Highveld Biological

The specificity of the antigen/antibody reaction has long been used as a diagnostic procedure for either identifying an unknown antigen by means of a known antibody (A) or identifying an unknown antibody by means of a known antigen (B).

The immunofluorescence staining method is an important method for visualizing the site of an antigen/antibody reaction. A specifically equipped fluorescence microscope is essential. For the direct fluorescent antibody method (FA), specific antibody molecules are chemically linked to a fluorescent dye such as fluorescein isothionate (FITC). In the case of the indirect method (IFA) the antigen/antibody complex is linked to a FITC labeled second antibody which is directed against the species of the first antibody (i.e. goat anti-human antibody). Structures to which fluorescent antibodies are bound emit a bright fluorescent green, yellow or red color dependent on the dye used.

The attachment of an enzyme to antibody molecules creates an immunological tool which is both highly specific and highly sensitive. This enzyme-linked immunsorbent assay (ELISA) makes use of specific antibodies to which enzymes have been covalently bound in such a manner that the enzyme's catalytic properties and the antibody's cognitive properties are preserved; enzymes catalyze reactions whose products are colored and can be measured in very low amounts by special spectrophotometers which read the absorbance of such products.

Two ELISA methods have been developed, one for detecting antigen (direct ELISA) and the other for detecting either antigen or antibody (indirect ELISA): To detect antigen such as virus particles in blood or faeces of a patient, the specimen is added to the wells of a microtitre plate which have been coated with specific antibody. If antigen is present in the patient's specimen, it will be trapped by the antibody on the plastic surface; unbound (non-specific) material is washed away and a second specific antibody to which enzyme is covalently bound, is added. This complex will bind to the remaining exposed antigenic sites and after more washings the enzyme activity in each well is determined by adding substrate to the enzyme and measuring the intensity of the resulting color which is proportional to the amount of antigen present in the test material.

To detect antibodies in patient's serum, an indirect ELISA system is used: in this case the wells of a microtitre plate are coated with antigen to which the test serum is added. The antibody will bind specifically to the antigen and after washing, a second antibody such as goat or rabbit anti-human IgG covalently linked to an enzyme, is added. When a substrate is added to this, color will develop and the amount of circulating antibody to the specific antigen is quantitated by measuring its intensity.

Both, the immunofluorescence method and the ELISA technique are used extensively in today's serological laboratories besides other important test systems like the radioimmunoassay assay (RIA) and precipitation - and agglutination assays. The merits of one method over another are determined by the type of specimen, the available laboratory facilities, the patient's requirements and the cost per test.

A heterogeneous population of autoantibodies to nuclear constituents occur in rheumatic diseases; they are termed antinuclear antibodies (ANA) and are formed against any antigenic site on DNA, RNA or transformation molecules. Indirect immunofluorescence using a human derived epidermoid carcinoma cell line (Hep-2) as fixed antigen is the method of choice to detect such antibodies and to a degree assess antibody specificity. The following patterns of fluorescence may be seen:

Mixed patterns of multiple antibodies are generally observed when performing ANAs particularly on patients with rheumatic diseases; an individual pattern may become evident after the serum is further diluted. Antinuclear antibodies are mostly of the IgG or IgM class of immunoglobulins but may be of any of the five classes.


Autoantibody FA Screen (AA's)

Substrate slides for autoantibody screen (composite slides) are assembled cross sections of rat liver, rat kidney and mouse stomach. These substrates will detect the presence of the following antibodies in the patient’s serum: anti-nuclear antibodies (ANA), parietal cell antibodies (PCA), mitochondrial antibodies (MA), smooth muscle antibodies (SMA), antibodies to liver/kidney (L/KA) and ribosomal antibodies (RA).

In order to obtain a differential diagnosis, the autoantibody screen (AAS) should be used in conjunction with specific substrate slides e.g. anti-nuclear antibody, thyroid antibody (TA1001), native DNA antibody (Crithidia nDNA - CL1010), striated muscle/skin antibody (ST0633), adrenal antibody, pancreatic antibody, skin antibody oesophagus antibody and ovarian antibody.


Native DNA Antibody FA Test (Crithidia nDNA Test)

Antibodies directed against the patient's own deoxyribonucleic acid (DNA) are autoantibodies which react with antigenic sites on the DNA molecule. There are two kinds of anti DNA antibodies: firstly antibodies reacting with double stranded native DNA (nDNA) and secondly antibodies reacting with single stranded denatured DNA (ssDNA).

In order to differentiate between Systemic Lupus Erythematosus (SLE) and other vascular collagen diseases, the test for autoantibodies against native double stranded DNA (nDNA) is recommended. As antibodies directed against nDNA are not organ - or species specific, Crithidia lucillae, a hemoflagellate which contains nDNA, is used as a substrate.


