Mumps IgM, Page 1
Atlas Link
12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
MUMPS IgM ELISA
For in vitro diagnostic use
Catalog No. 1411
PRINCIPLE
Enzyme-Linked Immunosorbent Assays (ELISA) rely on the ability of biological materials, (i.e. antigens) to adsorb to plastic surfaces such as polystyrene (solid phase). When antigens bound to the solid phase are brought into contact with a patient's serum, antigen specific antibody, if present, will bind to the antigen on the solid phase forming antigen-antibody complexes. Excess antibody is removed by washing. This is followed by the addition of goat anti-human IgM globulin conjugated with horseradish peroxidase which then binds to the antibody-antigen complexes. The excess conjugate is removed by washing, followed by the addition of chromogen/substrate tetramethylbenzidine (TMB). If specific antibody to the antigen is present in the patient's serum, a blue color develops. When the enzymatic reaction is stopped with 1N H2SO4, the contents of the wells turn yellow. The color, which is proportional to the concentration of antibody in the serum, can be read on a suitable spectrophotometer or ELISA microwell plate reader .
MATERIALS SUPPLIED
1.Purified Antigen Coated Wells - 12 X 8 well strips in holder, dried, ready-to-use. Assembled plate contains ALTERNATING strip wells of inactivated ANTIGEN and CONTROL ANTIGEN . Allow the wells to equilibrate to room temperature (21-25°C) in the pouch to protect from condensation. When stored at 2-8°C, coated strips are stable until the labeled expiration date.
2.Calibrator Serum - one vial, 0.25 mL, human serum containing 0.1% sodium azide and 0.01% pen/strep added as preservatives. Dilute Calibrator 1:41 in Serum Diluent prior to use. When stored at 2-8°C the liquid Calibrator is stable until the labeled expiration date. Factor for the Calibrator is listed on the packing list included in the kit.
3.Negative Control Serum - one vial, 0.25 mL, human serum containing 0.1% sodium azide and 0.01% pen/strep as preservatives. Dilute the Control 1:41 in Serum Diluent prior to use. When stored at 2-8°C, the liquid Control is stable until the labeled expiration date. Value range for the Control is listed on the packing list included in the kit.
4.Positive Control Serum - one vial, 0.25 mL, human serum containing 0.1% sodium azide and 0.01% pen/strep as preservatives. Dilute the Control 1:41 in Serum Diluent prior to use. When stored at 2-8°C, the liquid Control is stable until the labeled expiration date. Value range for the Control is listed on the packing list included in the kit.
5.Horseradish-peroxidase (HRP) Conjugate, goat anti-human IgM - two bottles, 16 mL each. Ready to use, containing 0.1% proclin as preservative. When stored at 2-8°C, the liquid Conjugate is stable until the labeled expiration date. Bring to room temperature prior to test use. If the solution appears turbid, do not use.
6.Serum Diluent - two bottles, 30 mL each. Ready to use, containing 0.1% proclin as preservative. When stored at 2-8°C, the Serum Diluent is stable until the labeled expiration date.
7.Wash Buffer (20X concentrate) - one bottle, 60 mL. The buffer contains TBS, Tween and 0.1% proclin as preservative. Dilute the buffer 1 + 19 by pouring the contents of the bottle into a container and add dH2O to 1200 mL final volume. Mix thoroughly and store the 1X solution at room temperature (21-25°C). This is stable for 1 week. Before reconstitution, this buffer is stable until the expiration date on the label, when stored at 2-8°C. If the buffer is turbid, do not use.
8.Chromogen/Substrate Solution - two bottles, 16 mL each. Tetramethyl-benzidine (TMB), ready-to-use. When stored at 2-8°C it is stable until the labeled expiration date.
9.Absorbent Solution - two bottles, 12 mL each. Ready-to-use. Contains goat/sheep anti-human IgG with protein stabilizers and 0.1% proclin as preservative. When stored at 2-8°C, the solution is stable until the labeled expiration date.
The following components are not kit lot # dependent and may be used interchangeably within the DAI ELISA assays: Serum Diluent, Chromogen/Substrate Solution, Wash Buffer.
