What is Enzymes Kits

Enzymatic Kits:

Reagent kits (for the quantitative determination of serum 25-OH vitamin D, Alkaline Phosphatase and calcium) will be purchased from market.

Subjects:

These will be divided into three groups with fifty subjects in each group. First group will contain 50 healthy volunteer subjects (as control) with no current disease symptoms and medical history of arthritis or joints pain. Second group will contain 50 patients with clinically diagnosed and established Rheumatoid arthritis while third group will contain 50 patients with confirmed Gout.

Experimental design:

150 subjects will be divided into three groups.

  Sr.#          Groups         No. of subjects              Disease condition

1                   A                       50              Healthy (Controls)

2                   B                       50              Rheumatoid arthritis

3                   C                       50              Gout

Estimation of 25-OH vitamin D:

Principle:

The IDS 25-hydroxy vitamin D kit is an enzyme immunoassay for the quantitation of 25-OH D andotherhydroxylated metabolites in serum or plasma. Calibrators, controls and samples are diluted with Biotin labeled 25-OH D. the diluted samples are incubated in microtitre wells which are coated with a highly specipic sheep 25-OH D antibodies for 2 hours at room temperature before aspiration and washing. Enzyme (horseradish peroxidase) labeled avidin, is added and binds selectively to complexed biotin and, following a further wash step, colour is developed using a chromogenic substrate (TMB).The absorbance of stopped reaction mixtures are read in a microtitre plate reader, colour intensity developed being inversely proportional to the concentration of 25-OH D. (Hyponen et al 2007)

Estimation of Alkaline Phosphatase:

Principle:

Alkaline Phosphatase (ALP) hydrolyzes p-nitrophenyl phosphate into p-nitrophenol and phosphate. The rate at which p-nitrophenol is formed is directly proportional to alkaline phosphatase activity. (tietz et al 1983 )

        p-nitophenyl phosphate + H2O                     ALP            phosphate + p-nitrophenol

Estimation of serum calcium

Principle:

Breuer & Breuer diagnostic kit (Germany) to measure serum calcium will be used. Cresolphthalein complex one reacts with calcium ion in alkaline medium forming a red violet colour. Interference by magnesium is eliminated by addition of 8-hydroxyquinoline. Absorbance is directly proportional to the concentration of calcium in serum. (Baginski et al 1973)