rev:January 28, 1998

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Promotional information:see actual kit insert for batch specific information
-strictly for in vitro research use only-not for use in diagnostic procedures in USA

The following Elisa kits are manufactured by IBL (Immuno Biological Laboratories,
Hamburg Germany) and distributed by RDI Divison of researchd Industries Intl for in vitro
research use only-not for use in or on humans or animals, not for use in

Catalogue No : RDI-CG59221 $688.00/kit $625.00/kit 2+ Product group : Autoimmunity Product name : Circulating Immunecompl. C3d ELISA 96 Method : ELISA Incubation time: 30 min, 15 min, 15 min Standard curve : Sample/Prep. : 50 �l Serum Isotope/Substr.: TMB, 450 nm


Introduction Contents of the Kit Principle of the Test Test Procedure Performance Characteristics Expected Values Alternative Applications (More) Clinical Background Sales Arguments Product Literature Miscellaneous


Back to Contents Circulating immune complexes (CIC) are present in many individuals with systemic lupus erythematosus (SLE) and Rheumatoid Arthritis (RA), especially with any of the vasculitides complications. Levels of CICs have been reported to show correlation with disease activity in that higher levels are reported during active phases of the disease. Many tests have been developed for the detection of CICs, including PEG precipitation, radial immunodiffusion and cellular based assays, such as the Raji cell assay. No single procedure appears to detect all types of CIC: however, those procedures which detect CICs containing fragments of complement (e.g. C1q and C3d) appear to detect clinically relevant events. The IBL test system for C3d circulating immune complexes detects immune complexes containing both C3d and IgG. The concentration is expressed as �g/ml heat aggregated human globulin (HAG) equivalents. IBL also offers a test kit for the determination of C1q and IgG containing circulating immune complexes.

Contents of the Kit

Back to Contents 1. Microtiterstrips 4 x 3 x 8 wells incl. frame, coated with murine monoclonal anti-C3d antibody. 2. Enzyme Conjugate 1 vial 12 ml, ready to use, Anti-human-IgG conjugated with peroxidase, pink coloured. 3. Standard Level 1 (low) 2 vials lyophilised, for dilution see chapter 8, For levels, see vial labels. 4. Standard Level 2 (high) 2 vials lyophilised, for dilution see chapter 8, For levels, see vial labels. 5. Control Serum, positive 1 vial 0.2 ml, dilute 1:5, (see certificate of analysis for value). 6. Control Serum, negative 1 vial 0.2 ml, dilute 1:5, (see certificate of analysis for value). 7. Wash Buffer 2 vials 25 ml, concentrate, dilute concentrate with distilled water so the final volume is 600 ml. The diluted wash buffer is stable for 1 year at 2 - 8�C. 8. Sample Diluent 1 vial 25 ml each, ready to use, blue coloured. 9. Substrate 1 vial 12 ml, ready to use, TMB solution. 10. Stop solution 1 vial 6 ml, ready for use, 0.25 M H2SO4, Caution acid!

Principle of the Test

Back to Contents The assay for C3d containing circulating immune complexes is a solid phase immunosorbent assay in which the analyte is indicated by a colour reaction of an enzyme conjugate. The assay wells are coated with a monoclonal antibody specific for the C3d component of complement. On adding diluted serum to the wells, any C3d containing CICs present bind to the antigen. After incubating and washing away unbound material, horseradish peroxidase conjugated anti-human IgG monoclonal antibody is added which binds to C3d and IgG containing CICs. Following incubation and washing, the substrate (tetramethyl benzidine) is added to each well. The presence of the {conjugate - cic - antigen} complex turns the substrate to a dark blue colour. Addition of stop solution turns the colour to yellow. The colour intensity is proportional to the amount of C3d containing CICs present in the original sample. A calibrator, standardised in �g/ml heat aggregated human globulin, is used to quantitate the test samples.

