Indirect immunofluorescence: an easy and modern method

Principle of the test

  • For the determination 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.).

Indirect immunofluorescence: a standardised technique for the determination of autoantibodies and antibodies against infectious agents

Pattern homog. Anti-dsDNS? Anti-Histones?

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Pattern fine-granular. Anti-SS-A? Anti-SS-B?

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Pattern nucleolar. Anti-PM-Scl?

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Pattern cytoplasmic. AMA-M2?

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  • 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 substrate 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.

EUROIMMUN’s innovations for the standardisation and modernisation of indirect immunofluorescence

BIOCHIP Technology and Mosaics.

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  • 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".

Chemically activated cover glasses for histochemistry

Fixation of frozen tissue sections to glass surfaces by covalent bonding.

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  • 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 are applied to the chemically activated cover glasses (Stöcker, W: European 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.

Determination of low-avidity antibodies

low-avide Ab against EBV-CA

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high-avide Ab against EBV-CA; without urea(left); with urea(right)

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  • 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 or more) by urea treatment.
  • The following test kits for avidity determination are available: Toxoplasma gondii, Rubella virus, West-Nile virus, EBV-CA, EBV-EA, VZV, CMV

 

ANA Global : Indirect Immunofluorscence patterns & clinical association

 

Pattern

Clinical Significance

Homogenous

SLE, Drug induced Lupus, Rheumatoid arthritis, Systemic Sclerosis.

Homogenous with nuclear ring

SLE, particularly when the disease active.

Coarse Speckled

SLE, Mixed connective tissue disease, Sharp syndrome.

Fine speckled

SLE, Sjogren’s Syndrome, Sub acute cutaneous Lupus, Scleroderma.

Nucleolar

Polymyositis, scleroderma overlap Syndrome

Cytoplasm Filamentous

CAH : Chronic active auto immune hepatitis and other un known clinical association.

Centromere

Progressive systemic Sclerosis, limited form.

Cytoplasmic granular

PBC: Primary Liver cirrhosis, PM : Polymyositis and other unknown clinical association.

Centerioles

Progressive systemic Sclerosis

Golgi apparatus

SLE, Sjogren’s Syndrome,Rheumatoid arthritis.

Nucleoplasm dots

PBC: Primary Liver cirrhosis, Rheumatic disease

Nuclear dots

PBC: Primary Liver cirrhosis, Rheumatic disease, SLE, Sjogren’s Syndrome

Several nuclear dots

PBC: Primary Liver cirrhosis, Rheumatic disease

Spindle fibers

Rheumatic disease

Midbody

Un known clinical association

Anti nuclear membrane

CFS: Chronic Fatigue Syndrome, CAH: Chronic active auto immune hepatitis, colegenoses.