immunoglobulins (IgM, IgG, IgA and IgD), complement components and other serum proteins.

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2 .Double immunodiffusion by Ouchterlonyis used more often. Antigen and antibody are allowed to diffuse towards each other in an agar medium, e.g. from separate wells cut in an agar plate or in a Petri dish. When antigen and antibody

meet in optimal proportions they produce a thin line of precipitate. Position of the precipitate line depends on

concentrations of both antigen and antibody and on their diffusion coefficient.

This reaction is used for diagnosing various bacterial, viral, fungal and autoimmune diseases, for recognizing toxin

production by Corynebacterium diphtheria

d/ Immunoelectrophoresisis a combination of electrophoresis and gel - diffusion precipitation. Antigens (most usually serum proteins) are first divided by electrophoresis according to their electric charge (albumins are directed towards the anode and globulins towards the cathode) on an agar coated slide. After electrophoresis is finished the longitudinal troughs are cut in the agar parallel to the axis of electrophoresis and filled with antibody. Diffusion then takes place. When antigen and antibody meet precipitate lines of single immunoglobulin classes occur. The lines are read after staining by amid black dye. Immunoelectrophoresis is a delicate technique for analyzing complicated mixtures of antigens and antibodies, e.g. serum immunoglobulins. e/ Countercurrent immunoelectrophoresisis a rapid and more sensitive variant of double diffusion method in which an electric current is used to drive the antigen towards the antibody in negatively charged gel. This method was used to detect hepatitis B surface antigen. It is used for the rapid detection of bacterial antigens in clinical specimens, alfa-1-fetoprotein, etc. This method is being replaced by the ELISA methods.

Immune assay

Immunoassays are chemical tests used to detect or quantify a specific substance, the analyze, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. High sensitivity is achieved by using an indicator system (e.g., enzyme label) that results in amplification of the

measured product .

Immunoassays may be qualitative (positive or negative) or quantitative (amount measured). An example of a qualitative assay is an immunoassay test for pregnancy. Pregnancy tests detect the presence of human chorionic gonadotropin (hCG) in urine or serum. Highly purified antibodies can detect pregnancy within two days of fertilization. Quantitative immunoassays are performed by measuring the signal produced by the indicator reaction. This same test for pregnancy can be made into a

quantitative assay of hCG by measuring the concentration of product formed .

The purpose of an immunoassay is to measure (or, in a qualitative assay, to detect) an analyte

for measuring analytes normally present at very low concentrations that cannot be

determined accurately by other less expensive tests .

Qualitative immunoassays are often used to detect antigens on infectious agents and antibodies that the body produces

to fight them .

For example, immunoassays are used to detect antigens on Hemophilic, Cryptococcus , and Streptococcus organisms in the cerebrospinal fluid (CSF) of meningitis patients. They are also used to detect antigens associated with organisms that are difficult to culture, such as hepatitis B virus and Chlamydia trichromatic . Immunoassays for antibodies produced in viral hepatitis, HIV, and

Lyme disease are commonly used to identify patients with these diseases .

There are several different methods used in immunoassay tests .

Immunoprecipitation. The simplest immunoassay method measures the quantity of precipitate, which forms after the reagent antibody (precipitin) has incubated

with the sample and reacted with its respective antigen to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative .

Particle immunoassays. By linking several antibodies to the particle, the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of antibodies that are markers of such diseases, as infectious mononucleosis and

rheumatoid arthritis .

Immunonephelometry. The immediate union of antibody and antigen forms immune complexes that are too small to precipitate. However, these complexes will scatter incident light and can be measured using an instrument called a nephelometer. The antigen concentration can be determined within minutes of the reaction

Radioimmunoassay (RIA) is a method employing radioactive isotopes to label either the antigen or

antibody. This isotope emits gamma rays are, which are usually measured following removal of unbound (free)

radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal

detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by

the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced

in routine clinical laboratory practice by enzyme immunoassay

Enzyme (EIA) immunoassay was developed as an alternative to radioimmunoassay (RIA). These methods use an enzyme to label either the antibody or antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes.

One of the most widely used EIA methods for detection of infectious diseases is the enzyme-linked immunosorbent assay

(ELISA) .

Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

Chemiluminescent immunoassays utilize a chemiluminescent label.

Chemiluminescent molecules produce light when they are excited by chemical energy. These emissions are measured by a light

detector .

Enzyme Immunoassays (EIA)There are two general approaches to diagnosing diseases by immunoassays: testing for specific antigens or testing for antigen-specific antibodies. Enzyme linked immunosorbent assays (ELISA), also known as enzyme immunoassays (EIA), are tests designed to detect antigens or antibodies by producing an

enzyme triggered color change .

All of the EIAs performed in the Diagnostic Serology Section are known as solid-phase assays. This assay requires the immobilization of antigens or antibodies on solid surfaces such as

plastic beads or the wells of microtiter plates .

EIAs for Antigen Detection

The only tests performed in the Diagnostic Serology Section for antigen detection are Hepatitis B EIAs. They are direct EIAs which have four steps:

*Antigen-specific antibody is attached to a solid-phase surface (plastic beads)

*Test specimen is added, which may or may not contain the antigen

*An enzyme-labeled antibody specific to the antigen is added (conjugate)

*Chromogenic substrate is added, which in the presence of the enzyme, changes color. The amount of color that develops is proportional to the amount of antigen in the test specimen.

Noncompetitive EIA

Specific antigen is attached to a solid-phase surface (plastic bead or microtiter well)

Test specimen is added, which may or may not contain the antibody

An enzyme-labeled antibody specific to the test antibody is added (conjugate)

Chromogenic substrate is added, which in the presence of the enzyme, changes color. The amount of color that develops is proportional to the

amount of antibody in the test specimen .

Competitive EIA

Specific antigen is attached to a solid-phase surface (plastic bead or microtiter well)

Test specimen, which may or may not contain the antibody, and an enzyme-labeled antibody specific to the test antibody (conjugate) are added together

Chromogenic substrate is added, which in the presence of the enzyme, changes color. The amount of color that develops is inversely proportional to the amount of antibody in the test

specimen .

Capture EIA

A capture EIA is designed to detect a specific type of antibody, such as IgG or IgM.

Antibody specific for IgG or IgM is attached to a solid-phase surface (plastic bead or microtiter well)

Test specimen containing IgG or IgM is addedSpecific antigen is added

An enzyme-labeled antibody specific to the test antigen is added (conjugate)

Chromogenic substrate is added, which in the presence of the enzyme, changes color. The amount of color that develops is proportional to the amount of antigen-specific IgG or IgM in the test specimen

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