Striated Muscle / Skin FA Test

Antibodies to striated muscle are present in 35% of patients with Myastenia gravis, a disease characterized by muscular weakness particularly of the cranial region. Also antibodies to structures of skin i.e. basement membrane and intercellular substance occur in several forms of Pemphigus, a potentially fatal skin disease of unknown etiology. As these antibodies are not organ - or species specific, satisfactory substrate slides comprise rat skeletal muscle, cardiac muscle, sections of oesophagus and skin and thus facilitate the differentiation of both auto-immune disorders on one test well.


Thyroid Antibody FA Test (TA Test)

Tests for autoantibodies against thyroid are indicated in the differential diagnosis of Grave's disease and Hashimoto's thyroid disease. Using the indirect immunofluorescence method, and monkey thyroid as substrate, two distinct organ specific autoantibodies are detected:

Low titers of these antibodies may be found in normal individuals and in patients with a variety of thyroid disorders like carcinoma of the thyroid and primary myxoedema and in patients with pernicious anemia and diabetes mellitus.

In order to establish true (thyroid) organ specificity, a specimen showing thyroid epithelial fluorescence should be tested on a composite slide (rat liver - rat kidney - mouse stomach): true thyroid antimicrosomal antibodies will not show any fluorescence in the autoantibody screen - AAS.


Parasitic Diseases

ELISA Test for Amoebiasis

Amoebiasis including amoebic colitis, amoebic dysentery and hepatic amoebiasis are infectious diseases caused by Entamoeba histolytica. In temperate climates the overall infection rate is between 1 and 10% while in tropical zones, the infection rate may be over 50%. The infective form of the parasite is the cyst which appears in the stool of infected animals and man; after accidental ingestion of contaminated food or water, cysts develop into trophozoites which multiply in the lumen and tissues of the digestive tract.

Amoebiasis is often asymptomatic or presents a non-specific clinical picture particularly in temperate climates. Amoebic dysentery however is common in the tropics with often enormous numbers of motile forms of E.histoytica in the diarrhoic and dysenteric stools. Through histolysis and phagocytosis the trophozoites can penetrate the mucus membranes and reach the liver, pleura, lung or pericardium where large abscesses may develop.


Gel Diffusion Test for Amoebiasis

The diagnostic precipitin test is based on Ochterlony's principle of diffusion of antibody in semi-solid medium and precipitation with antigen at the line of coalescence. This serological test for Amoebiasis is simple to carry out and very economical because it does not require sophisticated equipment.


Fluorescent Antibody Test for Bilharzia (Cercaria FAT)

The frozen and fixed larval stage (cercaria) of Schistosoma mansoni is used as antigen in the indirect fluorescence test for the serodiagnosis of bilharzia (schistosomiasis). The test is sensitive and specific. However as most patients come from endemic areas where they have been exposed to the parasite many times, the presence of antibodies reflects exposure rather than clinical encounter and results can only be expressed in positive or negative terms.


Bilharzia ELISA Test

The Enzyme-Linked Immunosorbent Assay (ELISA) is highly sensitive for the diagnosis of Bilharzia caused by Schistosoma mansoni. The test is positive in exposed individuals who do not show clinical symptoms and also in infected individuals before eggs appear in the stool. There are no false negatives; the level of antibodies does not bear any relationship to the intensity of the infection. High levels of antibodies may persist for several years.


Cysticercosis ELISA Test

If people live in close proximity to farm animals, in particular pigs, a condition called Cysticercosis will occur. This follows incidental ingestion of the ova of the tape worm Taena solium which will settle preferentially in the liver and brain and grow into large cysts. The ELISA test is a very specific test for the detection of antibodies to this serious condition which most often affects children.


Detection of Micro-organisms

Brucella ELISA Test

Brucellosis is an infectious disease which occurs world wide and is characterized by an acute stage with high fever without specific clinical manifestations and a chronic stage with relapsing fever, sweats, and general feeling of malaise. The causative micro-organism belongs to the genus Brucella and is associated with cattle (abortis), swine (suis) and sheep and goats (melitensis). Human brucellosis stems from direct contact with secretions and excretions and from ingestion of meat and milk of infected animals. The disease is rarely transmitted from person to person. Brucellosis is an occupational disease which affects people who deal with live animals like farmers, veterinarians and of abattoir workers. The ELISA test is very specific and will differentiate Brucellosis from other diseases which cause fever of unknown origin like typhoid, malaria, influenza, tuberculosis and infectious mononucleosis.