Indirect Immunofluorescence: An Easy and Modern Method

Principle of the Test

For the determation of autoantibodies or antibodies against infectious agents, cells, tissue sections or purified, biochemically characterized substances are used as antigen substrates.
If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to a solid phase.
In a second step, the attached antibodies are stained with fluorescein-labelled anti-human antibodies and visualized with the fluorescence microscope.
Positive samples can be titrated in steps. The most suitable titration interval is provided by the dilution factor 3.162 (square root of 10). In this way, every second step represents in its denominator an integral power of 10 (1:10, 1:32, 1:100, 1 : 320, 1 : 1000, 1 : 3200, 1 : 10000 etc.).

High specificity: positive and negative samples produce a large difference in signal strength. Each bound antibody shows a typical fluorescence pattern depending on the location of the individual antigens.
The entire antigen spectrum of the original subtrate is available, thus allowing the detection of a large number of antibodies and achieving a higher detection rate.
Immunofluorescence enables simultaneous detection of antibodies against several biochemically different antigens on one single biological substrate.
The indirect immunofluorescence test is the analytical method of choice when it would be too difficult or too complicated to prepare the test antigens individually for enzyme immunoassays.

Activation Technique: physically or chemically activated cover glasses are coated with cultured cells or tissue sections. Frozen tissue sections are fixed to the glass surface by covalent bonding, increasing adhesion more than 100 times and thus preventing the substrates from being detached.
BIOCHIP Technology: cover glasses coated with biological substrates are cut into millimetre-sized fragments (BIOCHIPs) on a machine. This makes it possible to obtain ten or more first-class preparations of homogeneous quality per tissue section, in the case of cultured cell substrates even several thousands.
BIOCHIP Mosaics™: using several BIOCHIPs coated with different substrates side by side on one and the same reaction field, antibodies against various organs or infectious agents can be investigated simultaneously. Detailed antibody profiles can thus be established with comparatively little effort, allowing the reciprocal determination of the results on different substrates.
TITERPLANE™ Technique: samples or reagents are applied to the reaction fields of a reagent tray. The BIOCHIP Slides are then placed into the recesses of the reagent tray, where all BIOCHIPs come into contact with the fluids, and the individual reactions commence simultaneously. As the fluids are confined in a closed space, there is no need for the use of a conventional "humidity chamber".

For diagnostics of organ-specific or tissue-specific autoantibodies frozen tissue sections of various organs are used. However, formerly, the morphology of tissues suffered during incubation in aqueous medium, tissue parts occasionally became detached from slides, and the interpretation of results was difficult.
Using the activation technique for the first time in histology, we have applied solid phase techniques. Firstly, the surface of cover glasses is coated with spontaneously reactive aldehyde groups. In a second step, the tissue sections (Stöcker, W: European are applied to the chemically activated cover glasses Patent No. 0117262; U.S. Patent No. 4,647,543). Free amino groups of the tissue sections, especially of the hydroxylysine contained in the collagen, bind to the carrier material by covalent bonding.
This results in an increased adhesion of frozen tissue sections more than a hundredfold and prevents them from being detached during incubation.
Furthermore, in some cases the activation technique results in a significantly better conservation of tissue structures, especially in organs which previously exhibited a generally low level of adhesion. Therefore, the tests can be evaluated with considerably greater confidence.

An alternative principle for the serological diagnosis of fresh infections has been established by investigating the antibody avidity.
The first reaction of the immune system following an infection is the formation of low-avidity antibodies. As the infection proceeds, increasingly antigen-adapted IgG is formed, and avidity grows. As long as high-avidity IgG is not yet detected in the serum, it can be assumed that the infection is still in an early stage.
To identify low-avidity antibodies in a patient’s serum, two immunofluorescence tests are performed in parallel: one test is carried out in the conventional way, the other one includes urea treatment between incubations with patient’s serum and peroxidase-labelled anti-human IgG, resulting in the detachment of low-avidity antibodies from the antigens.
Low-avidity antibodies are present if the fluorescence intensity is significantly reduced (two intensity levels ore more) by urea treatment.
The following test kits for avidity determination are available: Toxoplasma gondii, Rubella virus, West-Nile virus, EBV-EA, EBV-CA.

: EUROPLUS™: BIOCHIP combination of tissue sections/cell substrates (left) and antigen dots (right).

In EUROPLUS™ immunofluorescence tests antibody detection is performed using both tissue sections/cell substrates and monospecifically reacting antigen dots.
Antibodies detected in IFT screening tests can therefore be differentiated or confirmed with one and the same reaction field. In some cases, the antigen dots help to extend the antigen spectrum, offering a wider range for screening.
BIOCHIPs coated with purified or recombinant antigens in droplet form are used as monospecific substrates.
A positive result shows green circular areas of antigen dots in front of a dark background under the microscope.
In some EUROPLUS™ test systems several different antigens are coated on one BIOCHIP in separate antigen rows. In this manner, several monospecific analyses can be performed using a single BIOCHIP.
Many different BIOCHIP combinations for various diagnostic investigations are available.