Intended Use
The Cell Replacement Enzyme Immunoassay measures immune complexes containing C3 activation fragments in human serum and plasma.
Contents of Kit
The CIC-Raji Cell Replacement EIA kit contains the following:
1. Standards A-E: 1 each, 2 mL
Each contains a known quantity of serum-treated heat-aggregated human gamma globulin (HAGG) in PBS, 2.5% stabilizers, 0.01% thimerosal.
2. Low Control: 3 each
(Lyophilized) Contains low levels of HAGG, 20 mM EDTA, 0.01% thimerosal
3. High Control: 3 each
(Lyophilized) Contains high levels of HAGG, 20 mM EDTA, 0.01% thimerosa
4. Microassay Plate: 12 each
96-well with retainer and holder consisting of 12 eight-well strips coated with mouse anti-human C3 fragments in a resealable foil pouch
5. Stop Solution: 6 mL
Contains 250 mM oxalic acid
6. 20× Wash Solution Concentrate: 2 each, 50 mL
Contains phosphate buffered saline (PBS), 1.0% Tween-20 and 0.035% Proclin 300
7. Complement Specimen Diluent: 50 mL
Contains PBS, 2.5% stabilizers, 0.035% ProClin 300
8. Substrate Diluent: 25 mL
Contains 0.1M citrate buffer and 0.05% H2O2
9. Substrate Concentrate: 1.5 mL
Contains 0.7% 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), diammonium salt
10. CIC-Raji Conjugate:2 each, 3 mL
Contains peroxidase-conjugated (mouse) anti-human IgG suspended in an HRP stabilizing buffer with preservative
11. CIC-Raji Confirmation Diluent: 12 mL
Contains PBS, 2.5% Stabilizers, anti-human C3 fragment antibody. 0.35% ProClin 300
Storage
Store unopened kit at 2°C to 8°C. After the kit is opened, the 20× Wash Solution Concentrate may be stored at 2°C to 30°C.
After selecting the reagents or materials to be used in the assay, return the unused reagents immediately to their appropriate storage temperatures. Bring reagents and materials to room temperature (15°C to 30°C) before use.
INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS
The Substrate Concentrate may range in color from colorless to pale or dark green. This condition will not influence performance. However, the freshly prepared Substrate Solution should be colorless to pale green. A dark green color indicates that the prepared Substrate Solution has deteriorated, must be discarded, and new Substrate Solution prepared in clean glassware.
Cloudiness or discoloration of the diluted Wash Solution indicates a deterioration of this reagent. If this occurs, the solution should be discarded.
Performance Characteristics
Accuracy
A World Health Organization (WHO) Immune Complex Standard, i.e., tetanus toxoid-anti-tetanus toxoid complexes pre-incubated in fresh normal human serum, was used to standardize the assay. To test the accuracy of the assay, five dilutions of the WHO Standard were tested in triplicate in nine runs in the CD kit.
The assayed concentrations showed a correlation of 0.99 with the known values.
Sensitivity
The CIC-Raji Cell Replacement EIA measures at least 4 μg Eq/mL or greater of CIC analyte based on comparison to the WHO Standard.
General Description
The importance of circulating immune complexes (CIC) and their relationship to various diseases has been the subject of investigation for a number of years. Formation of immune complexes is a protective, on-going, and usually benign process of a normally functioning immune system. CIC are removed from the circulation in the normal host by a number of complex biochemical, enzymatic, and cellular processes. The effective clearance of many CIC requires the activation of complement. Complement activation will result in C3 fragment deposition within the immune complex followed by enhanced elimination by phagocytic cells of the reticuloendothelial system. Under certain disease conditions, which are not fully understood, immune complexes may not be efficiently eliminated from the body. In these diseases, the immune complexes may accumulate and initiate complement-dependent injury in various organs and tissues. This activation of complement may begin a series of potentially destructive events in the host including anaphylatoxin production, cell lysis, leukocyte stimulation, and activation of macrophages and other cells. When immune complexes become fixed to vessel walls or cell membranes, destruction of normal tissue can occur, as in some cases of glomerulonephritis.
Certain properties of CIC influence their potential pathogenicity. Of particular importance are: (1) nature, size and concentration of the antigen; (2) nature, size and concentration of the antibody; (3) rate of formation and clearance of the immune complexes.
Circulating immune complexes have been measured in a variety of conditions: for example, infections, autoimmune disorders, trauma, and neoplastic proliferative diseases. Current studies suggest that CIC determinations can be important in the evaluation of certain diseases and, sometimes, in monitoring efficiency of therapy. This is especially true in systemic lupus erythematosus (SLE) and some forms of rheumatoid arthritis (RA). The first disease state linked to the formation of immune complexes was serum sickness, described in the early 1900' s by von Pirquet. Since that time elevated levels of CIC have been described in autoimmune diseases (SLE, SLE-related syndrome, RA), glomerulonephritis, neoplastic disease (Hodgkin' s, leukemia), bacterial infections (subacute bacterial endocarditis, leprosy), parasitic infections (malaria, schistosomiasis) and viral infections (hepatitis, mononucleosis).
Over 40 assay techniques to detect or quantitate CIC have been described. Such tests as the Raji Cell assay, C1q deviation test, conglutinin test, fluid phase C1q binding procedures, rheumatoid factor assay, PEG precipitin test, and solid phase C1q assays have been described. Since the size and physiochemical properties of CIC vary markedly, none of these assays has been accepted as a standard. A collaborative study sponsored by the World Health Organization in 1978 determined that no single method was appropriate in all suspected disease states and recommended that at least two different assay techniques be performed to detect and measure CIC adequately.
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