ELISA/assay

Dengue NS1 Antigen ELISA Kit

DEIABL14

96T

Dengue NS1 Antigen ELISA Kit

DENV NS1

Dengue NS1 Antigen ELISA Kit

sELISA

DENV NS1

Human

Qualitative

human serum

96T

Stable at 2-8°C until the expiration date.

Fifty-eight (58) antigen or viral load positive samples that tested positive for other potentially cross-reactive pathogens were evaluated with the DENV NS1 ELISA in order to determine potential cross-reactivity. West Nile virus samples ranged 100-43,000 copies/mL by nucleic acid testing; hepatitis B virus (HBV) levels ranged 500-5,000,000,000 by chemiluminescent immunoassay; hepatitis C virus (HCV) viral loads ranged 3.9×10 6 -3.8×10 8 by nucleic acid testing; human immunodeficiency virus (HIV) viral loads ranged 3.8×10 3 -4.4×10 6 by nucleic acid testing and signal/cut-off ratios ranged 8.55-16.87 in enzyme immunoassay (EIA); SLE samples were all >1:100 by IFA. In addition, forty-two (42) contrived samples that consisted of cultured micro-organisms spiked into normal human sera were also evaluated with the DENV NS1 ELISA. Clinically relevant levels of viruses at 10 5 pfu/mL and bacteria at 10 6 cfu/mL were tested. VZV was tested at only 5×10 3.43 U/mL, due to low concentration of stock obtained. For EBV, both 10 7 and 10 8 copies/mL (as determined by quantitative RT-PCR) of EBV were tested in this study.

DENV NS1

Human

virus; DENV; dengue virus; nonstructural protein 1; NS1; Dengue Virus NS1; DENV type1; DENV type2; DENV type3; DENV type4; DENV type1 NS1; DENV type2 NS1; DENV type3 NS1; DENV type4 NS1; DENV NS1; Dengue virus Nonstructural Protein 1

Dengue virus NS1 Antigen ELISA Kit for early detection of Dengue virus (DENV), is an ELISA assay system for the detection of NS1 antigen in human serum. It is not intended to screen blood or blood components, and is for investigational use only. Not for use in diagnostic procedures.

Dengue Fever (DF) is an acute viral disease of man, which is transmitted by Aedes aegypti mosquitoes. DF is characterized clinically by biphasic fever, rash and hematopoietic depression, and by constitutional symptoms such as malaise, arthralgia, myalgia and headache. Infrequently, more severe disease is seen, manifested by hemorrhagic fever which may progress to lethal shock. It is endemic in the tropics and subtropics, worldwide, where an estimated 100,000,000 cases occur annually. It has been estimated that about 50 to 100 million cases of DF occur every year with about 250,000 to 500,000 cases of Dengue Hemorrhagic Fever (DHF). During 2002, more than 30 Latin American countries reported over 10,000,000 DF cases with large number of DHF cases. This has been followed by extensive epidemics of DHF in several parts of India during 2003 through 2005. In the Americas, the reported incidence has more than tripled from 1996 to 2002. The incidence of Dengue outbreak has been reported in Hawaii, and in Laredo, Texas. Dengue NS1 (non-structural) protein is a secreted protein and is believed to play a role in viral RNA replication. NS1 is strongly immunogenic, eliciting antibodies with complement fixing activity. NS1 antigen can be detected in circulating blood during acute Dengue infection. The Dengue virus NS1 Antigen ELISA Kit can detect NS1 antigen in serum samples within 1 to 2 days following infection.

The Dengue virus NS1 Antigen ELISA Kit is an enzymatically amplified "two-step" sandwich-type immunoassay to detect low levels of NS1 in serum. In this assay, controls and unknown serum samples are diluted in sample dilution buffer, containing secondary antibody, and incubated in microtitration wells. These wells have been coated with a highly effective NS1 antibody and then blocked. NS1 antigens present in the samples are then "sandwiched" between the capture and secondary antibodies. The presence of NS1 antigen is confirmed by the colorimetric response obtained using an antibody-HRP conjugate and liquid 3,3', 5,5'-tetramethylbenzidine (TMB) substrate. Once the reaction is stopped, using an acidic solution, the enzymatic turnover of the substrate is determined by absorbance measurement at 450 nanometers. The values obtained for the kit controls serve as guidelines as to determining if a sample contains NS1 antigen.

