Datasheets

RICKETTSIA CONORI ELISA

CANINE IGG ANTIBODY DETECTION

INTENDED USE

The Rickettsia conori IgG Antibody Kit is intended for the qualitative detection of canine IgG class serum antibody to Rickettsia conori.

PRINCIPLE OF ASSAY

Canine sera for testing are diluted to 1:100 and allowed to react with Rickettsia conori antigens coated on specially treated micro-wells. After appropriate incubation, the wells are washed to remove unreacted serum proteins, and an enzyme-labeled anti-dog IgG (conjugate) is then added to react with and tag the antigen-antibody complexes. Following another incubation period, the wells are again washed to remove unreacted conjugate. A urea peroxide substrate with TMB as chromogen is added to start color development. Development of a blue color indicates a positive reaction while negative reactions appear colorless or with a trace of blue. The reaction is interrupted with a stop solution. Positive reactions remain a dark blue while negative reactions remain colorless or with a hint of blue. Color intensity (absorbance) is read at a wavelength of 450nm on a spectrophotometer or ELISA reader. Visual determinations can be made with the aid of the color chart provided in this insert.

REAGENTS AND MATERIALS SUPPLIED

This kit supplies sufficient materials for 96 determinations. All liquid components are provided ready to use and contain 0.01% thimerosal as a preservative.

Rickettsia conori ELISA microplates

96-wells (12 strips) containing Rickettsia conori antigens and packaged with desiccant.

Conjugate, 2 x 6 ml – Brown cap

Affinity-purified horseradish peroxidase (HRP)-labeled rabbit anti-dog IgG (heavy chain). Protect from light.

Positive Control, 1.0 mL - Red cap

Dog serum reactive with Rickettsia conori.

Negative Control, 1.0 mL - White cap

Dog serum non-reactive with Rickettsia conori.

Wash Buffer, 1 packet

Phosphate-buffered saline (PBS) with Tween 20, pH 7.4 and 0.05% Tween 20 when reconstituted to 1L with distilled water

TMB Substrate, 2 x 6 mL - Blue cap

A solution containing urea peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB). Protect from light. Non-carcinogenic.

Stop Solution, 2 x 6 mL - Yellow cap

Diluted phosphoric acid.

WARNING

1. DO NOT INTERCHANGE COMPONENTS BETWEEN KITS AND DIFFERENT LOTS OF THE SAME TEST.

2. The control sera have not been screened for infectious agents. Since no testing can assure the absence of infectious agents, however, these reagents, as well as the serum specimens and equipment coming in contact with these specimens, should be handled with good laboratory practices to avoid skin contact and ingestion.

3. The coated microwells are prepared with inactivated antigens. However, they should be considered potentially infectious and handled accordingly.

Storage and Handling

Kit components should be stored at 2-8° C. Bring them to room temperature (96-25° C) before opening bottles and plate pouches. TMB substrate and stop solution are also stable at room temperature.

Specimen Collection

Blood samples should be collected using approved venipuncture techniques by qualified personnel. Allow sample to clot and separate serum by centrifugation. Transfer serum aseptically to a tightly closing sterile container. Store at 2-8°C. If testing is to be delayed longer than 5 days, freezing the sample at -96°C or colder is recommended. Acute specimens should be drawn at the onset of illness; convalescent specimens should be obtained 1-2 weeks later.

Materials Required But Not Supplied

1. Test tubes or microtiter plate for preparing serum dilutions

2. Precision pipette(s) (2uL to 1000uL) for making and delivering dilutions

3. Adhesive cover for microplates.

4. Optional if optical density reading is preferred: ELISA reader equipped with a 450nm filter. A program for data reduction would be helpful.

Precautions

1. Do not use components past expiration date.

2. HRP-labeled conjugate and TMB-substrate are photosensitive and are packaged in opaque plastic vials for protection. Store in the dark and return to storage after use.

ASSAY PROCEDURE

IMPORTANT: BRING WHOLE KIT TO ROOM TEMPERATURE FOR AT LEAST 30 MINUTES BEFORE USE. FAILURE TO DO SO MAY RESULT IN REDUCED SENSITIVITY (LOWER OPTICAL DENSITY READING)

1. Prepare wash buffer by adding 1 packet of powder to 1L of distilled water.

2. Prepare 1:100 dilutions for all patient serum specimens in wash buffer, e.g. 5µL in 500µL.

3. For each patient serum dilution to be tested, add 100 µL to each micro-well and record the location for later reference. For each assay run, include a Positive Control (3 drops per well) and a Negative Control (3 drops per well). Controls are provided ready to use, so please do not dilute. Incubate plate for 15 minutes at ambient temperature (20-25oC). Incubation for 30 minutes can enhance sensitivity.

4. Wash plates 3 - 4 times with a gentle stream of wash buffer from a wash bottle or a plate washer. Tap plates on a stack of absorbent paper towels to remove residual buffer.

5. Add the rabbit anti-dog IgG Conjugate (2 drops) to each well. Incubate for 15 minutes at ambient temperature.

6. Wash plates as in step 4 above.

7. To each microwell, add TMB Substrate Solution (2 drops) and allow reaction to proceed for 10 minutes at ambient temperature. A blue color indicates positive reaction and can be recorded at this point or stopped as described below.

8. Stop reaction by adding Stop Solution (2 drops) to each well.

9. Evaluate final color of reaction by comparing with Positive and Negative Controls or read absorbance (OD) on a microplate reader equipped with a 450nm filter. Reaction mixture turning blue to yellow and more intense than the negative control indicates a definite positive. A very light yellow or clear mixture is negative. Please use color chart provided to determine reactivity.

QUALITY CONTROL

This assay is designed to detect IgG antibodies to Rickettsia conori in dogs that reside in endemic areas for ticks carrying this organism and hence a background level of antibodies is expected due to natural exposure.

1. If the sample color is bright yellow after reaction has been stopped, report the result as POSITIVE. If thesample color is clear or light yellow after the reaction has been stopped, report the result as NEGATIVE.

2. The positive controls consist of pooled sera from dogs and are designed to give readings that are clearly positive, although not always at high titers.

Out of Control (OOC) statement: If controls do not react to expected colors (Positive=bright blue, Negative=clear or light blue), the run should be repeated with new controls.

LIMITATIONS

1. The level of antibody detected is influenced by the endemicity of Rickettsia conori organisms. In an area of low endemicity, low levels of antibody may be of significance. In an area of high endemicity, low levels of antibody may not be correlated with disease state.

2. Antibody levels measured in this assay must be correlated with clinical findings for diagnostic utility.

QUALITY ASSURANCE

This assay is compliant with the Quality Assurance policies and protocols implemented at Helica Biosystems, Inc.