Incubation

The indirect immunofluorescence test performed using the TITERPLANE Technique

The Titerplane Technique was developed by EUROIMMUN in order to standardize immunological analyses: Samples or labelled antibodies 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 of the slide come into contact with the fluids, and the individual reactions commence simultaneously. Position and height of the droplets are exactly defined by the geometry of the system. As the fluids are confined in a closed space, there is no need for the use of a conventional ”humidity chamber”. It is possible to incubate any number of samples next to each other and simultaneously under identical conditions.

Prepare: Check the reagent tray: Are the reaction fields hydrophiIic and the surrounding coating hydrophobic? If not, rub with a wet paper towel, using normal household detergent or Extran MA 01 (Merck) if necessary, and rinse thoroughly with water. For occasional disinfection, immerse for 1 h in 3% Sekusept Extra (Henkel) in water. Open the individual packets containing the BIOCHIP Slides only after they have reached room temperature. Do not touch the BIOCHIPs. Mark BIOCHIP Slides as required with a felt pen.

Dilute: Dilute serum samples according to the user’s test protocol. Include positive and negative controls with every test procedure. Mix control sera before use.

Pipette: Apply the diluted serum to each reaction field of the reagent tray, avoiding air bubbles. Transfer all samples to be tested before starting the incubation (up to 200 droplets). Use a polystyrene pipetting template.

Pipetting volumes:
10 µl per field (3 x 3 mm)
30 µl per field (5 x 5 mm)
70 µl per field (7 x 9 mm)

Incubate: Start reactions by fitting the BIOCHIP Slides into the corresponding recesses of the reagent tray. Ensure that each sample makes contact with its BIOCHIP and that the individual samples do not come into contact with each other. Incubate for 30 min at room temperature.

Wash: Rinse the BIOCHIP Slides with a flush of PBS-Tween using a beaker, and immerse them immediately afterwards in a cuvette containing PBS-Tween for at least 5 min.

1 s flush
5 min cuvette

Pipette: Apply fluorescein-labelled anti–human immunoglobulin (conjugate) onto each reaction field of a clean reagent tray. Add all drops (reagent for a maximum of 50 slides) before continuing incubation. Use a stepper pipette. The labelled anti-human serum should be mixed with a pipette before use. To save time, conjugate can be pipetted onto separate reagent trays during incubation with the diluted serum.

Pipetting volumes:
10 µl per field (3 x 3 mm)
25 µl per field (5 x 5 mm)
65 µl per field (7 x 9 mm)

Incubate: Remove one BIOCHIP Slide from the PBS-Tween and within five seconds blot only the back and the long edges with a paper towel and immediately put the BIOCHIP Slide into the recesses of the reagent tray. Do not dry the areas between the reaction fields. Check for correct contact between the BIOCHIPs and liquids. Then continue with the next BIOCHIP Slide. From now on, protect the slides from direct sunlight. Incubate for 30 min at room temperature.

Wash: Rinse the BIOCHIP Slides with a flush of PBS-Tween using a beaker and put them in a cuvette containing PBS-Tween for at least 5 min.

1 s flush
5 min cuvette

Embed: Place drops of the glycerol/PBS onto a cover glass. Use a polystyrene embedding template. Remove one BIOCHIP Slide from the PBS-Tween and dry the back and all four edges as well as the surface around, but not between, the reaction fields with a paper towel. Put the BIOCHIP Slide, with the BIOCHIPs facing downwards, onto the prepared cover glass. Check immediately that the cover glass is properly fitted into the recesses of the slide. Correct the position if necessary. Now proceed in the same way with the next BIOCHIP Slide.

Embedding volumes:
10 µl per field (3 x 3 mm)
10 µl per field (5 x 5 mm)
20 µl per field (7 x 9 mm)

Evaluate: Read the fluorescence under the microscope.

Please see also the automation options.

When performing the test, the current valid test instruction is to be used.