Diagnostic techniques used in pathological analysis

المؤلف:  اعداد المرجع الالكتروني للمعلوماتية

المصدر:  almerja.com

الجزء والصفحة: 

16-2-2016

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Diagnostic techniques used in pathological analysis

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA.

The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used.

The test requires:

• the antibodies fixed to a solid surface, such as the inner surface of a test tube;
• a preparation of the same antibodies coupled to an enzyme. This is one (e.g., galactosidase) that produces a colored product from a colorless substrate.

Performing the Test

1. The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules.
2. The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody " sandwich".
3. After washing away any unbound conjugate, the substrate solution is added.
4. After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.

ELISA can also be adapted to measure the concentration of antibodies. In this case,

1- The wells are coated with the appropriate antigen.

2- The solution (e.g., serum) containing antibodies is added.

3- After they have had time to bind to the immobilized antigen,

4- an enzyme-conjugated anti-immunoglobulin is added, consisting of an antibody against the antibodies being tested for. For example, if human anti-HIV antibodies are being assayed, then antibodies (raised in a goat or rabbit against human immunoglobulins) are conjugated to o the enzyme.

5- After washing away unreacted reagent, the substrate is added.

6- The intensity of the color produced is proportional to the amount of enzyme-labeled antibodies bound (and thus to the concentration of the antibodies being assayed).

Literally hundreds of ELISA kits are manufactured for

• research
• human and veterinary diagnosis

Some examples:

• screening donated blood for evidence of viral contamination by

          -  HIV-1 and HIV-2 (presence of anti-HIV antibodies)

          -  hepatitis C (presence of antibodies)

          -  hepatitis B (testing for both antibodies and a viral antigen)

          - HTLV-1 and -2 (presence of antibodies)

• measuring hormone levels

          - HCG (as a test for pregnancy)

          - LH (determining the time of ovulation)

          -TSH, T3 and T4 (for thyroid function)

          - hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes

• detecting infections

             - sexually-transmitted agents like HIV, syphilis, and chlamydia o          hepatitis B and C

            - Toxoplasma gondii

•  detecting allergens in food and house dust
•  measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like lupus erythematosus
• measuring toxins in contaminated food
• detecting illicit drugs, e.g.,

          - cocaine

         - opiates

         - ∆-9-tetrahydrocannabinol, the active ingredient in marijuana

Immunofluorescence

Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. This technique is often used to visualize the sub cellular distribution of biomolecules of interest. Immunofluorescent-labeled tissue sections or cultures are studied using a fluorescence microscope or by confocal microscopy.

A-Direct fluorescent antibody

Direct fluorescent antibody (DFA or dFA) (also known as "Direct immunofluorescence") is a laboratory test that uses antibodies tagged with fluorescent dye that can be used to detect the presence of microorganisms. This method offers straight-forward detection of antigens using fluorescently labeled antigen-specific antibodies. Because detection of the antigen in a substrate of patient sample (cellular smear, fluid or patient- inoculated culture medium) is the goal, DFA is seldom quantitative. This is the main test used to detect rabies in animals and requires the examination of brain tissue.

B-Indirect Fluorescent Antibody

A form of fluorescent antibody technique commonly used to detect serum antibodies and immune complexes in tissues and microorganisms in specimens from patients with infectious diseases. The technique involves formation of an antigen-antibody complex which is labeled with fluorescein-conjugated anti-immunoglobulin antibody.

Radioimmunoassay

A highly sensitive and specific assay method that uses the competition between radiolabeled and unlabeled substances in an antigen-antibody reaction to determine the concentration of the unlabeled substance; it can be used to determine antibody concentrations or to determine the concentration of any substance against which specific antibody can be produced. Radioimmunoassay (RIA) is a very sensitive technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use a bioassay. Although the RIA technique is extremely sensitive and extremely specific, it requires specialized equipment and is costly. It also requires special precautions, since radioactive substances are used. Therefore, today it has been largely supplanted by the ELISA method, where the antigen-antibody reaction is measured using colorimetric signals instead of a radioactive signal. The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.

Agglutination

A 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- Latex agglutination

Inert 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.

B- haemagglutination

is 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 erythrocytal 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 pneumoniae (agglutination of human group 0 red blood cells at low temperatures).

Precipitation

When a specific antibody - precipitin combines with a coloidal antigen - precipitinogen in solution or in gel the antigen - antibody complex is thrown out of solution - precipitate. The precipitate is most heavy in the equivalence zone, when antigen and antibody are fully combined. In some tests optimum relation between antigen and antibody must be kept up to carry out the reaction - the so called flocculation. Flocculation test is used for the quantitative measurement of toxin, toxoid or antitoxin. Precipitation reactions may be carried out in various ways:

A -Gel - diffusion precipitation

Antigen and antibody meet in an agar medium and a thin line of precipitate is produced there (antigen - antibody complex).

I. 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.

II. Double immunodiffusion by Ouchterlony

is 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 diphtheriae.

III- Immunoelectrophoresis

is 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 amidoblack dye. Immunoelectrophoresis is a delicate technique for analyzing complicated mixtures of antigens and antibodies, e.g. serum immunoglobulins.