rev: June 18, 2004
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Human IgM Rheumatoid Factor ELISA Kit (in USA-for in vitro research use only-not for use in diagnostics) Manufactured by CLB Reagents (Netherlands Red Cross)
CAT#: RDI-M1950clb PRICE: $312.00/kit
Cat#RDI-M1950clb US$312.00/kit / November 2003
Sample insert see batch specific data with each kit
ELISA Human IgM Rheumatoid Factor kit M1950
- ELISA kit for quantitative determination of human IgM Rheumatoid Factor in serum
In serum of patients with rheumatoid arthritis (RA), commonly rheumatoid factors (RF) can be detected. RF are antibodies directed to the Fc part of antibodies of the human IgG class (1-5). In most cases RF belong to the IgM class (IgM-RF), occasionally RF of the IgG class (IgG-RF) can be found together with IgM-RF. Rarely sera with circulating IgG-RF and no IgM-RF were found. Close examination of sera from RA patients and healthy individuals demonstrated equal frequencies of the occurrence of IgG-RF in both populations (1, 8). Consequently, it was concluded that the quantification of IgG-RF did not improve the serological diagnosis of RA.
Testing for IgM-RF has a high diagnostic value, as their detection or exclusion can support or place doubt on a tentative diagnosis based on case history data and clinical findings (6). Since there is substantial evidence indicating a role of IgM-RF in the pathogenesis of RA and the formation of complement activating immune complexes (1?3), a seropositive IgM-RF generally has a more unfavourable prognosis than the seronegative form. IgM-RF can be detected not only in RA but also in a number of other connective tissue diseases, therefore the results of IgM-RF determination should always be assessed in connection with clinical and other laboratory findings.
II. TECHNICAL PRINCIPLE
The IgM Rheumatoid Factor ELISA kit is a "sandwich-type" of enzyme assay. The kit contains microwell strips coated with human immunoglobulins. Test samples, calibrator and controls are incubated in the respective wells. IgM-RF will bind to the solid phase and non- bound material is removed by washing. Next peroxidase conjugated anti-human IgM is added to each well and non-bound conjugate is removed by washing. After incubation with substrate solution (ABTS) and H2O2, the reaction is stopped with an acid buffer. The green colored reaction product is measured by absorbance and the concentration of IgM-RF in the test sample calculated relative to the values of a reference curve.
IgM-RF controls are assayed to check the validity of the calibration curve and the accuracy of the IgM-RF determinations.
The IgM-RF level in the calibrator was determined using the CLB RELARES calibrator (7).
III. STORAGE AND STABILITY
The IgM-RF ELISA kit should be stored upright at 2-8 °C. It can be used until the expiration date shown on the label.
For all components stability after opening is 1 week when stored at 2-8 °C.
Transport conditions may differ from storage conditions.
IV. CONTENTS OF THE KIT
The IgM-RF ELISA kit contains sufficient reagents for 96 tests, including calibrators, controls and blank. See the Table at the end of this package insert.
The human IgG used as a coat is a purified IgG fraction (Sanquin).
The calibrator and controls are lyophilized human plasma's.
The monoclonal antibody was purified from tissue culture medium, using column chromatography (ion exchange and affinity chromatography) and labelled to horseradish peroxidase (HRP).
V. ADDITIONAL MATERIALS REQUIRED
- Distilled water for dilution of wash- dilution- and substrate buffers and for reconstitution of the calibrator and controls.
- Pipetting devices for accurate delivery of volumes.
- An incubator (37 ± 2°C).
- A standard ELISA washer or a 500 mL plastic squirt bottle for automatic or manual washing of the strips.
- A standard ELISA reader for measuring absorbance at 414 nm or 405 nm.
- Log linear paper.
VI. TEST SAMPLE HANDLING
Only serum samples should be tested. If samples are to be run within 24 hours they may be stored at 2-8°C, otherwise samples should be stored frozen. The samples should be manually diluted before use (see Vll ASSAY PROTOCOL).
VII. ASSAY PROTOCOL
- Bring all reagents to room temperature (18-25°C) and mix thoroughly. Avoid bubbles or foam.
- It is advised to test all samples, controls and calibrator dilutions in duplicate.
The IgM-RF ELISA kit provides the flexibility to use partial plates on separate occasions.
