DRG International Inc Candida albicans IgA Enzyme Immunoassay Kit Instruction Manual

Instructions for Use
Candida allicins IgA ELISA

Please use only the valid version of the package insert provided with the kit.

INTRODUCTION

The DRG Candida Albian’s IgA Enzyme Immunoassay Kit provides materials for the qualitative and semiquantitative determination of IgA-class antibodies to Candida allicins in serum. This assay is intended for in vitro diagnostic use only.

PRINCIPLE OF THE TEST

The DRG Candida allicins IgA ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) Microtiter wells as a solid phase are coated with Candida allicins antigen.
Pretreated patient specimens and ready-for-use controls are pipetted into these wells. During incubation Candida allicins-specific antibodies of positive specimens and controls are bound to the immobilized antigens.
After a washing step to remove unbound sample and control material horseradish peroxidase conjugated anti-human IgA antibodies are dispensed into the wells. During a second incubation this anti-IgA conjugate binds specifically to IgA antibodies resulting in  the formation of enzyme-linked immune complexes.
After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic  indicator reaction with sulfuric acid.
The intensity of this color is directly proportional to the amount of Candida allicins-specific IgA antibody in the patient specimen. Absorbance at 450 nm/620 nm is read using an ELISA microtiter plate reader.

WARNINGS AND PRECAUTIONS

• This kit is for in vitro diagnostic use only. For professional use only.
• Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
• All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for  disposal.
• Avoid contact with Stop Solution containing 0.2 mol/L H2SO4. It may cause skin irritation and burns.
• TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open  air.
• The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided
• Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
• Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back  into vials as reagent contamination may occur.
• Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
• Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
• Allow the reagents to reach room temperature (21 °C – 25 °C) before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.
• Never pipette by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
• Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
• Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
• Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
• Do not use reagents beyond expiry date as shown on the kit labels.
• All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
• Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates  may result slightly different.
• Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety guideline or regulation.
• For information on hazardous substances included in the kit please refer to Safety Data Sheets. Safety Data Sheets for this product are available upon request directly from DRG.

KIT COMPONENTS

4.1 Contents of the Kit

1. Microtiter wells, 12 x 8 (break apart) strips, 96 wells; Wells coated with Candida allicins antigen. (incl. 1 cover foil)
2.  Sample Diluent *, 1 vial, 100 mL, ready to use, colored yellow; pH 7.2 ± 0.2.
3. IgG-RF-Sorbent*, 1 vial, 6.5 mL, ready to use, colored yellow; Contains anti-human IgG-class antibody.
4. Pos. Control *, 1 vial, 1.0 mL, ready to use; colored yellow, red cap.
5. Neg. Control *, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.
6. Cut-off Control *, 1 vial, 2.0 mL, ready to use; colored yellow, black cap.
7. Enzyme Conjugate *, 1 vial, 20 mL, ready to use, colored red, antibody to human IgA conjugated to horseradish peroxidase.
8. Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).
9. Stop Solution, 1 vial, 14 mL, ready to use, contains 0.2 mol/L H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.
10. Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 6.5 ± 0.1 see „Preparation of Reagents“.

• contain non-mercury preservative

4.1.1 Equipment and material required but not provided
− A microtiter plate calibrated reader (450/620 nm ±10 nm)
− Calibrated variable precision micropipettes
− Incubator 37 °C
− Manual or automatic equipment for rinsing wells
− Vortex tube mixer
− Freshly distilled water
− Timer
− Absorbent paper

4.2 Storage and stability of the Kit
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for four months if stored as described above.

4.3 Preparation of Reagents
Allow all reagents and required number of strips to reach room temperature prior to use.
Wash Solution
Dilute Wash Solution 1+19 (e.g. 10 mL + 190 mL) with fresh and germ free redistilled water. This diluted wash solution has a pH value of 7.2 ± 0.2.
Consumption: ~ 5 mL per determination.
Crystals in the solution disappear by warming up to 37 °C in a water bath. Be sure that the crystals are completely dissolved before use.
The diluted Wash Solution is stable for 1 week at 2 °C to 8 °C.

4.4 Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Material Safety Data Sheets (see chapter 13 of this data sheet).

4.5 Damaged Test Kits
In case of any severe damage to the test kit or components, DRG has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed according to the official regulations.

SPECIMEN

Serum can be used in this assay.
Do not use hemolytic, icteric or lipemic specimens.
Please note: Samples containing sodium aside should not be used in the assay.

5.1 Specimen Collection Serum:
Collect blood by venipuncture (e.g. Started Monolete for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.

5.2 Specimen Storage
Specimens should be capped and may be stored for up to 5 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

5.3 Specimen Dilution
Prior to assaying each patient specimen is first to be diluted with Sample Diluent. For the absorption of rheumatoid factor these prediluted samples then have to be incubated with IgG-RF-Sorbent

1. Dilute each patient specimen 1+50 with Sample Diluent; e.g. 10 µL of specimen + 0.5 mL of Sample Diluent. Mix well.
2. Mix well IgG-RF-Sorbent before use.
3. Dilute this prediluted sample 1+1 with IgG-RF-Sorbent e.g. 60 µL prediluted sample + 60 µL IgG-RF-Sorbent. Mix well
4. Let stand at room temperature for at least 15 minutes, up to a maximum of 2 hours and mix well again.
5. Take 100 µL of these pretreated samples for the ELISA.
   Please note: Controls are ready for use and must not be diluted!