The Dengue virus NS1 Antigen ELISA Kit contains sufficient reagents for one plate of 96 wells (12×8 strips) each. Warning: Do not use any reagents where damage to the packaging has occurred. Dengue virus NS1 Antigen ELISA Kit supplied materials: 1. Coated Microtiter Strips: ELISA strip holder in a ziplock foil pouch with desiccant, containing 96 polystyrene microtiter wells coated with anti-NS1 antibody in each well. Stable at 2-8°C until the expiration date. 2. Negative Control: One vial, 300μL containing heat-inactivated negative control serum. The negative control will aid in verifying the validity of the kit. Stable at 2-8°C until the expiration date of the kit. 3. Positive Control: One vial, 300μL containing recombinant NS1 in a phosphatebased buffer with 0.05% Proclin-300. The positive control will aid in verifying the validity of the kit. Stable at 2-8°C until the expiration date. 4. Cut-off Control: One vial, 300μL containing recombinant NS1 in a phosphate-based buffer with 0.05% Proclin-300. The Cut-off Control will aid in determining the cut-off value for the ELISA. Stable at 2-8°C until the expiration date. 5. Sample Dilution Buffer: One bottle, 15mL, containing the secondary antibody in a Tris-based buffer with 0.02%-0.05% Proclin-300. Stable at 2-8°C until the expiration date. 6. 100× Conjugate: One vial, 150μL, containing horseradish peroxidase-labeled polyclonal antibody in a Tris-based buffer with 0.03% - 0.05% Proclin-300. Stable at 2-8°C until the expiration date. 7. Conjugate Diluent: One bottle, 12mL. This contains the diluent solution for the 100× Conjugate in a Tris-based buffer with 0.01% Thimerosal as a preservative. The 100× conjugate is diluted directly into this solution. After diluting 100× Conjugate into this solution, the now ready-to-use conjugate may be stored up to 2 weeks at 2-8°C before it should be discarded. Stable at 2-8°C until the expiration date. 8. 10× Wash Buffer: One bottle, 120 mL. 10× concentrated phosphate buffered saline with Tween 20 (pH 6.8-7.0). Stable at 2-8°C until the expiration date. 9. Liquid TMB Substrate: One bottle, 12mL, ready to use. Contains 3, 3', 5, 5'-tetramethylbenzidine (TMB) and hydrogen peroxide in a citric acid-citrate buffer (pH 3.3-3.8). Stable at 2-8°C until the expiration date. Note: The substrate should always be stored in the light protected bottle provided. 10. Stop Solution: One bottle, 6mL, read to use 1N Sulfuric Acid. Used to stop the reaction. Stable at 2-8°C until the expiration date. Warning: Strong acid. Wear protective gloves, mask and safety glasses. Dispose all materials according to all applicable safety rules and regulations.

1. ELISA spectrophotometer capable of absorbance measurement at 450 nm 2. Biological or high-grade water 3. Appropriately sized beakers and stir bars 4. Vacuum pump 5. Automatic plate washer 6. 37°C incubator without CO 2 supply 7. 1-10 μL single-channel pipettors, 50-200 μL single- and multichannel pipettors 8. Polypropylene tubes or 96 well dilution plates 9. Parafilm or plastic plate cover 10. Timer 11. Vortex

1. Only human serum should be used for this assay, and the usual precautions for venipuncture should be observed. Blood obtained by venipuncture should be allowed to clot at room temperature (20-25°C) for 30 to 60 minutes and then centrifuged according to the Clinical and Laboratory Standards Institute (CLSI Approved Guideline – Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests; GP44). 2. Testing should be performed as soon as possible after collection. Do not leave sera at room temperature for prolonged periods. Separated serum should remain at 20-25°C for no longer than 8 hours. If assays are not completed within 8 hours, serum should be refrigerated at 2-8°C. If assays are not completed within 48 hours, or the separated serum is to be stored beyond 48 hours, serum should be frozen at or below -20°C. 3. Avoid repeated freezing and thawing of samples more than four times as this can cause analyte deterioration. Frost-free freezers are not suitable for sample storage. 4. Frozen samples should be thawed to room temperature and mixed thoroughly by gentle swirling or inversion prior to use. Always quick spin before use. 5. If sera are to be shipped, they should be packed in compliance with Federal Regulations covering transportation of infectious agents. 6. Do not use sera if any indication of microbial growth is observed.

