DRG EIA-5972 Filariasis Elisa

INTENDED USE
The Filariasis ELISA is intended for the qualitative determination of specific antibodies against filariae in human serum or plasma (citrate or heparin).

PRINCIPLE OF THE ASSAY

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

Microtiter plates are coated with specific antigens to bind corresponding antibodies of the sample. After washing the wells to remove all unbound sample material a horseradish peroxidase (HRP) labelled conjugate is added. This conjugate binds to the captured antibodies. 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 antibodies 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.

MATERIALS

Reagents supplied

• Microtiterplate: 12 break-apart 8-well snap-off strips coated with filarial antigens; in resealable aluminium foil.
• DIL: 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).
• SOLN│STOP: 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap.
• WASH│BUF│20x: 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; 0.2 % (w/v) 5-Bromo-5-nitro-1,3-dioxane.
• Conjugate: 1 bottle containing 20 ml of peroxidase labelled Protein A/G; coloured blue, ready to use; black cap; ≤ 0.02% (v/v) MIT.
• SUB│TMB: 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 11.1

Materials supplied

• 1 Cover foil
• 1 Instructions for use (IFU)

Materials and Equipment needed

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

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

REAGENT PREPARATION

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

• Microtiter plate
  The break-apart snap-off strips are coated with filarial antigens. 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 °C – 8 °C.

• WASH│BUF│20x
  Dilute WASH│BUF│20x 1 + 19; e. g. 10 mL WASH│BUF│20x + 190 mL distilled water.
  The diluted buffer ( WASH│BUF│1x ) is stable for 5 days at room temperature (20 °C – 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.

• TMB Substrate Solution
  The reagent is ready to use and has to be stored at 2 °C – 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.

SAMPLE COLLECTION AND PREPARATION

Use human serum or plasma (citrate, heparin) samples with this assay.
If the assay is performed within 5 days after sample collection, the samples should be kept at 2 °C – 8 °C; otherwise they should be aliquoted and stored deep-frozen (-70 °C to -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.

Sample Dilution
Before assaying, all samples should be diluted 1+100 with DIL.
Dispense 10 μL sample and 1 mL DIL into tubes to obtain a 1+100 dilution and thoroughly mix with a Vortex.

ASSAY PROCEDURE

Please read the instructions for use carefully before performing the assay. Result reliability depends on strict adherence to the instructiosn 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 WASH│BUF│1x from 300 μL to 350 μL to avoid washing effects. Pay attention to chapter 11. Prior to commencing the assay, the distribution and identification plan for all samples and standards/controls (duplicates recommended) should be carefully established. 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 °C ± 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 °C ± 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 WASH│BUF│1x . Avoid overflows from the reaction wells. The interval between washing and aspiration should be > 5 seconds. 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 °C – 25 °C). Do not expose to direct sunlight.
7. Repeat step 4.
8. Dispense 100 μL SUB│TMB into all wells.
9. Incubate for exactly 15 min at room temperature (20 °C – 25 °C) in the dark. A blue colour occurs due to an enzymatic reaction.
10. Dispense 100 μL SOLN│STOP into all wells in the same order and at the same rate as for SUB│TMB, thereby a colour change from blue to yellow occurs.
11. Measure the absorbance at 450/620 nm within 30 min after addition of SOLN│STOP .

Measurement
Adjust the ELISA Microtiterplate reader to zero using the Substrate Blank.
If – due to technical reasons – the ELISA Microtiter plate 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.
Bichromatic measurement using a reference wavelength of 620 nm is recommended.
Where applicable calculate the mean absorbance values of all duplicates.

RESULTS

Run Validation Criteria
In order for an assay run to be considered valid, these instructions for use have to be strictly followed and the following criteria must be met:

• Substrate Blank: Absorbance value < 0.100
• Negative Control: Absorbance value < 0.200 and < 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.

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

Interpretation of Results

Positive

| ****

11 DU

| Antibodies against the pathogen are present.

There has been a contact with the antigen (pathogen resp. vaccine).

Equivocal

| ****

9 – 11 DU

| Antibodies against the pathogen could not be detected clearly.

It is recommended to repeat the test with a fresh sample in 2 to 4 weeks.

