The Aspergillus GM ELISA Kit

1-INTENDED USE

The Aspergillus GM ELISA kit is an immunoenzymatic sandwich microplate assay for the detection of Aspergillus galactomannan antigen in adult and pediatric serum samples and bronchoalveolar lavage (BAL) fluid samples. The Aspergillus GM ELISA kit is a test which, when used in conjunction with other diagnostic procedures such as microbiological culture, histological examination of biopsy samples and radiographic evidence can be used as an aid in the diagnosis of Invasive Aspergillosis.

2-SUMMARY AND EXPLANATION

Aspergillus infections usually start in the lung as the port of entry following inhalation of Aspergillus spores which are present in the environment. Invasive forms, which have been on the increase for the past 10 years, constitute the most serious infections. They mainly occur in immunocompromised patients, such as acute leukemia, bone marrow stem cell transplantation, solid organ transplantation, and malignant tumor chemotherapy. The incidence of clinically IA has been on the rise over the past few decades as the number of patients with immunodeficiency, transplantation or high dose hormone therapy has increased. Aspergillus is rarely isolated from blood culture. The diagnosis is often based on nonspecific diagnostic or radiological evidence (clinical symptoms, CT scan, chest x-ray, etc.) The test for soluble galactomannan antigen in serum appears to be a serological method able to aid in the diagnosis of Invasive Aspergillosis.

3-PRINCIPLE OF THE PROCEDURE

The Aspergillus GM ELISA kit is a immunoenzymatic sandwich microplate assay which detects galactomannan in human serum and BAL fluid. The assay uses rat antibodies, which are directed against Aspergillus galactomannan. The antibodies are used, (1) to coat the wells of the microplate and bind the antigen, and (2) to detect the antigen bound to the sensitized microplate (conjugate reagent: peroxidase-linked monoclonal antibodies). Serum or BAL fluid samples are heat-treated in the presence of EDTA in order to dissociate immune complexes and to precipitate proteins that could possibly interfere with the test. The treated samples and conjugate are added to the wells coated with antibodies, and incubated. A monoclonal antibody – galactomannan- antibody / peroxidase complex is formed in the presence of galactomannan antigen. The strips are washed to remove any unbound material. Next, the Chromogen TMB solution is added, which will react with the complexes bound to the well to form a blue color reaction. The enzyme reaction is stopped by the addition of acid, which changes the blue color to yellow. The absorbance (optical density) of specimens and controls is determined with a spectrophotometer set at 450 and 620 nm wavelength.

4-REAGENTS

Aspergillus GM ELISA kit:  Store the kit at 2-8° C. Bring all reagents to room temperature (18-25° C) for at least 30 minutes before use. Return all reagents to 2-8° C immediately after use. Return unused strips/plates to pouch and reseal.

5-WARNINGS FOR USERS

1.For in vitro diagnostic use.

2.For professional use only.

3.Use of this test kit with samples other than human serum and BAL fluid is not recommended.

4.Wear protective clothing, including lab coat, eye/face protection and disposable gloves (synthetic, non-latex gloves are recommended) and handle the kit reagents and patient samples with the requisite Good Laboratory Practices. Wash hands thoroughly after performing the test.

5.Do not pipette by mouth.

6.Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled.

7.Avoid splashing samples or solutions

CAUTION: Do not place solutions containing bleach in the autoclave.

6-SPECIMEN COLLECTION

This test is performed on serum or BAL fluid.

I. SERUM

Collect blood samples according to standard laboratory procedures. Serum samples must be uncontaminated with fungal spores and/or bacteria. Transport and store samples in sealed tubes, unexposed to air. Unopened samples can be stored at 2-8° C for up to 5 days prior to testing. After initial opening, samples may be stored at 2-8° C for 48 hours prior to testing. For longer storage, store the serum at -70° C. Serum samples can be subjected to a maximum of 4 freezing / thawing cycles. Previously frozen specimens should be thoroughly mixed after thawing prior to testing. The results are not affected by samples containing 20 mg/L of bilirubin, lipemic samples containing the equivalent of 2 g/L of triolein (triglyceride) or hemolyzed samples containing 500 mg/dL of hemoglobin. Interferences related to excess albumin have not been tested. Do not decomplement sera.

II. BAL FLUID

Collect BAL fluid samples according to standard laboratory procedures. BAL fluid samples must be collected in sterile saline and may be tested on neat samples (as is) or supernatants from centrifuged samples (10,000 rpm for 10 min) before proceeding to treat the sample per Section 10. BAL fluid samples must be uncontaminated with fungal spores and/or bacteria. Transport and store samples in sealed tubes, unexposed to air. After initial opening, samples may be stored at 2-8° C for up to 24 hours. For longer storage, store the BAL samples frozen (-20° C or less) up to 5 months. BAL samples can be subjected to a maximum of 4 freezing/thawing cycles. Previously frozen specimens should be thoroughly mixed after thawing prior to testing.

