Anti-Factor H ELISA Assay Kit

$460.00

The Eagle Biosciences Anti-Factor H ELISA Assay Kit is used for the quantitative determination of IgG antibodies to complement factor H in human serum for the diagnosis of atypical haemolytic-uraemic syndrome (HUS).The Anti-Factor H ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Anti-Factor H ELISA Assay Kit

For Research Use Only

Size: 1×48 wells
Sensitivity: 1.0 U/m
Dynamic Range: 10 – 300 U/mL
Incubation Time: 2 hours
Sample Type: Serum
Sample Size: 10 µL

Additional Information

Assay Background


Hemolytic-uremic syndrome (HUS) is a disease of small blood vessels, characterized by hemolytic anemia, thrombocytopenia and acute renal failure. Most common cause is an infection with toxin forming Escherichia coli bacteria (Shiga toxin, Vero toxin). First symptom mainly is watery, sometimes bloody diarrhea, later extra intestinal manifestations are possible. Beside acute renal insufficiency neurological and cardiac complications may occur. Up to 10% of critical cases are lethal.

About 5% of HUS patients do not show diarrheal symptoms or other symptoms of E. coli infection. This so-called atypical HUS is based on a disorder of complement regulation, caused by genetic mutations or antibodies to complement factor H.

Assay Principle


The Eagle Biosciences Anti- Factor H ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of IgG antibodies to complement factor H. The antibodies of the calibrators, controls, and diluted patient samples react with human recombinant complement factor H immobilized on the solid phase of microtiter plates. Following an incubation period of 60 min at room temperature (RT), unbound serum 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 RT 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 standard curve is established by plotting the antibody concentrations of the calibrators (x-axis) and their corresponding OD values (y-axis) measured. The concentration of antibodies of the specimen is directly read off the standard curve.

Assay Procedure


  1. Bring all reagents to room temperature (18-25°C) before use. Mix gently without causing foam.
  2. Dispense 100 μl calibrators 0 – 4 100 μl controls P, N (N optionally) 100 μl diluted patient samples into the respective wells.
  3. 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) to each well.
  6. 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.

Typical Standard Curve


Manual

Product Manual


Publications

References


  • Dragon-Durey MA, Loirat C, Cloarec S, Macher MA, Blouin J, Nivet H, Weiss L, Fridman WH, Fremeaux-Bacchi V: Anti-Factor H Autoantibodies associated with Atypical Hemolytic Uremic Syndrome. J Am Soc Nephrol 16: 555-563, 2005
  • Dragon-Durey MA, Sidharth KS, Bagga A, Blanc C, Blouin J, Ranchin B, Andre JL, Takagi N, Cheong HI, Hari P, Le Quintrec M, Niaudet P, Loirat C, Fridman WH, Fremeaux-Bacchi V: Clinical Features of Anti-Factor H Autoantibody-Associated Hemolytic Uremic Syndrome. J Am Soc Nephrol 21: 2180-2187, 2010
  • Loirat C, Fakhouri F, Ariceta G, Besbas N, Bitzan M, Bjerre A, Coppo R, Emma F, Johnson S, Karpman D, Landau D, Langman CB, Lapeyraque AL, Licht C, Nester C, Pecoraro C, Riedl M7, van de Kar NC, Van de Walle J, Vivarelli M, Frémeaux-Bacchi V; for HUS International. An international consensus approach to the management of atypical hemolytic uremic syndrome in children. Pediatr Nephrol 31(1): 15-39, 2016