Vetline DIRO Ag, Elisa kit

1. INTRODUCTION

2. INTENDED USE

3. PRINCIPLE OF THE ASSAY

The qualitative immunoenzymatic determination of specific antigens is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique.

Microtiterplates are coated with specific antibodies to bind corresponding antigens of the sample. After washing the wells to remove all unbound sample material a horseradish peroxidase (HRP) labelled antibody conjugate is added. This conjugate binds to the captured antigens. In a second washing step unbound conjugate is removed. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product.

The intensity of this product is proportional to the amount of specific antigens in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA Microtiterplate reader.

4. MATERIALS

4.1. Reagents supplied

• Microtiterplate: 12 breakapart 8-well snap-off strips coated with anti-Dirofilaria antibodies; in resealable aluminium foil.
• Sample Dilution Buffer: 1 bottle containing 100 mL of phosphate buffer (10 mM) for sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap; ≤ 0.0015 % (v/v) CMIT/MIT (3:1).
• Stop Solution : 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap.
• Washing Buffer (20x conc.): 1 bottle containing 50 mL of a 20-fold concentrated phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap.
• Conjugate: 1 bottle containing 15 mL of peroxidase labelled anti-Dirofilaria antibodies; coloured red, ready to use; white cap; ≤ 0.02 % (v/v) MIT.
• TMB Substrate Solution: 1 bottle containing 15 mL 3,3',5,5'-tetramethylbenzidine (TMB), < 0.1 %; ready to use; yellow cap.
• Positive Control: 1 vial containing 2 mL control; coloured yellow; ready to use; red cap; ≤ 0.02 % (v/v) MIT.
• Cut-off Control: 1 vial containing 3 mL control; coloured yellow; ready to use; green cap; ≤ 0.02 % (v/v) MIT.
• Negative Control: 1 vial containing 2 mL control; coloured yellow; ready to use; blue cap; ≤ 0.0015 % (v/v) CMIT/MIT (3:1).

For hazard and precautionary statements see 12.1

For potential hazardous substances please check the safety data sheet.

4.2. Materials supplied

• 1 Cover foil
• 1 Instruction for use (IFU)
• 1 Plate layout

4.3. Materials and Equipment needed

• ELISA Microtiterplate reader, equipped for the measurement of absorbance at 450/620 nm
• Incubator 37 °C
• Manual or automatic equipment for rinsing Microtiterplate wells
• Pipettes to deliver volumes between 10 and 1000 µL
• Vortex tube mixer
• Distilled water
• Disposable tubes
• Heating block
• Centrifuge

5. STABILITY AND STORAGE

Store the kit at 2...8 °C. The opened reagents are stable up to the expiry date stated on the label when stored at 2...8 °C.

6. REAGENT PREPARATION

It is very important to bring all reagents and samples to room temperature (20…25 °C) and mix them before starting the test run!

6.1. Microtiterplate

The break-apart snap-off strips are coated with anti-Dirofilaria antibodies. Immediately after removal of the strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2...8 °C.

6.2. Washing Buffer (20x conc.)

Dilute Washing Buffer 1 + 19; e. g. 10 mL Washing Buffer + 190 mL distilled water. The diluted buffer is stable for 5 days at room temperature (20…25 °C). In case crystals appear in the concentrate, warm up the solution to 37 °C e.g. in a water bath. Mix well before dilution.

6.3. TMB Substrate Solution

The reagent is ready to use and has to be stored at 2...8 °C, away from the light. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away.

7. SAMPLE COLLECTION AND PREPARATION

Use veterinary serum samples with this assay. If the assay is performed within 5 days after sample collection, the samples should be kept at 2...8 °C; otherwise they should be aliquoted and stored deep-frozen (-70…-20 °C). If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. Heat inactivation of samples is not recommended.

7.1. Sample Dilution and Heat Incubation

Before assaying, all samples should be diluted 1+1 with Sample Dilution Buffer. Dispense 250 μL sample and 250 μL Sample Dilution Buffer into tubes to obtain a 1+1 dilution and thoroughly mix with a Vortex.

In case of a suspected false negative result the sample should be treated with heat incubation to inactivate intrinsic antibodies. Example protocol: The diluted sample (at least 500 μL) should be incubated for 10 minutes at 104 °C on a heating block. Then centrifugate the sample at 16.000 x g for 5 minutes. The supernatant can be used as sample material for further testing.

8. ASSAY PROCEDURE

Please read the instruction for use carefully before performing the assay. Result reliability depends on strict adherence to the instruction for use as described. The following test procedure is only validated for manual procedure. If performing the test on ELISA automatic systems we recommend increasing the washing steps from three up to five and the volume of Washing Buffer from 300 µL to 350 µL to avoid washing effects. Pay attention to chapter 12. Prior to commencing the assay, the distribution and identification plan for all samples and standards/controls (duplicates recommended) should be carefully established on the plate layout supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder.