7-PROCEDURE

Bring all reagents to room temperature for at least 30 minutes before use.

1. Remove the plateholder and microwell strips from the plate pouch. Return any strips that will not be used to the pouch, with the desiccant, and reseal the pouch.

2. Mix the contents of the Conjugate bottle  by inverting before use. Add 50 µL of Conjugate to each well. Next, add 50 µL of treated serum/BAL supernatant to each well, as designated above. Do not add serum/BAL fluid samples to the wells before the conjugate.

3.Cover plate with plate sealer, or other means to prevent evaporation, ensuring that entire surface is covered and watertight.

4.Incubate the microplate in a dry microplate incubator for 90 ± 5 minutes at 37° C (± 1° C).

5.Remove the plate sealer. Aspirate the contents of all wells into a waste container (containing sodium hypochlorite). Wash the plate 5 times with a microplate washer (using 800 µL of Working Washing Solution). After the last wash, invert the microplate and gently tap on absorbent paper to remove remaining liquid.

6.Rapidly add 200 µL of the Chromogen TMB Solution to each well, avoiding exposure to bright light.

7.Incubate the microplate in the dark at room temperature (+18-25° C) for 30 ± 5 minutes. Do not use adhesive film during this incubation step.

8.Add 100 µL of Stopping Solution to each well, utilizing the same order for addition of Chromogen TMB Solution. Mix well.

9.Thoroughly wipe the bottoms of each plate.

10.Read the optical density of each well at 450 nm (reference filter of 620/630 nm). Microplates must be read within 30 minutes of addition of Stopping Solution.

8- QUALITY CONTROL (VALIDITY CRITERIA)

Cut-off Control: The O.D. of each Cut-off Control Serum must be ≥ 0.300 and ≤ 0.500.

Positive Control: The index of the Positive Control Serum must be greater than 0.6. I = OD Positive Control > 0.6 Mean Cut-off Control OD

Negative Control: The index of the Negative Control Serum must be less than 0.40. I = OD Negative Control < 0.40 Mean Cut-off Control OD

9-INTERPRETATION OF RESULTS

Using the concentration of the standard sample as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper; According to the OD value of the sample, find out the corresponding concentration from the standard curve and multiply it by the dilution multiple. Or calculate the linear regression equation of the standard curve according to the concentration of the standard sample and the OD value. Then calculate the concentration of the sample with the OD value of sample and the equation, and multiply by the dilution multiple to obtain the actual concentration of the sample.

10– Sensitivity and Specificity

A. Sensitivity

~80%

B. Specificity

~90%

11– LIMITATIONS OF THE PROCEDURE

1. A negative test from serum and/or BAL samples cannot rule out the diagnosis of Invasive Aspergillosis. Serum samples from patients at risk for Invasive Aspergillosis should be tested twice a week.

2. Failure to add specimen or reagent as instructed in the procedure could result in a falsely negative test. Repeat testing of additional samples should be considered where there is clinical suspicion of Invasive Aspergillosis or procedural error.

3. Contamination of negative patient specimen wells by positive control/patient specimen wells is possible if the contents of one well spill over into another well due to rough handling of the microplate or poor pipetting technique while adding reagents.

4. Can not been evaluated with neonatal samples.

5. May exhibit reduced detection of galactomannan in patients with chronic granulomatous disease (CGD) and Job’s syndrome .

6. The concomitant use of mold-active anti-fungal therapy in some patients with Invasive Aspergillosis may result in reduced sensitivity.

7. Cannot been evaluated for use with plasma or other sample types such as urine or CSF.

8.  Cross-reactivity of BAL fluid samples with Mycoplasma pneumoniae or anaesthetic drugs/lubricants used to numb the neck/throat area for the aspiration process has not been evaluated.

9. In some particular cases, specific factors should be taken into account when interpreting the test:

a. Positive test results with no clinical signs have been reported, especially in young children.

b. Galactofuranose has been demonstrated in various foods, particularly cereals, cereal products and cream desserts. Dietary factors must therefore be taken into account in interpretation of the course of antigenemia in young children, and more generally in all patients with an altered intestinal barrier.

c. There have been reports of positive galactomannan test results in patients receiving piperacillin/tazobactam.

Aicon Biotech,Inc

8194 SW DurhamRd, Tigard, OR, 97224