Test Procedure

Back to Contents 1. Select sufficient microwells. Duplicate determinations are recommended. Wash plate three times with diluted Wash Buffer immediately prior to commencing assay, remove excess solution tapping the inverted plated on a paper towel but do not allow the plate to dry out! 2. Pipette 100�l of diluted Standards, Controls and Samples into the wells of the microtiter strips. To achieve blanking on the ELISA reader a 'no serum' control of 100 �l of sample diluent should be used. 3. Incubate at room temperature (18 - 25�C) for 30 minutes. 4. After the first incubation, invert the plate and briskly shake out the well contents. Wash the wells three times with diluted Wash Buffer using a multichannel pipette or an automatic microplate washing system. Then remove excess solution tapping the inverted plate on a paper towel. 5. Pipet 100�l of the Enzyme Conjugate into the wells. 6. Incubate at room temperature (18 - 25�C) for 15 minutes. 7. Repeat the washing procedure as described in 9.4. 8. Pipet 100 �l of Substrate Solution into each well. 9. Incubate for 15 minutes at room temperature (18 - 25�C). 10. Stop the reaction by adding 50 �l of Stop Solution to each well. 11. Read the optical density at 450 nm within 15 minutes of stopping using a microplate reader. Collection of Specimens and Storage Collect whole blood according to accepted medical techniques. Allow the specimen to clot at room temperature for 2 hours before the serum is removed. Do not allow the specimen to clot at refrigerator temperature (2-8 �C). Do not store the clotted specimens at 2-8 �C. Do not use anticoagulated specimens collected as plasma and then clotted. Do not use heat inactivated serum specimens. Do not use plasma. Serum may be stored up to 24 hours at 2-8 �C in a sealed container. If longer storage is required, store at - 70 �C. Freeze and thaw only once. Preparation of Samples and Reagents All Reagents are ready to use except for the following: Wash Buffer Concentrate Dilute the whole contents of the wash buffer concentrate vial with distilled water so the final volume is 600 ml. Store this solution at 2 - 8�C if it is not to be used at once. The diluted wash buffer is stable for 1 year at 2 - 8�C. Controls and Patient Samples Dilute controls and Patient Samples 1:5 with Sample Diluent (50 �l sample plus 200 �l diluent). Reconstitution of Lyophilised Standards Reconstitute one vial of each standard (level 1 and 2) by adding 0.15 ml of double distilled or deionised water to each vial. Gently resuspend the powder. Allow the rehydrated standards to stand at room temperature for at least 10 minutes prior to use. Do not vortex or mix vigorously, as this could cause denaturation of serum IgG and result in abnormally high values. Reconstituted standards are stable for 5 days if stored at 2-8 �C. Dilution to Working Strength of Reconstituted Standards Dilute the reconstituted standards 1:5 (50 �l plus 200 �l) with sample diluent prior to testing. The diluted standards should be used within 48 hours.

Performance Characteristics

Back to Contents Calculation of Results Plot the blank connected optical densities (ODs) of the standards against the concentration value, using a linear Y-axis (OD) and linear X-axis (concentration). The concentration value of the patients samples can be determined from this calibration curve. The dilution of the samples has already been considered in the concentration of the standards. Alternatively, if curve fitting software is available, the data may be processed using this. The curve produced using the C3d containing CIC assay is best described by a 2 point linear regression fit with linear axes. In order for the assay to be declared valid, the following criteria should be met: slope: 0.008-0.020 Y-intercept: < 0.500 Typical Results A typical example of a standard curve with the C3d CIC ELISA is listed below. OD1 OD2 Mean OD Mean OD Result Blank (�g/l) Reagent Blank 0.057 0.055 0.056 - 0 Standard 1 0.343 0.257 0.300 0.244 8 Standard 2 1.231 1.308 1.270 1.214 114 Sample 1 1.163 1.211 1.187 1.131 96.1 Sample 2 0.763 0.837 0.800 0.744 55.8 Slope 0.009589 Y-Intercept 0.171 Sensitivity The sensitivity of the C3d containing CIC assay is 0.7 �g/ml HAG equivalents. Precision The within batch reproducibility of the assay was as based on the analysis of three separate plates as follows: Within Batch Variation (values in �g/ml) Sample Average CV (%) 1 76.5 16.6 2 51.4 6.3 3 15.6 9.9 The between batch reproducibility of the assay was based on the variation of three controls on two production batches as follows: Between Batch Variation (values in �g/ml) Sample Average CV (%) 1 78.5 11.1 2 52.8 3.7 3 14.2 2.5 Limitations of use Grossly haemolysed, lipaemic or microbiologically contaminated samples should not be used. A negative result should not be used as a sole criterion to rule out SLE, RA or other autoimmune disease but must be taken in relation to other clinical observations and diagnostic tests. It should be noted that C3d containing CICs do occur in other autoimmune or non-autoimmune conditions. Therefore, all other clinical observations and diagnostic tests should be taken into account for clinical diagnosis.

