DiaMetra CEA Elisa Kit

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

CLINICAL SIGNIFICANCE
Carcinoembryonic antigen (CEA) is a glycoprotein, with a molecular weight of 180 kD, involved in cell adhesion. It is normally produced during fetal development, but the production of CEA stops before birth. Therefore, it is not usually present in the blood of healthy adults, although levels are raised in heavy smokers. CEA was identified in human colon cancer tissue extracts. It was later found that serum from individuals with colorectal and other carcinomas had higher levels of CEA than healthy individuals and can be used to monitor the response to colon cancer treatment.
CEA and related genes make up the CEA family belonging to the immunoglobulin superfamily.
The most frequent cancer which causes an increased CEA is cancer of the colon and rectum. Others include cancers of the pancreas, stomach, breast, lung, and certain types of thyroid and ovarian cancer. Benign conditions which can elevate CEA include smoking, infections, inflammatory bowel disease, pancreatitis, cirrhosis of the liver, and some benign tumors in the same organs in which an elevated CEA indicates cancer. Chemotherapy and radiation therapy can cause a temporary rise in CEA due to the death of tumor cells and release of CEA into the blood stream. Benign tumours does not usually cause an increase above 10 ng/mL.

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.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems, it is recommended to increase the number of washes.
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.

QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of CEA 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. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the calibration curve for run-to-run reproducibility. 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
Note
The optical densities (O.D.s) of some calibrators and samples may be higher than 2.0, in such a case, they could be out of the measurement range of the microplate reader. It is therefore necessary, for O.D.s higher than 2.0, to perform a reading at 405 nm (=wavelength of peak shoulder) in addition to 450 nm (peak wavelength) and 620 (reference filter for the subtraction of interferences due to the plastic).
For microplate readers unable to read the plate at 3 wavelengths at the same time, it is advisable to proceed as follows:
Read the microplate at 450 nm and at 620 nm.
Read again the plate at 405 nm and 620 nm.
Find out the wells whose ODs at 450 nm are higher than 2.0
Select the corresponding ODs read at 405 nm and multiply these values at 405 nm by the conversion factor 3.0 (where OD 450/OD 405 = 3.0), that is: OD 450 nm = OD 405 nm x 3.0.
Warning: The conversion factor 3.0 is suggested only. For better accuracy, the user is advised to calculate the conversion factor specific for his own reader.
The OD of calibrator 5 should be 1.3.

Mean Absorbance
Calculate the mean of the absorbance (Em) for each point of the calibration curve (C0-C5) and of each sample.
Calibration curve
Plot the mean value of absorbance (Em) of the Calibrators (C0-C5) against concentration. Draw the best-fit curve through the plotted points. (es: Four Parameter Logistic).
Calculation of Results
Interpolate the values of the samples on the calibration curve to obtain the corresponding values of the concentrations expressed in ng/mL.