INTENDED USE

The Mouse Anti-SCL70 Total Ig ELISA Kit is an immunoassaysuitable for quantifying or titering total antibody activity (IgG, IgAand IgM) specific for scl70 in serum or plasma. Other biologicalfluids, including tissue culture medium, may be validated for use.

GENERAL INFORMATION

Antibodies reactive with autologous nuclear components, suchas DNA and histones, can represent an autoimmune basis forpathological conditions such as systemic lupus erythematosis(SLE) in humans. In mice homozygous for thelymphoproliferation spontaneous mutation (Faslpr), a systemicautoimmunity develops with age which includes elevated levelsof anti-dsDNA and other anti-nuclear antibodies (ANA), includinganti-scl70 (topoisomerase I). Also, mice with the tight skin gene(Tsk1,2/+), a model for connective tissue disease includingsystemic sclerosis, develop elevated levels of anti-scl70antibodies.The expanded use in the drug industry of biological modifiers hasbeen associated with production of autoantibodies, of whichmice, and possibly also other hosts such as humans andmonkeys, are susceptible. A prototype disease in mice is lupuscaused by the drug minocycline, with elevated anti-dsDNAamong other autoantibodies and pathological conditions.Recent investigations have focused on the role of innate immunemechanisms, including Toll-like receptors (TLRs) and TH2immunity, responding to the damage-associated molecularpatterns of dying cells, as underlying cause of autoimmunity;these may be induced by drugs, including vaccines andadjuvants, with aging, or with other health conditions.

PRINCIPLE OF THE TEST

The Mouse Anti-SCL70 Total Ig ELISA kit is based on thebinding of mouse anti- scl70 IgG, IgA and IgM in samples toscl70 immobilized on the microwells, and total anti-scl70antibody is detected by anti-mouse IgG+IgA+IgM (H+L) specificantibody conjugated to HRP (horseradish peroxidase) enzyme.After a washing step, chromogenic substrate (TMB) is added andcolor is developed by the enzymatic reaction of HRP on thesubstrate, which is directly proportional to the amount of antiscl70 Ig present in the sample. Stopping Solution is added toterminate the reaction, and absorbance at 450nm is thenmeasured using an ELISA microwell reader. The activity of totalmouse antibody in samples is calculated relative to mouse antiscl70 calibrators.

Specificity

Purified bovine scl70 is used to coat the microwells; thus theassay is specific for antibodies directed to scl70. The anti-MouseIgG+IgA+IgM (H+L) HRP conjugate reacts with mouse IgG, IgAand IgM class antibodies that bind to scl70 on the plate. IgEantibody would not be measured above background signals.Assay SensitivityThe scl70 coating level, HRP conjugate concentration and LowNSB Sample Diluent are optimized to differentiate anti- scl70 Igfrom background (non-antibody) signal with mouse serumsamples diluted 1:100.Calibrator ValuesThe calibrators are dilutions of mouse antibody reactive to scl70.Values are assigned as arbitrary anti-scl70 activity units (seeLimits of the Assay).

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