CAUTION: The test procedure must be strictly followed. Any deviations from the procedure may produce erroneous results. Bring all reagents and specimens to room temperature (~25°C) before use. Thoroughly mix the reagents and samples before use by gentle inversion. NOTE: For long-term storage, serum samples should not be repeatedly thawed and frozen more than four times. Sera should be further divided into small aliquots and stored at -20°C or below. 1. Preparation of 1× Wash Buffer Dilute the 10× Wash Buffer to 1× using Biological or High-Grade Water. To prepare a 1× wash buffer solution, mix 120 mL 10× wash buffer with 1080 mL distilled (or deionized) water. Mix thoroughly to ensure that any precipitate is dissolved and that the solution is uniform. Once diluted to 1×, the solution can be stored at room temperature for up to 6 months. Properly label the 1× wash buffer solution and carefully note the expiration date on the label. Check for contamination prior to use. Discard if contamination is suspected. 2. Microtiter Strip Wells Select the number of coated wells required for the assay. The remaining unused wells should be repackaged immediately with the supplied desiccant and stored at 2-8°C until ready to use or expiration. 3. Preparation of Conjugate Solution Add 120μL of 100× Conjugate directly to the 12mL bottle of Conjugate Diluent (1 part : 100 parts). Alternatively, use a clean pipette to remove the required volume of Conjugate Diluent and add the necessary volume of 100× Conjugate into a clean polypropylene test tube in order to maintain the 1:100 ratio. Mix by inverting the solution several times. This solution may be stored for up to 2 weeks if stored at 2-8°C. After 2 weeks, this conjugate solution should be discarded and no longer used in this assay.

1. Positive, negative, and cut-off controls should be assayed in duplicate (and run on every plate, each time an assay is performed). Unknown serum samples may be tested in singlet. (However, it is recommended to run samples in duplicate until the operator is familiar with the assay.) Up to ninety test specimens can be tested in singlet with an entire plate. Immediately place any unused ELISA plate wells back into the original foil packaging with the provided desiccant, properly seal, and store at 2-8°C. 2. Using a single channel or multichannel pipettor, aliquot 50μL of Sample Dilution Buffer for the Dengue virus NS1 Antigen ELISA Kit into each of the required wells. 3. Add 50μL of each undiluted sera (test samples and control samples) directly to the center of the wells containing the Sample Dilution Buffer. Use a clean disposable pipette tip for each control or test sample. Gently rock the plate by hand from side to side 5 times to ensure the samples are well mixed. 4. Cover the top of the plate with parafilm (or plastic plate cover) and remove any excess parafilm from the edges of the plate. Note: This is to make sure the temperature distribution is evenly spread out in all wells from bottom and sides; any extra parafilm can be cut off once the top is sealed to block evaporation. Note: Do not stack plates on top of each other. They should be spread out as a single layer. This is very important for even temperature distribution. Do not use CO 2 or other gases. Do not place plates in contact with any wet substances such as wet paper towels etc. 5. Incubate the plate at 37°C for 1 hour in an incubator. 6. After the incubation, wash the plate 6 times with an automatic plate washer using 1× Wash Buffer. Use 300 μL per well in each wash cycle. 7. Prepare the Conjugate Solution (120μL of 100× Conjugate: 12mL of Conjugate Diluent) and add 100μL per well of this Conjugate Solution into all wells using a multi-channel pipettor. Discard the remaining Conjugate Solution or store for up to 2 weeks at 2-8°C. 8. Cover the plate with parafilm or plastic plate cover, as shown above, and incubate at 37°C for 30 minutes in an incubator. 9. After the incubation, wash the plate 6 times with the automatic plate washer using 1× Wash Buffer. 10. Add 100μL per well of Liquid TMB substrate into all wells using a multi-channel pipettor. 11. Incubate the plate, uncovered at room temperature in the dark, for 20 minutes. 12. Add 50μL per well of Stop Solution into all appropriate wells using a multi-channel pipetter. Make sure to add the Stop Solution in the same order and at approximately the same speed at which the TMB was applied. ( Note: As the TMB substrate produces an enzymatic reaction with the HRP-conjugate, it is critical this incubation time point is followed as closely as possible ). Let the plate stand, uncovered at room temperature, for 1 minute. 13. Read the optical density at 450nm (OD450) with a microplate reader. DO NOT SUBTRACT OR NORMALIZE ANY BLANK VALUES OR WELLS.