Expected Values

Back to Contents A value above 24 �g/ml HAG equivalents is considered as positive. A value between 16 - 24 �g/ml can be regarded as equivocal. Values below 16 �g/ml are considered as negative. Any positive results should be interpreted in view of the clinical situation. These reference values are only a guideline. It is recommended that each laboratory establish its own reference range.

Alternative Applications

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(More) Clinical Background

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Sales Arguments

Back to Contents Circulating Immune Complexes Clinical Application: - Vasculitides associated with SLE, rheumatoid arthritis, polyarthritis, Schoenlein-Henoch purpura, post- and parainfectious immune complex diseases Sample Collection: - Allow blood specimen to clot at room temperature for 2 hours before serum is removed. Store for max. 24 h at 2-8 �C or freeze at -70 �C; freeze and thae only once. Do not use plasma or heat-inactivated serum samples. Comparison to other ELISAs: IBL QUIDEL/LD Imtec Medac C1q- / C3d-CIC C1q- / C3-CIC C1q- / C3d-CIC C1q- / C3d-CIC Antigen mAb to C1q C1q, C1q, mAb to C1q, mAb to C3d Mouse-anti-C3 C3d (coming mAb to C3d soon) Sample 50 �l serum 10 �l serum 10 �l serum 100 �l serum (1:5) (1:50) (1:100) (1:5) Incuba- prewash; prewash; 2 x 2 h; prewash; tions 1 x 30 min; 1 x 1 h; 1 x 10 min 2 x 1 h; 2 x 15 min 2 x 30 min 1 x 30 min. Enz./Sub POD / TMB POD/*1 POD / TMB POD / OPD-Tabl. Standards 2 x 2; 2 x 3; 3 x 4; 4 x 3; lyophilised lyophilised lyophilised lyophilised Controls pos./neg.(1:5) none pos., lyoph. none other Wash Buffer Wash Buffer Wash Buffer Wash Buffer reagents Substrate Sample Diluent Sample Diluent to Substrate Conjugate prepare*� Substrate *1) 2,2-Azino-di-(3-ethylbenzothiazoline sulfonic acid)-diammonium salt *�) all other reagents are ready for use Argumentation for selling the IBL Assay: - use of monoclonal antibodies to C1q and C3d; therefore detection of clinically relevant immune complexes containing complement - fast and easy performance - almost all reagents ready to use - controls provided with the kit - material saved by two-point standard curve Autoimmune Diseases New ELISAs - IgA Rheumatoid Factors - dsDNA Antibodies - IgG Rheumatoid Factors - ssDNA Antibodies* - IgM Rheumatoid Factors - Thyroglobulin Antibodies - ENA-6 Profile - Thyroid Peroxidase Antibodies - ENA-6 Screen - Cardiolipin Antibodies - Sm Antibodies - Phosphatidylserine Antibodies - RNP Antibodies - Mitochondria Antibodies - Ro (SS-A) Antibodies - C1q CIC - La (SS-B) Antibodies - C3d CIC - Jo-1 Antibodies - Proteinase 3 Antibodies* - Scl-70 Antibodies - Myeloperoxidase Antibodies* - GPC Antibodies - GBM Antibodies* * under development YOUR ADVANTAGES: - common test performance - results within one hour - ready to use reagents - exchangeable reagents and buffers - two kit controls - excellent reproducibility - suitable for automation Pipetting scheme: Pipette standards, samples and controls Incubate for 30 min. at room temperature Wash 3 x Add enzyme conjugate Incubate for 15 min. at room temperature Wash 3 x Add TMB Incubate for 15 min. at room temperature Add stop solution and measure OD at 450nm Please call us for more detailed information!
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