DiaMetra CH50 Elisa Kit

CLINICAL SIGNIFICANCE 
The primary utility of the CH50 in the practice of an allergist-immunologist is to screen for complementdeficiency associated immunodeficiency (primarily classic or terminal complement component deficiencies). Absent or significantly reduced individual complement components may result in infections, Neisserial meningitis, or sepsis.
A reduced CH50 in this situation warrants quantification and functional assays of individual complement components.
Reduction of the CH50 occurs when individual complement component(s) are deficient or consumed.

PRINCIPLE
The complex β-galactosidase/anti- β -galactosidase, is solubilized by serum through the deposition of C3b molecules. The formation of C3b quantity necessary for the solubilization is mediated by alternative pathway, but it is accelerated from activity of C3- convertasi by classic way.
The quantity of complex β-galactosidase/Anti βgalactosidase dissociated from the antibody, detectable by enzymatic activity in the supernatant at the end of the reaction, is a measure of serum capacity to form C3b molecules.
The o-nitrophenilgalactopiranoside (o-NPG) is used as substrate and the measure of reagent product (onitrophenol) is read at 420nm (or 405 nm).

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated. The reagents are stable until the expiry date when stored and handled as indicated. • Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately tested.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate.
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of the Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence to eliminate any time deviation during the reaction.
Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera.
Maximum precision is required for reconstitution and dispensation of reagents.
Samples microbiologically contaminated, highly lipemeic or haemolysed should not be used in the assay.

QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of CH50 for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations.

RESULTS
The result can be expressed as: 
a. CH50 value or
b. CH50% 
The CH50 Value of Reference Calibrator is Lotdependent and is reported on the CoA.

Determine the results using the following formula:
a. OD (sample) / OD (Reference Calibrator) x CH50 (value of Reference Calibrator) = CH50 Value of sample b. OD (sample) / OD (Reference Calibrator) x CH50% (% of Reference Calibrator) = CH50% of sample

Example:
CH50 Value of Reference Calibrator = 100 
CH50% of Reference Calibrator = 50 
Absorbance of Reference Calibrator = 0.350 
Absorbance of Sample = 1.108
a. CH50 Value of Sample = 1.108/0.350 × 100 = 316
b. CH50% of sample = 1.108/0.350 × 50 = 158%