Each kit contains positive, negative and cut-off controls. An acceptable Discrimination Capacity (R PC/NC ) must be obtained to ensure assay validity. The negative and positive controls are intended to monitor for substantial reagent failure. The positive control will not ensure precision at the assay cutoff. The test is invalid and must be repeated if the (R PC/NC ) value is too low or if the control samples do not meet the specifications. If the test is invalid, the results cannot be used. Quality control (QC) requirements must be performed in conformance with local, state, and/or federal regulations or accreditation requirements and your laboratory's standard Quality Control procedures. It is recommended that the user refer to CLSI C24 and 42 CFR 493.1256 for guidance on appropriate QC practices. The results below are given strictly for guidance purposes only and applicable for spectrophotometric readings only. First, calculate the mean (average) negative, positive and cut-off control raw OD450 values as shown in the following examples. Next, calculate the ratio between the positive and negative controls as shown in the following example. Finally, verify that the quality control requirements, listed in the table below, are fulfilled. Summary: The results on the table above must be obtained for the assay to be considered valid. Non-fulfillment of these criteria is an indication of deterioration of reagents or an error in the test procedure and the assay must be repeated.

The assay cut-off value was determined by testing one hundred forty-three (143) dengue-positive and thirty-seven (37) dengue-negative by RT-PCR with the Dengue virus NS1 Antigen ELISA Kit. The raw OD values and sample status were used for generating the ROC curves. The optimal ISR cut-off was defined so that equal weight was given to both specificity and sensitivity. A rudimentary bootstrap method was applied to minimize bias by any potential outliers in the sample set. The status of the unknown sample is determined by first calculating the cut-off of the assay (shown in Quality Control Example 3 ), followed by calculating the ratio of the optical density (OD450) divided by the cut-off. Calculate Immune Status Ratio (ISR): The immune status ratio (ISR) is calculated from the ratio of the optical density (OD) obtained with the test sample divided by the calculated Cut-Off Value. Calculate the ISR for each test sample. If unknown samples were tested in duplicate, then calculate the average optical density (OD450) before dividing by cut-off to determine ISR. SAMPLE INTERPRETATION CHART

Endemic Population Serum samples from 505 patients displaying signs and symptoms characteristic of dengue infections were prospectively collected in Colombia, Argentina, and Sri Lanka. These samples were collected within 7 days (inclusive) after the onset of clinical symptoms. The reactivities of the Dengue virus NS1 Antigen ELISA Kit with this endemic population are shown in Table, below. Expected results from an endemic region with individuals displaying symptoms for Dengue

The precision study of the Dengue virus NS1 Antigen ELISA Kit was performed at 3 different sites by 2 different individuals (6 operators total) on 5 separate days. All samples (including controls) were run in triplicate. Four serum specimens (Panels 1-4) using clinical specimens diluted in an analyte-negative matrix, plus a positive control, a negative control and cut-off control, were used. The four serum specimens included a positive specimen, two weakly positive specimens and a negative specimen. The ISR total precision %CV varied from 3.7-11.9%, depending on the sample. The results are shown in the following table.

The analytical reactivity was performed to determine the limits of detection (LODs) of Dengue virus NS1 Antigen ELISA Kit when tested with native NS1 from viral culture supernatants representing all four serotypes of dengue virus. Dilutions were tested in replicates of 20, and the lowest concentrations at which ≥95% of replicates tested positive were considered the LOD for each serotype. The LOD of Dengue virus NS1 Antigen ELISA Kit ranges from 82-611 pfu/mL, depending upon DENV serotype, as shown in Table below.

Samples were evaluated to assess microbial interference in the presence of dengue virus. Fifty-eight viral load positive samples that tested positive for other potentially cross-reactive pathogens were evaluated, as well as forty-two contrived samples that consisted of cultured micro-organisms diluted into normal human sera. Samples were then spiked with cultured dengue virus (ISR 2.0-3.2) just prior to testing with Dengue virus NS1 Antigen ELISA Kit. The tables below summarize the results of this study. Dengue virus NS1 Antigen ELISA Kit microbial interference with positive clinical specimens Dengue virus NS1 Antigen ELISA Kit microbial interference with spiked samples Interference by endogenous substances in the Dengue virus NS1 Antigen ELISA test was evaluated using a panel of four simulated clinical specimens (a strongly positive specimen, two weakly positive specimens and a negative specimen). Interfering substances at the levels indicated were tested as described in CLSI EP07-A2. There was no inhibition at the following concentrations of interferents that were tested. Endogenous Interfering Substances Study

This kit contains reagents made with human serum or plasma. The serum or plasma used has been heat-inactivated unless otherwise stated. Handle all sera and kits used as if they contain infectious agents. Observe established precautions against microbiological hazards while performing all procedures and follow the standard procedures for proper disposal of specimens.