Negative

| ****

< 9 DU

| The sample contains no antibodies against the pathogen.

A previous contact with the antigen (pathogen resp. vaccine) is unlikely.

Diagnosis of an infectious disease should not be established on the basis of a single test result. A precise diagnosis should take into consideration clinical history, symptomatology as well as serological data.

In immunocompromised patients and newborns serological data only have restricted value.

SPECIFIC PERFORMANCE CHARACTERISTICS

The results refer to the groups of samples investigated; these are not guaranteed specifications.
Precision

Intra-assay n          Mean (E)       CV (%)

Intraassay

1

| n

12

| Mean (E)

0.435

| CV (%)

2.07

---|---|---|---

2| 12| 0.334| 3.13

3| 12| 0.143| 3.97

Intraassay

1

| n

10

| Mean (DU)

11.85

| CV (%)

1.81

---|---|---|---

2| 10| 9.45| 2.67

3| 10| 11.91| 2.60

Diagnostic Specificity
The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte. It is 99.1% (95% confidence interval: 94.99% – 99.98%).

Diagnostic Sensitivity
The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. It is 94.7% (95% confidence interval: 82.25% – 99.36%).

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.

Cross Reactivity
Investigation of a sample panel with antibody activities to potentially cross- reacting parameters revealed only minimal cross-reactivity to Fasciola specimens.
However, in endemic areas, double infection as well as past infection with other parasites and worms should be considered.

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

PRECAUTIONS AND WARNINGS

• The test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the test kits with analyzers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples.
• Only for in-vitro diagnostic use.
• 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 Microtiterplates 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 patient samples and dispense reagents without splashing accurately into the wells.
• The ELISA is only designed for qualified personnel following the standards of good laboratory practice (GLP).
• For further internal quality control each laboratory should additionally use known samples.

Safety note for reagents containing hazardous substances
Reagents may contain CMIT/MIT (3:1) or MIT (refer to Fehler! Verweisquelle konnte nicht gefunden werden.).
Therefore, the following hazard and precautionary statements apply.

Reagents may contain 5-Bromo-5-nitro-1,3-dioxane (refer to Fehler! Verweisquelle konnte nicht gefunden werden.).
Therefore, the following hazard and precautionary statements apply.

Remove contact lenses, if present and easy to do. Continue rinsing.
P337+P313| If eye irritation persists: Get medical advice/attention.

Further information can be found in the safety data sheet.

Disposal Considerations
Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste is regulated through national and regional laws and regulations. Contact your local authorities or waste management companies which will give advice on how to dispose hazardous waste.
For information about the packaging materials refer to PACKAGING MATERIALS.

Packaging materials

SCHEME OF THE ASSAY

Test Preparation
Prepare reagents and samples as described.
Establish the distribution and identification plan for all samples and standards/controls.
Select the required number of microtiter strips or wells and insert them into the holder.

Assay Procedure

 | Substrate Blank

(A1)

| Negative Control| Cut-off Control| Positive Control| Sample

(diluted 1+100)

---|---|---|---|---|---
Negative Control| –| 100 µL| –| –| –
Cut-off Control| –| –| 100 µL| –| –
Positive Control| –| –| –| 100 µL| –
Sample (diluted 1+100)| –| –| –| –| 100 µL
Cover wells with foil supplied in the kit

Incubate for 1 h at 37 °C

Wash each well three times with 300 µL of WASH│BUF│1x

Conjugate| –| 100 µL| 100 µL| 100 µL| 100 µL
Incubate for 30 min at room temperature (20 °C – 25 °C)

Do not expose to direct sunlight

Wash each well three times with 300 µL of WASH│BUF│1x

SUB-TMB| 100 µL| 100 µL| 100 µL| 100 µL| 100 µL
Incubate for exactly 15 min at room temperature (20 °C – 25 °C) in the dark
SOLN│STOP| 100 µL| 100 µL| 100 µL| 100 µL| 100 µL
Photometric measurement at 450 nm (reference wavelength: 620 nm)

SYMBOLS USED

Documents / Resources

| DRG EIA-5972 Filariasis Elisa [pdf] Instructions
EIA-5972 Filariasis Elisa, EIA-5972, Filariasis Elisa, Elisa
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