DBC Beta2-Microglobulin Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabelled antigen (present in standards, controls and patient samples) and an enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed is inversely proportional to the concentration of β2-M in the sample. A set of standards is used to plot a standard curve from which the amount of β2-M in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
β2-Microglobulin (β2-M) is a single polypeptide chain containing 100 amino acids and is found on the surface of nucleated cells. β2 -M is continuously secreted into the circulatory system and therefore maintains a balanced serum level. Clinical Trends: 
• Decreases in the glomerular fi ltration rate gives rise to increases in the level of β2-M. 
• β2 -M is a helpful marker in the diagnosis of kidney disease and active rheumatoid arthritis. 
• Patients with acquired immune defi ciency syndrome (AIDS) show an increased level of β2-M. 
β2-M has a low concentration in serum. We have found in a normal unselected population that in serum the highest level for β2 -M is 3.8 mg/L. The total number of serum samples tested was 92, showing little differences in the normal level in males, pre- and postmenopausal females. The average for male samples = 1.582 mg/L, for premenopausal female = 1.457 mg/L, for postmenopausal female = 1.608 mg/L and fi nally for young people = 1.13 mg/L.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Draw a calibrator curve on semi-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the unknowns directly off the calibrator curve. 
5. If a sample reads more than 10 mg/L then dilute it with calibrator A at a dilution of no more than 1:20. The result obtained should be multiplied by the dilution factor.