DiaMetra LH Elisa Kit

CLINICAL SIGNIFICANCE
Luteinising hormone (LH) is a glycoprotein consisting of two subunits with a molecular mass of 30,000 Daltons. The αsubunit is similar to other pituitary hormones [follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (HCG)] while the βsubunit is unique. The β-subunit confers the biological activity to the molecule. The α-subunit consists of 89 amino acid residues while the β-subunit contains 129 amino acids. The carbohydrate content is between 15% and 30%. The clinical usefulness of the measurement of luteinising hormone (LH) in ascertaining the homeostasis of fertility regulation via the hypothalamic - pituitary - gonadal axis has been well established1,2. In addition, the advent of in vitro fertilisation (IVF) technology to overcome infertility associated problems has provided the impetus for rapid improvement in LH assay methodology from the technically demanding bioassay3 to the procedurally simple and rapid immunoenzymometric assays.

PRINCIPLE OF THE METHOD
In this method, the LH calibrators, the patient specimens and/or the controls (containing the native antigen) are first added to streptavidin coated wells. Then monoclonal biotinylated and enzyme labelled antibodies are added and the reactants mixed: these antibodies have high affinity and specificity and are directed against distinct and different epitopes of LH. A reaction between the various LH antibodies and native LH occurs in the microwells without competition or steric hindrance forming a soluble sandwich complex.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit control provided in the kit should be tested as unknown and is intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of each control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate.
DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit, including Calibrator 0 is required. Other curve fitting algorithms are not recommended. Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve.
Read the mean absorbance value of each unknown sample off the curve. In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark.
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label.
Once opened, the calibrators are stable at 2 – 8°C for 6 months.
The diluted wash solution is stable for 30 days at 2 – 8°C.
Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.