Perform all assay steps in the order given and without any delays.

A clean, disposable tip should be used for dispensing each standard/control and sample.

Adjust the incubator to 37 ± 1 °C.

• 1. Dispense 100 µL standards/controls and diluted samples into their respective wells. Leave well A1 for the Substrate Blank.
• 2. Cover wells with the foil supplied in the kit.
• 3. Incubate for 1 hour ± 5 min at 37 ± 1 °C.
• 4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 300 µL of Washing Buffer. Avoid overflows from the reaction wells. The interval between washing and aspiration should be > 5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is important! Insufficient washing results in poor precision and false results.
• 5. Dispense 100 µL Conjugate into all wells except for the Substrate Blank well A1.
• 6. Incubate for 30 min at room temperature (20...25 °C). Do not expose to direct sunlight.
• 7. Repeat step 4.
• 8. Dispense 100 µL TMB Substrate Solution into all wells.
• 9. Incubate for exactly 15 min at room temperature (20...25 °C) in the dark. A blue colour occurs due to an enzymatic reaction.
• 10. Dispense 100 µL Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution, thereby a colour change from blue to yellow occurs.
• 11. Measure the absorbance at 450/620 nm within 30 min after addition of the Stop Solution.

8.1. Measurement

Adjust the ELISA Microtiterplate reader to zero using the Substrate Blank .

If - due to technical reasons - the ELISA Microtiterplate reader cannot be adjusted to zero using the Substrate Blank, subtract its absorbance value from all other absorbance values measured in order to obtain reliable results!

Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard/control and sample in the plate layout.

Bichromatic measurement using a reference wavelength of 620 nm is recommended.

Where applicable calculate the mean absorbance values of all duplicates.

9. RESULTS

9.1. Run Validation Criteria

In order for an assay to be considered valid, the following criteria must be met:

• Substrate Blank : Absorbance value < 0.100
• Negative Control : Absorbance value < Cut-off
• Cut-off Control : Absorbance value 0.150 – 1.300
• Positive Control : Absorbance value > Cut-off

If these criteria are not met, the test is not valid and must be repeated.

9.2. Calculation of Results

The Cut-off is the mean absorbance value of the Cut-off Control determinations.

Example: Absorbance value Cut-off Control 0.44 + absorbance value Cut-off control 0.42 = 0.86 / 2 = 0.43

Cut-off = 0.43

9.2.1. Results in Units [NTU]

$\frac{\mathrm{Sample\; (mean)\; absorbance\; value\; x\; 10}}{\mathrm{Cut-off}}$ = [NovaTec-units = NTU]

Ejemplo: $\frac{1591\times 10}{0.43}$ = 0,37 NTU

9.3. Interpretation of Results

10.SPECIFIC PERFORMANCE CHARACTERISTICS

For further information about the specific performance characteristics please contact NovaTec Immundiagnostica GmbH.

10.1. Precision

10.2. Diagnostic Specificity

10.3. Diagnostic Sensitivity

10.4. Interferences

Interferences with hemolytic, lipemic or icteric samples are not observed up to a concentration of 10 mg/mL hemoglobin, 5 mg/mL triglycerides and 0.5 mg/mL bilirubin.

10.5. Cross Reactivity

11. LIMITATIONS OF THE PROCEDURE

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the absorbance values.

12.PRECAUTIONS AND WARNINGS

• For research use only!
• All materials of human or animal origin should be regarded and handled as potentially infectious.
• All components of human origin used for the production of these reagents have been tested for anti-HIV antibodies, anti-HCV antibodies and HBsAg and have been found to be non-reactive.
• Do not interchange reagents or strips of different production lots.
• No reagents of other manufacturers should be used along with reagents of this test kit.
• Do not use reagents after expiry date stated on the label.
• Use only clean pipette tips, dispensers, and lab ware.
• Do not interchange screw caps of reagent vials to avoid cross-contamination.
• Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
• After first opening and subsequent storage check conjugate and standard/control vials for microbial contamination prior to further use.
• To avoid cross-contamination and falsely elevated results pipette samples and dispense reagents without splashing accurately into the wells.
• The ELISA is only designed for qualified personnel who are familiar with good laboratory practice.

12.1. Safety note for reagents containing hazardous substances

Reagents may contain CMIT/MIT (3:1) or MIT (refer to 4.1)

Therefore, the following hazard and precautionary statements apply.

13. ORDERING INFORMATION

Prod. No.: DIRVT4760 VetLine Dirofilaria Antigen ELISA (96 Determinations)

ABBREVIATIONS / ABKÜRZUNGEN / ABRÉVIATIONS / ABBREVIAZIONI / ABREVIACIÓNES /ABREVIATURAS