Before opening the plastic pouch, determine the number of strips required to test the desired number of samples plus 20 wells needed for running calibrators, controls and blanks in duplicate. Remove strips that will not be used from the plate-frame and re-pack them in the plastic pouch containing the desiccant and store at 2-8°C.
2. BUFFER PREPARATIONS
Wash buffer: Prepare the wash buffer by adding the total content of the bottle of the wash buffer concentrate to 950 mL distilled water. The diluted wash buffer must be stored at 2-8°C and remains stable for 1 week.
Note: The concentrated buffer may contain salt crystals. Before preparing the working-strength buffer, warm the concentrated buffer BRIEFLY to 37°C to dissolve the crystals.
Dilution buffer: Calculate the quantity of dilution buffer required (approximately 2 mL undiluted buffer per microwell strip) and prepare a working-strength solution by diluting the buffer 10 fold in distilled water.
3. PREPARATION OF THE CALIBRATOR AND CONTROLS
For concentrations see Table 2 and 3 of the enclosed information leaflet. For assay ranges see Table 1 of the enclosed information leaflet.
Reconstitute each vial with 200 mL distilled water. Leave for 30 minutes at room temperature (18-25°C) and mix gently.
Label three tubes, one for the positive control, one for the negative control and one for the highest standard point. Pipette 990 mL working-strength dilution buffer into each tube and add 10 mL of reconstituted IgM-RF standard and controls in their respective tube and mix well.
Label six tubes, one tube for each calibration curve dilution. Subsequently, pipette 250 mL of dilution buffer into these tubes. Transfer 250 mL from the first tube into the second tube and mix well. Repeat the serial dilution five more times by adding 250 mL of the previous tube of diluted calibrator to the 250 mL of diluent in the labelled tubes.
4. PREPARATION OF SAMPLES
Dilute the test samples 1:100 in dilution buffer. For results that lie outside the given ranges (see Table 1 of the enclosed information leaflet), the test should be repeated with a different sample dilution.
5. FIRST WASH STEP
Wash the required microwells in the plate-frame four times with washing buffer. For manual washing, completely fill the wells (> 300 µL) with washing buffer and discard, repeat this procedure three times. Finally, the wells should be completely empty. Subsequent reagent should be added immediately, do not let the wells stand dry for extended period of time.
6. FIRST INCUBATION STEP
Add 100 µL of the diluted calibrators, controls and samples into the appropriate wells. Leave the blank wells empty.
Cover plate with adhesive seal, gently agitate by tapping the edge of the microtitreplate for a few seconds to mix contents of each well.
Incubate for 1 hour at 37°C.
Just before washing, prepare next incubation reagent as described in point 8.
7. SECOND WASH STEP
Aspirate supernatant from the wells and wash the plate as described in point 5.
8. INCUBATION WITH HRP-CONJUGATED ANTIBODY TO HUMAN IgM
Dilute the conjugate 1:500 (2 µl conjugate per microwell strip) in dilution buffer.
Add 100 mL to each well. Leave the blank wells empty.
Cover the plate with adhesive seal, gently agitate by tapping the edge for a few seconds to mix the contents of each well.
Incubate for 1 hour at 37°C.
Just before washing prepare next incubation reagent as described in point 10.
9. THIRD WASH STEP
Aspirate supernatant from the wells and wash the plate as described in point 5.
10. INCUBATION WITH ABTS-SUBSTRATE
Calculate the amount of substrate solution (approximately 0.9 mL per microwell strip will be needed).
Add the equivalents of 200 µL hydrogen peroxide stock solution and 400 µL ABTS stock solution to 20 mL of working strength substrate buffer (2 mL substrate stock solution + 18 mL distilled water).
Add 100 µL of substrate solution to all wells.
Gently agitate by tapping the edge of the microtitreplate for a few seconds to mix the contents of each well.
Incubate for 30 minutes at room temperature (18-25°C).
11. STOP ENZYMATIC REACTION
Add 100 µL of stop solution to all wells.
12. PLATE READ-OUT
Read within 1 hour at 414 (preferably) or 405 nm in an ELISA reader.
VIII. RESULTS AND INTERPRETATION OF DATA
- Record the absorbance at 414 (preferably) or 405 nm for each well and average the duplicate values.
- For each sample, duplicates should not differ more than 15% from the mean value. If duplicates vary more, the assay should be repeated.