PRECAUTIONS
1. The human serum components used in the preparation of the controls and calibrator in this kit have been tested by an FDA approved method for the presence of antibody to Human Immunodeficiency Virus Type-1 (HIV-1), as well as for Hepatitis B surface antigen and found negative. Because no test methods can offer complete assurance that HIV-1, Hepatitis B virus, or other infectious agents are absent, specimens and human-based reagents should be handled as if capable of transmitting infectious agents. NOTE: The Center for Disease Control and the Center for Devices and Radiological Health recommends that potentially infectious agents be handled at Biosafety Level 2.
2.The components in this kit have been quality control tested as a Master Lot unit. Do not mix components from different lot numbers except Chromogen/Substrate and Wash Buffer. Serum Diluent supplied with IgG kits can be used only with other IgG kits and Serum Diluent supplied with IgM kits can only be used with other IgM kits. Do not mix with components from other manufacturers.
3.Do not use reagents beyond the stated expiration date marked on the package label.
4.All reagents must be at room temperature (21-25o C) before running assay. Remove only the volume of reagents that are needed. NOTE: Do not pour reagents back into vials as reagent contamination may occur.
5.Before opening Control and Calibrator vials, tap firmly on the bench top to ensure that all liquid is at the bottom of the vial.
6.Use only distilled or deionized water and clean glassware.
7.Do not let wells dry during assay; add reagents immediately after completing wash steps.
8.Avoid cross-contamination of Conjugate and Chromogen/Substrate solution. Wash hands before and after handling reagents.
9.If washing steps are performed manually, wells are to be washed three times. Up to five wash cycles may be necessary if a washing manifold or automated equipment is used.
10.Sodium azide inhibits Conjugate activity. Clean pipette tips must be used for the Conjugate addition so that sodium azide is not carried over from other reagents.
11. It has been reported that sodium azide may react with lead and copper in plumbing to form explosive compounds. When disposing, flush drains with water to minimize build-up of metal azide compounds.
12.Never pipette by mouth or allow reagents or patient sample to come into contact with skin.
13.If sodium hypochlorite (bleach) solution is being used as a disinfectant, do not expose to work area during actual test procedure because of potential interference with enzyme activity.
14.Avoid contact of sulfuric acid with skin or eyes. If contact occurs, immediately flush area with water.
15.For in vitro diagnostic use.
MATERIALS REQUIRED BUT NOT SUPPLIED
1.Stop solution - 1N Sulfuric Acid (H2SO4)- (One part H2SO4 (18M) to 35 parts deionized or distilled water).
2.Graduated cylinder (100 mL).
3.Flask (1L).
4.Timer - 0 to 60 minutes.
5.Micropipettes capable of accurately delivering 10-200 L volumes (less than 3% CV).
6.Deionized or distilled water.
7.Paper towels.
8.Wash bottle, semi-automated or automated wash equipment.
9.Single or dual wavelength microplate reader with 450 nm filter. If dual wavelength is used, set the reference filter to 600-650 nm. Read the operators' manual or contact the instrument manufacturer to establish linearity performance specifications of the reader.
10.Test tubes for serum dilution.
11.Disposal basin and disinfectant (e.g. 0.5% sodium hypochlorite).
NOTE: Use only clean, dry glassware.
STORAGE AND SHELF LIFE OF REAGENTS
Store unopened kit between 2o and 8o C. Precautions were taken in the manufacture of this product to protect the reagents from contamination and bacteriostatic agents have been added to the liquid reagents. Care should be exercised to protect the reagents in this kit from contamination. If constant storage temperature is maintained, reagents and substrate will be stable for the dating period of the kit. Refer to package label for expiration date.
SPECIMEN COLLECTION
1.Handle all blood, plasma and serum as if capable of transmitting infectious agents.
2.Optimal performance of the DAI IgM ELISA kit depends upon the use of fresh serum samples (clear, non-hemolyzed, non-lipemic, non-icteric). A minimum volume of 50 L is recommended, in case repeat testing is required. Specimens should be collected aseptically by venipuncture. Early separation from the clot prevents hemolysis of serum.
