ANA Pro (Antinuclear Antibody) ELISA Assay Kit

$340.00

The Eagle Biosciences ANA pro ELISA Assay Kit is used for the separate semi-quantitative determination of autoantibodies to nuclear and cytoplasmic antigens (dsDNA, RNP, Sm, SS-A, SS-B, Scl-70, CENP, Jo-1) in human serum or plasma for the differential diagnosis of systemic rheumatic inflammatory diseases. The ANA pro ELISA Assay Kit is for research use only and not for diagnostic procedures.

SKU: ANP31-K01 Categories: , ,

ANA Pro (Antinuclear Antibody) ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL

Additional Information

Assay Background

Systemic autoimmune diseases such as systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjögren’s syndrome, dermatomyositis, mixed connective tissue disease are characterized by the appearance of a variety of autoantibodies directed against components of the cell nucleus or plasma.

Although significance and pathological relevance of some auto-antibodies are not completely revealed yet, the detection of auto-antibodies is widely established and plays an important role in the diagnosis of systemic autoimmune diseases (1,2,3).

ANApro allows both the detection of autoantibodies to dsDNA as well as autoantibodies to extractable nuclear and cytoplasmic protein antigens.

ANApro offers a rapid and handsome opportunity for the determination of the whole autoantibody pattern in systemic autoimmune diseases on one test plate. The use of specified recombinant antigens in combination with selected highly purified ones guarantees a maximum of specificity for these parameters.

Assay Principle

ANApro is an enzyme immunoassay for the separate semi-quantitative determination of IgG antibodies to nuclear and cytoplasmic antigens.

The antibodies of the calibrator and diluted patient samples react with nuclear and cytoplasmic antigens immobilized on the solid phase of microtiter plates. Highly purified dsDNA, SS-A and Sm as well as recombinant SS-B, RNP (68 kDa, A, C), Scl-70, Jo-1 and CENP-B guarantees the specific binding of autoimmune antibodies of the specimen under investigation. Following an incubation period of 60 min at room temperature (RT), unbound sample components are removed by a wash step.

The bound IgG antibodies react specifically with anti-human-IgG conjugated to horseradish peroxidase (HRP). Within the incubation period of 30 min at RT, excessive conjugate is separated from the solid-phase immune complexes by the following wash step.

HRP converts the colorless substrate solution of 3,3’,5,5’-tetramethyl-benzidine (TMB) added into a blue product. The enzyme reaction is stopped by dispensing an acidic solution into the wells after 15 min at room temperature turning the solution from blue to yellow.

The optical density (OD) of the solution at 450 nm is directly proportional to the amount of specific antibodies bound. The cut-off is established by multiplying the OD of the calibrator with the respective factor of each antigen of the kit. Patient ratios are calculated by dividing the respective OD of the specimen with the calculated cut-off OD of the respective antigen.

Assay Procedure

  1. Bring all reagents to room temperature (18-25°C) before use. Mix gently without causing foam.
  2. Dispense 100 µl calibrator (Ca)100 µl negative control (N) 100 µl diluted patient samples into the respective wells.
  3. Cover plate, incubate 60 min at room temperature (18-25°C).
  4. Decant, then wash each well three times using 300 µl wash solution (made of B).
  5. Add 100 µl of conjugate (D) solution to each well.
  6. Cover plate, incubate 30 min at room temperature (18-25°C).
  7. Decant, then wash each well three times using 300 µl wash solution (made of B).
  8. Add 100 µl of substrate (E) to each well.
  9. Incubate 15 min protected from light at room temperature (18-25°C).
  10. Add 100 µl of stop solution (F) to each well and mix gently.
  11. Read the OD at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.

Manual

Product Manual


Publications

Citations

  • von Mühlen CA, Tan EM: Autoantibodies in the diagnostic of systemic rheumatic diseases. Semin Arthritis Rheum 1995 24:323-358
  • Smeenk RJT: Antinuclear antibodies: cause of disease or caused by disease? Rheumatol 2000 39:581-584