- Calculate the net values by substracting the blank value.
- Plot the average absorbances of the calibrators (y-axis) versus the related IgM-RF concentration in IU/mL (x-axis) on log-linear paper and draw the best fitting curve.
- The IgM-RF levels of the controls should lie within the range(s) given in Table 3 of the enclosed information leaflet.
- Interpolate the average absorbances value for each sample on the calibration curve.
- Test samples which show a mean absorbance outside the range of the calibration curve dilutions, should be diluted appropriately.
IX. ASSAY RANGES
Consult Table 1 of the enclosed information leaflet for the kit specific assay ranges.
X. REFERENCE RANGES
Measurements in a group of normal healthy blood donors in the Netherlands (n=665), yielded IgM-RF values under 12.5 IU/mL in 96% of the cases. If a serum contains more than 12.5 IU/mL of IgM-RF, this serum should be considered as positive (8). As reference ranges are subject to many influencing variables which may differ for each population investigated, each laboratory should establish its own reference range.
XI. SPECIFIC PERFORMANCE CHARACTERISTICS
|IU/mL||Intra-assay variation (%)||Inter-assay variation (%)|
The values cited for specific performance characteristics of the test represent typical results and are not to be viewed as specifications for this kit.
1. User should be trained and familiar with ELISA assays and test procedure.
2. Grossly haemolysed or lipaemic samples should not be used. Unexpected results may be obtained for samples containing high bilirubin levels, or other circulating immune complexes. These samples should be analysed by other method.
3. Out of range samples should be repeated using different dilutions.
4. The finding of an increased level of the IgM-RF can never provide a definite diagnosis, but should rather be considered as an indication of a disturbance of the immune system, requiring further diagnostic investigation.
5. Controls should always be used to check the validity of the calibration curves. When the controls are out of range, the results of the test samples are not reliable. The test should be repeated.
6. Reagents from different batches are not interchangeable.
7. Rests of reagents (e.g. dead volume) should not be mixed with contents of freshly opened vials.
8. Caps and vials are not interchangeable. Caps should be replaced on the corresponding vials.
9. Although the human calibrator and control sera have been tested for the markers of specific disease transmitting agents in accordance with current EU guidelines to GMP and found to be nonreactive, all components of human origin should be considered as potentially infectious.
10. Preservative: Thiomersal® 0,001%.
11. Use new plate seals for each incubation/fixation step in the ELISA-experiment to avoid cross contamination. Do not use aluminium foil.
12. Use disposable pipette tips for each transfer to avoid cross contamination.
13. Each time the kit is used, fresh dilutions of calibrators, conjugate and buffers should be made.
14. Do not use other reagents and microtitre strips than those supplied with the kit.
15. Sodium azide inactivates HRP, do not use sodium azide-containing solutions, nor add sodium azide to the supplied buffers.
16. The waste-disposal should be performed according to your laboratory regulations.
1.Dorner,R.W. et al (1987) Clin.Chim.Acta 167: 1
2.Harris,E.D. (1990) N.Engl.J.Med. 322: 1277
3.Mannik,M. (1992) J.Rheumatol. 19: 46
4.RF review issue (1988) Scand.J.Rheumatol. 75: 1
5.Williams,R.C. (1992) J.Rheumatol. 19: 42
6.Silman,A.J. (1987) Br.J.Rheumatol. 27: 342
7.Klein,F. et al (1987) Ann.Rheum.Dis. 46: 674
8.Vademecum CLB diagnostisch onderzoek 2003 : 154-155
ELISA IgM-RF kit
REF Contents of the kit
M195002 1 12x8 wells Precoated human IgG
M195003 1 0.2 mL Calibrator (lyophilized)
M195005 1 0.2 mL Negative control (lyophilized)
M195004 1 0.2 mL Positive control (lyophilized)
M195006 1 0.05 mL Horseradish peroxidase conjugated anti-human IgM 1:500
M1805 1 50 mL Wash buffer 1:20
M1806 1 60 mL Dilution buffer 1:10
M1808 1 5 mL Substrate buffer 1:10
M1810 1 1.0 mL ABTS solution 1:50
M1809 1 0.5 mL Hydrogen peroxide solution 1:100
M1807 1 20 mL Stop buffer
- 4 - Plate seals
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