3.Store serum between 2 o and 8 o C if testing will take place within two days. If specimens are to be kept for longer periods, store at -20 o C or colder. Do not use a frost-free freezer because it may allow the specimens to go through freeze-thaw cycles and degrade antibody. Samples that are improperly stored or are subjected to multiple freeze-thaw cycles may yield spurious results.
PREPARATION OF REAGENTS
1. Allow pouched plate, reagents, calibrators, buffers and patient sera to equilibrate to room temperature (21-25°C) to avoid condensation.
2. Wash Buffer should be diluted 1:20 to 1.2 liters with distilled and/or deionized water.
3. Calibrator, Control and patient sera should be diluted 1:41 (i.e.: 10 L + 400 L) with Serum Diluent.
SERUM TREATMENT
Solid phase immunoassays for the detection of virus-specific IgM are known to be sensitive to interfering factors. This kit overcomes interferences by treating samples prior to running the assay. The Absorbent solution diminishes competing virus-specific IgG, which would be responsible for false negative reactions. False positives are similarly minimized by removing the IgG, thus neutralizing the bound rheumatoid factor in the samples.
Procedure for Serum Absorption
A. In a set of test tubes, dilute Calibrator, Controls and patient samples 1:41 in Serum Diluent (i.e., 400 L Diluent + 10 L of Calibrator, Control or serum sample).
B. For each assay, run the positive Calibrator in duplicate, in both the antigen and control antigen wells. Add 250 L of the 1:41 dilution (step A.) to 250 L Absorbent Solution. Mix well. This will give enough for 2 antigen wells and 2 control antigen wells. (Final dilution 1:81).
C. For the Controls and test samples, add 150 L of the 1:41 dilution (step A) to 150 L Absorbent Solution. Mix well. This will give enough for 1 antigen well and 1 control antigen well. (Final dilution 1:81).
D. Incubate all absorbent dilutions at room temperature (21-25°C) for 20 minutes + 5 min.
PROCEDURE
1. Bring all reagents and microwells to room temperature (21-25°C).
2. Remove desired number of strips and strip retainer from the sealed plastic package. Replace unused strips back in the pouch with desiccant and seal tightly. Store unused strips and reagents at 2-8°C.
3. The Calibrator, Positive & Negative Controls and each patient serum must be run in both an antigen and control antigen coated well. Check software and reader requirements for the correct Calibrator/Control configurations.
Example Configuration:
-reagent blank well (A-1) to zero instrument
-Negative Control well
-2 Calibrator wells
-Positive Control well
-patient sera wells
4. Record all steps of assay and label test tubes with appropriate identification for kit Control, Calibrator and patient samples to be tested.
5. Pipette 100 L of diluted and absorbed patient, Calibrator and Control sera into corresponding wells of the antigen strip and control antigen strip. In reagent blank well (A-1) add 100 L of Serum Diluent.
6. Incubate all wells at room temperature (21-25°C) for 20 min. + 2 min.
7. At the end of the incubation, empty the contents of the wells and wash 3 to 5 times with 250 L Wash Buffer per well with a squeeze bottle or automated washing system. Aspirate liquid immediately. Do not leave Wash Buffer in wells. Remove residues by tapping wells inverted on a paper towel.**
8. Pipette 100 L Conjugate into all wells, including reagent blank well (A-1). Avoid air bubbles.
9. Incubate all wells at room temperature (21-25°C) for 20 + 2 min.
10. Repeat wash step 7**.
11. Pipette 100 L of Chromogen/Substrate solution into all wells, including reagent blank well (A-1).
12. Incubate all wells at room temperature (21-25°C) for 10 min.
13. Stop the reaction by adding 100 L of 1N H2SO4 Stop Solution into each well, including reagent blank well (A-1), at the same rate and sequence the substrate was added. Gently agitate the plate to mix the contents of the wells.
14. Wait a minimum of five (5) minutes and read. The plate may be held up to one (1) hour after addition of Stop Solution before reading. Zero the spectrophotometer on the blank well (A-1). Carefully wipe the bottom of the wells and measure and record the absorbances at 450 nm (reference filter 620-650 nm optional). The reagent blank must be less than 0.150 Absorbance at 450 nm. If the reagent blank is > 0.150 the run must be repeated.