1. All reactive samples must be confirmed by PCR or a serological assay. 2. Cross-reactivity with malaria and syphilis has not been evaluated with this assay. 3. The assay performance characteristics have not been established for visual result determination. 4. The assay performance characteristics have not been established for matrices other than serum. 5. Assay performance characteristics have not been established for testing cord blood, for testing neonates, for prenatal screening, or for general population screening. 6. False negatives may arise from patients co-infected with hepatitis B or with HIV. 7. Samples containing high levels of triglycerides or samples that are hemolyzed should be avoided for analysis with this assay. 8. HAMA does not show the false positive results, but may reduce ISR values for positive samples. 9. Results from immunosuppressed patients must be interpreted with caution. 10. Assay results should be interpreted only in the context of other laboratory findings and the total clinical status of the patient.

Dengue virus NS1 Antigen ELISA Kit for early detection of Dengue virus (DENV), is an ELISA assay system for the detection of NS1 antigen in human serum. This test will aid in the early diagnosis of Dengue virus in human serum even prior to the presence of IgM or IgG antibodies. It is not intended to screen blood or blood components, and is for investigational use only. Not for use in diagnostic procedures.

The Dengue virus NS1 Antigen ELISA Kit contains sufficient reagents for one plate of 96 wells (12×8 strips) each. Warning: Do not use any reagents where damage to the packaging has occurred. Dengue virus NS1 Antigen ELISA Kit supplied materials: 1. Coated Microtiter Strips for DENV Detect NS1 ELISA: ELISA strip holder in a ziplock foil pouch with desiccant, containing 96 polystyrene microtiter wells coated with anti-NS1 antibody in each well. Stable at 2-8°C until the expiration date. 2. Negative Control for DENV Detect NS1 ELISA: One vial, 300μL containing heat-inactivated negative control serum. The negative control will aid in verifying the validity of the kit. Stable at 2-8°C until the expiration date of the kit. 3. Positive Control for DENV Detect NS1 ELISA: One vial, 300μL containing recombinant NS1 in a phosphatebased buffer with 0.05% Proclin-300. The positive control will aid in verifying the validity of the kit. Stable at 2-8°C until the expiration date. 4. Cut-off Control for DENV Detect NS1 ELISA: One vial, 300μL containing recombinant NS1 in a phosphate-based buffer with 0.05% Proclin-300. The Cut-off Control will aid in determining the cut-off value for the ELISA. Stable at 2-8°C until the expiration date. 5. Sample Dilution Buffer for DENV Detect NS1 ELISA: One bottle, 15mL, containing the secondary antibody in a Tris-based buffer with 0.02%-0.05% Proclin-300. Stable at 2-8°C until the expiration date. 6. 100× Conjugate for DENV Detect NS1 ELISA: One vial, 150μL, containing horseradish peroxidase-labeled polyclonal antibody in a Tris-based buffer with 0.03% - 0.05% Proclin-300. Stable at 2-8°C until the expiration date. 7. Conjugate Diluent for DENV Detect NS1 ELISA: One bottle, 12mL. This contains the diluent solution for the 100× Conjugate in a Tris-based buffer with 0.01% Thimerosal as a preservative. The 100× conjugate is diluted directly into this solution. After diluting 100× Conjugate into this solution, the now ready-to-use conjugate may be stored up to 2 weeks at 2-8°C before it should be discarded. Stable at 2-8°C until the expiration date. 8. 10× Wash Buffer: One bottle, 120 mL. 10× concentrated phosphate buffered saline with Tween 20 (pH 6.8-7.0). Stable at 2-8°C until the expiration date. 9. Liquid TMB Substrate: One bottle, 12mL, ready to use. Contains 3, 3', 5, 5'-tetramethylbenzidine (TMB) and hydrogen peroxide in a citric acid-citrate buffer (pH 3.3-3.8). Stable at 2-8°C until the expiration date. Note: The substrate should always be stored in the light protected bottle provided. 10. Stop Solution: One bottle, 6mL, read to use 1N Sulfuric Acid. Used to stop the reaction. Stable at 2-8°C until the expiration date. Warning: Strong acid. Wear protective gloves, mask and safety glasses. Dispose all materials according to all applicable safety rules and regulations.

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