rev: 5/16/2008

Return (Index/Home Page)

Return (master kit index)

---

Promotion 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 distributed by RDI Division of Fitzgerald Industries Intl for in vitro
research use only-not for use in or on humans or animals, not for use in
diagnostics.




 
 Catalogue No : RDI-CG59211 $724.50/kit  
 

Product group : Autoimmunity

Product name : Circulating Immunecompl. C1q ELISA 96

Method : ELISA

Incubation time: 30 min, 15 min, 15 min

Standard curve :

Sample/Prep. : 50 µl Serum

Isotope/Substr.: TMB, 450 nm

CONTENTS

 
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
 
 

Introduction

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. 
 C1q binds with greatest avidity to immune complexes ranging in size from 
 19 to 27 S. In serum sickness, which is the prototype immune complex 
 disease, this size of complex has typically been found to be deposited in  
 tissues leading to damage. 
 The IBL test system for C1q circulating immune complexes detects immune 
 complexes containing both C1q and IgG. The concentration is expressed as 
 µg/ml heat aggregated human globulin (HAG) equivalents. 
 IBL also offers a test kit for the determination of C3d and IgG containing 
 circulating immune complexes. 
  
 
 

Contents of the Kit

Back to Contents
 
 1. Microtiterstrips 4 x 3 x 8 wells 
 8 wells each 
 coated with murine monoclonal anti-C1q 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 C1q 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 C1q 
 component of complement. On adding diluted serum to the wells, any C1q  
 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 C1q 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 C1q containing CICs 
 present in the original sample. A calibrator, standardised in µg/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 100µl 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 µl 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 100µl 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 µl of Substrate Solution into each well. 
 9. Incubate for 15 minutes at room temperature (18 - 25°C). 
 10. Stop the reaction by adding 50 µl 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 µl sample 
 plus 200 µl 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 µl plus 200 µl) 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 C1q 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: 0.004-0.012 Y-intercept: < 0.750 
 
 Typical Results 
  
 A typical example of a standard curve with the C1q CIC ELISA is listed 
 below. 
  
 OD1 OD2 Mean Mean OD Result 
 OD Blank (µg/ml) 
  
 Reagent Blank 0.060 0.117 0.088 - 0 
 Standard 1 0.827 0.831 0.829 0.741 13 
 Standard 2 1.709 1.790 1.750 1.662 135  
 
 Sample 1 1.081 0.910 0.998 0.910 37.0 
 Sample 2 1.107 1.102 1.105 1.017 51.1 
  
 Slope 0.0075 
 Y-Intercept 0.643 
  
 
 Sensitivity 
  
 The sensitivity of the C1q containing CIC assay is 10 µg/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 µg/ml) 
  
 Sample Average CV (%) 
 1 61.9 7.3 
 2 78.2 11.0  
 3 36.3 11.5 
 
 
 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 µg/ml) 
  
 Sample Average CV (%) 
 1 64.5 6.9 
 2 89.6 7.9  
 3 39.6 4.6 
 
 
 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 C1q 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 50 µg/ml HAG equivalents is considered as positive. A value 
 between 40 - 50 µg/ml can be regarded as equivocal. Values below 40 µg/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

Back to Contents
 
 

(More) Clinical Background

Back to Contents
 
 

Sales Arguments

Back to Contents
 
  
 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 
 
 Sample 50 µl serum 10 µl serum 10 µl serum 100 µl 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*ý  Substrate 
 
 
 *1) 2,2-Azino-di-(3-ethylbenzothiazoline sulfonic acid)-diammonium salt 
 
 *ý) 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 (C1q-CIC will be changed to 
 ready to use 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! 




 
  
 
Return (master kit index)
HOME (index Page)
 
RDI Division of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA 01742-3049 

USA

phone (978) 371-6446

or (800) 370-2222

fax      (978) 371-2266

EMAIL:Wien@researchd.com