**IMPORTANT NOTE: Regarding steps 7 and 11 - Insufficient or excessive washing will result in assay variation and will affect validity of results. Therefore, for best results, the use of semi-automated or automated equipment set to deliver a volume to completely fill each well (250-300 L) is recommended. A total of up to five (5) washes may be necessary with automated equipment. Please contact DAI, Inc. with any question regarding appropriate wash equipment. Complete removal of the Wash Buffer after the last wash is critical for the accurate performance of the test. Also, visually ensure that no bubbles are remaining in the wells.
QUALITY CONTROL
1. Calibrator and Controls must be run with each test run.
2.Reagent blank (when read against air blank) must be <0.150 Absorbance (A) at 450 nm.
3. Negative Control must be < 0.250 A at 450 nm (when read against reagent blank).
4. Each Calibrator must be > 0.300 A at 450 nm (when read against reagent blank).
5.Positive Control must be > 0.250 A at 450 nm (when read against reagent blank).
6. If above criteria are not met on repeat, contact DAI Technical Service.
7.DAI recommends that a Positive Control of known reactivity be included in each assay, run as part of the users quality control program.
CALCULATION OF RESULTS
For each patient sample and Controls/Calibrator, subtract the control antigen absorbance from the antigen well absorbance. This is the Delta value. Proceed with this Delta value to INTERPRETATION OF RESULTS.
INTERPRETATION OF RESULTS
1.Multiply the mean Delta absorbance of the Calibrators by the factor assigned. The factor is Master Lot specific and is recorded on the vial label. This is the Calibrator value.
2.The ISR value for each patient sample is calculated by dividing the Delta sample absorbance by the Calibrator value (obtained in Step 1).
ISR ValueResultsInterpretation
<0.90NegativeNo significant level of detectable IgM antibody.
0.91-1.09EquivocalSamples should be retested. See number 3 below.
>1.10PositiveSignificant level of detectable IgM antibody. Indicative of current or recent infection.
3.Samples that remain equivocal after repeat testing should be retested on an alternate method, e.g. immunofluorescence assay (IFA).
LIMITATIONS
1.The user of this kit is advised to carefully read and understand the package insert. Strict adherence to the protocol is necessary to obtain reliable test results. In particular, correct sample and reagent pipetting, along with careful washing and timing of the incubation steps are essential for accurate results.
2.The results of ELISA immunoassays performed on serum from immunosupressed patients must be interpreted with caution.
3.This device is not intended for the determination of immune status. It is intended for the determination of a person's immune response to indicate primary infection, reinfection or virus reactivation.
4.Samples that remain equivocal after repeat testing should be retested by an alternate method, e.g., immunofluorescence assay (IFA). If results remain equivocal upon further testing, an additional sample should be taken.
5.The absence of detectable IgM antibody does not rule out the possibility of recent or current infection. However, if infection is still suspected, obtain a second specimen 5-7 days later and repeat the testing. The IgM results in conjunction with IgG results for paired sera may prove useful in demonstrating current infection.
6.Specific IgG may complete with the IgM for sites and may result in a false negative. Conversely, rheumatoid factor in the presence of specific IgG may result in a false positive reaction. The Absorbent Solution diminishes competing virus-specific IgG and minimizes rheumatoid factor interference in samples. Studies indicate that the maximum amount of IgG which can be removed by the kit Absorbent Solution is in excess of the expected high end of the normal range for IgG > 1380 mg/dL. The highest titer of RF+ tested (1: 1280; 500 IU/mL) did not adversely affect the performance of the assay.
7.Some antinuclear antibodies have been found to cause a false positive reaction on some ELISA tests.
8.It is strongly recommended that neonate's and mother's serum samples be tested in parallel. The presence of IgM antibody in the neonate's serum can be considered indicative of congenital infection only if there has not been placental leakage. Additionally, if the infant has a congentital infection the IgM antibody (and IgG antibody) level may persist or rise, whereas if the source of the antibody is maternal, the neonate's antibody level will drop in parallel to the half-life of that immunoglobulin.
9.Results of this test should be interpreted by the physician in the light of other clinical findings and diagnostic procedures.
P/N # 2000-29 REV A 12/94
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664