DRG INTERNATIONAL EIA-3817 Ascaris Lumbricoides IgG ELISA Instruction Manual

DRG INTERNATIONAL EIA-3817 Ascaris Lumbricoides IgG ELISA

Product Information

Specifications

• Product Name: Ascaris lumbricoides IgG ELISA EIA-3817
• Intended Use: Qualitative determination of IgG class antibodies against Ascaris lumbricoides in human serum or plasma
• Materials: 12 break-apart 8-well snap-off strips coated with Ascaris lumbricoides antigens in resealable aluminum foil

Product Usage Instructions

Assay Procedure

1. Perform all assay steps in the given order without delays.
2. Use a clean, disposable tip for dispensing each standard/control and sample.
3. Cover wells with the foil supplied in the kit.
4. After incubation, remove the foil, aspirate well contents, and wash each well three times with >5 seconds aspiration per wash. Tap strips on tissue paper to remove remaining fluid.
5. Repeat step 4.
6. Measure absorbance at 450/620 nm within 30 minutes after adding SOLNSTOP.

Results

Run Validation Criteria

• Substrate Blank: Absorbance value should be less than 0.100
• Negative Control: Absorbance value should be less than 0.200 and less than Cut-off
• Cut-off Control: Absorbance value should be greater than Cut-off
• Positive Control: Absorbance value should be greater than Cut-off

Calculation of Results
The Cut-off is calculated as the mean absorbance value of the Cut-off Control determinations.

Interpretation of Results

Cut-off

• Positive: > 11 DU – Antibodies against the pathogen are present.
• Equivocal: 10 DU – Indicates contact with the antigen (pathogen or vaccine).

FAQ

What should I do if the assay run does not meet the validation criteria?
If the criteria are not met, the test is not valid and must be repeated following the instructions strictly.

Instructions for Use

Ascaris lumbricoides IgG ELISA

DRG Instruments GmbH, Germany
Frauenbergstraße 18, D-35039 Marburg

• Phone: +49 (0)6421-1700 0,
• Fax: +49 (0)6421-1700 50
• Website: www.drg-diagnostics.de
• E-mail: drg@drg-diagnostics.de

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081

• P hone: 973-564-7555, Fax: 973-564-7556
• Website: www.drg-international.com
• E-mail: corp@drg-international.com

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

The Ascaris lumbricoides IgG ELISA is intended for the qualitative determination of IgG class antibodies against Ascaris lumbricoides in human serum or plasma (citrate, heparin).

PRINCIPLES OF THE ASSAY

The qualitative immunoenzy matic 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 Ascaris lumbricoides 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 in phosphate buffer (10 mM); coloured blue, ready to use; black cap.

• 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!

Microtiterplate
The break-apart snap-off strips are coated with Ascaris lumbricoides 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.

SUB│TMB
The reagent is ready to use and has to be stored at 2 °C – 8 °C, away from the light.
SUB│TMB should be colourless or could have a slight blue tinge. If SUB│TMB 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 instructions 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 60 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 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 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.
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

Results in Units [DU]
Sample (mean) absorbance value × 10 = [DRG Units = DU] Cut-off

Example:
1.591 × 10 = 37 DU 0.43

Interpretation of Results

SPECIFIC PERFORMANCE CHARACTERISTICS

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

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