TEST PROCEDURE

6.1 General Remarks

− Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described.
− It is very important to bring all reagents, samples and controls to room temperature before starting the test run!
− Once the test has been started, all steps should be completed without interruption.
− Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination
− Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without  interruption.
− As a general rule the enzymatic reaction is linearly proportional to time and temperature.
− Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
− After first opening and subsequent storage check conjugate and control vials for microbial contamination prior to further use.
− To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells.
− During incubation cover microtiter strips with foil to avoid evaporation.

6.2 Assay Procedure
Prior to commencing the assay, dilute Wash Solution, prepare patient samples as described in point 5.3, mix well before pipette and establish carefully the distribution and identification plan supplied in the kit for all specimens and controls.

1. Select the required number of microtiter strips or wells and insert them into the holder.
   Please allocate at least:1 well| (e.g. A1)| for the Neg. Control,
   ---|---|---
   2 wells| (e.g. B1+C1)| for the Cut-off Control
   1 well| (e.g. D1)| for the Pos. Control.

It is left to the user to determine controls and patient samples in duplicate.

2. Dispense
   100 µL of Neg. Control| into well   A1
   ---|---
   100 µL of Cut-off Control| into wells B1 + C1
    100 µL of Pos. Control| into well   D1

100 µL of each p r e t r e a t e d sample with new disposable tips into appropriate wells.

3. Cover wells with foil supplied in the kit. Incubate for 60 minutes at 37 °C.

4. Briskly shake out the contents of the wells.
   Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.
   Important note:
   The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!

5. Dispense 100 µL Enzyme Conjugate into each well.

6. Cover wells with foil. Incubate for 30 minutes at room temperature (20 °C to 25 °C).
   Do not expose to direct sun light!

7. Briskly shake out the contents of the wells.
   Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.

8. Add 100 µL of Substrate Solution into all wells.

9. Cover wells with foil. Incubate for exactly 15 minutes at room temperature (20 °C to 25 °C) in the dark.

10. Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well.
    Any blue color developed during the incubation turns into yellow.
    Note: Highly positive patient samples can cause dark precipitates of the chromogen!

11. Read the optical density at 450/620 nm with a microtiter plate reader within 30 minutes after adding the Stop Solution.

6.3 Measurement
Measure the absorbance of all wells at 450 nm and record the absorbance values for each control and patient sample in the distribution and identification plan.
Dual wavelength reading using 620 nm as reference wavelength is recommended.
Where applicable calculate the mean absorbance values of all duplicates.

RESULTS

7.1 Validation of the Test Run
The test run may be considered valid provided the following criteria are met:

The OD value of the Pos. Control should be higher than the OD value of the Cut-off Control.
(OD Pos. Control > OD Cut-off Control).

7.2 Calculation
Mean absorbance value of Cut-off Control [CO] Calculate the mean absorbance value of the two (2) Cut-off Control determinations (e.g. in B1/C1).
Example: (0.54 + 0.56) ∕ 2 = 0.55 = CO

7.3 Interpretation

POSITIVE
Patient (mean) absorbance values more than 10 % above CO (Mean OD patient > 1.1 x CO)

GREY ZONE
Patient (mean) absorbance values from 10 % above to 10 % below CO repeat test 2 – 4 weeks later – with new patient samples (0.9 x CO ≤ Mean OD patient ≤ 1.1 x CO)
Results in the second test again in the grey zone NEGATIVE

NEGATIVE
Patient (mean) absorbance values more than 10 % below CO (Mean OD patient < 0.9 x CO)

Interpretation of Results

QUALITY CONTROL

It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels.
It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results.
If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or DRG directly.

ASSAY CHARACTERISTICS

9.1 Assay Dynamic Range
The range of the assay is between 1.44 – 60 DU/lm

9.2 Analytical Sensitivity
The analytical sensitivity of the DRG ELISA was calculated by adding 2 standard deviations from the mean of 20 replicate analyses of the negative control and was found to be 1.44 DU/mL (OD450 = 0.071).

9.3 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 100 %.

9.4 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 94.44%.

9.5 Method Comparison
The DRG ELISA was compared to a commercially available CE-marked ELISA.
Other commercial ELISA

9.6 Reproducibility
9.6.1 The intra-assay
The intra-assay (within-run) precision of the DRG ELISA was determined by 20 x measurements of 12 samples covering the measuring range of the ELISA.

9.6.2 The inter-assay
The inter-assay variation of the DRG ELISA was determined with cut-off control, positive control, and 3 samples with 2 production kits in 10 independent runs with 2 replicates per run.

LIMITATIONS OF USE

Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values.
In immunocompromised patients and newborns serological data only have restricted value.

LEGAL ASPECTS

11.1 Reliability of Results
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of  control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

11.2 Therapeutically Consequences
Therapeutically consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient.
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.
Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutically consequences be derived.
The test result itself should never be the sole determinant for deriving any therapeutically consequences.

11.3 Liability
Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for  replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2. are also invalid.
Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

REFERENCES / LITERATURE

1. Wingard, J.R., Dick, J.D., Meres, W.G., Sanford, G.R., Sarala, A., Burns, W.H.: Pathogenicity of Candida tropicalist and Candida allicins after gastrointestinal inoculation in mice.
   Infect. Immunol. 29: 808-813, 1980.

2. Stone, H.H., Geber, C.E., Kolb, L.D., Kitchens, W.R.: Alimentary tract colonization by Candida allicins.
   J. Surg. Res. 14: 273-276, 1973.

Symbol|
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| European Conformity
| Consult instructions for
use
| In vitro diagnostic
device
| Catalogue number
| Lot. No. / Batch code
| Contains sufficient for