Description

The ¹²⁵I-hCEA IRMA system provides a direct in vitro quantitative determination of human carcinoembryonic antigen (hCEA) in human serum in the range of 0-180 ng/ml. Each kit contains materials sufficient for 100 assay tubes permitting the construction of one standard curve and the assay of 41 unknowns in duplicate.

Introduction

Carcinoembryonic antigen (CEA) is a cell-surface glycoprotein with a molecular weight of 180-200 kD, that occurs in high levels in colon epithelial cells during embryonic development. Levels of CEA are significantly lower in colon tissue of adults, but can become elevated when inflammation or tumors arise in any endodermal tissue, including in the gastrointestinal tract, respiratory tract, pancreas and breast.

An overexpression of CEA protein has been detected in a variety of adenocarcinomas, including gastric, pancreatic, small intestine, colon, rectal, ovarian, breast, cervical and non-small-cell lung cancers. Carcinoembryonic antigen is also expressed by epithelial cells in several non-malignant disorders, including diverticulitis, pancreatitis, inflammatory bowel disease, cirrhosis, hepatitis, bronchitis and renal failure and also in heavy smokers.

Therefore CEA should not be regarded as a tumor-specific marker for the screening of general population for undetected cancers. However, the determination of carcinoembryonic antigen levels provides important information about patient prognosis, recurrence of tumours after surgical removal and effectiveness of therapy.

Principle of method

The technology uses two high affinity monoclonal antibodies in an immunoradiometric assay (IRMA) system.

The ¹²⁵I labelled signal-antibody binds to an epitope of the CEA molecule spatially different from that recognised by the biotin-capture-antibody. The two antibodies react simultaneously with the antigen present in standards or samples, which leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a “sandwich”.

During a 2-hour incubation period with shaking the immuno-complex is immobilized to the reactive surface of streptavidin coated test tubes. Reaction mixture is then discarded, test tubes are washed exhaustively, and the radioactivity is measured in a gamma counter.

The concentration of antigen is directly proportional to the radioactivity measured in test tubes. By constructing a calibration curve plotting binding values against a series of calibrators containing known amount of hCEA, the unknown concentration of hCEA in patient samples can be determined

Contents of the kit

Materials, tools and equipment required

Distilled water
Test tube rack
Precision pipettes with disposable tips (50, 200 and 2000 µl)
Vortex mixer
Shaker
Gamma counter
Plastic foil
Absorbent tissue

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf or other)
Dispenser with 1-L reservoir (instead of the 2 ml pipette)

Specimen collection and storage

Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Samples can be stored at 2-8 °C if the assay is carried out within 24 hours, otherwise aliquots should be prepared and stored deep frozen (-20 °C). Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens. Samples with a hCEA concentration higher than that of the most concentrated standard should be diluted and reassayed.

Preparation of reagents, storage

Store the reagents between 2-8 °C after opening. At this temperature each reagent is stable until expiry date. The actual expiry date is given on the package label and in the quality certificate.

Add the wash buffer concentrate (20 ml) to 700 ml distilled water to obtain 720 ml wash solution. Upon dilution store at 2-8 °C until expiry date.

Add 500 µl distilled water to the lyophilised control serum. Mix gently with shaking or vortexing (foaming should be avoided).

Ensure that complete dissolution is achieved, and allow the solution to equilibrate at room temperature for at least 20 minutes. Store at 2-8 °C until expiry date.

CAUTION! Equilibrate all reagents and serum samples to room temperature. Mix all reagents and samples thoroughly before use. Avoid excessive foaming.

Assay procedure

(For a quick guide, refer to Table 1.)

Table 1. Assay Protocol, Pipetting Guide (all volumes in microliters)

Table 2. Typical Assay Data

Figure 1
A typical standard curve
(Do not use to calculate unknown samples)

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 2. Calculate the average CPM for each pair of assay tubes.

Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

Using semi-logarithmic graph paper plot B/T (%) for each standard versus the corresponding concentration of CEA.

Determine the CEA concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range.

Out of fitting programs applied for computerized data processing logit-log, or spline fittings can be used.

Automated data processing systems are also available.

Characterization of the assay

Typical assay parameters

Specificity

No cross reactivity with NCA can be detected in normal physiological levels.

Sensitivity

The analytical sensitivity or minimum detectable limit is calculated by the interpolation of the mean counts of zero standard plus 2 standard deviation from the standard curve. Determination was carried out using 15 replicates of zero standard response.

The value of analytical sensitivity is 0,05 ng/ml measured using 4-week old tracer.

The functional sensitivity is a measure of the carcinoembryonic antigen concentration that is significantly different from zero as determined by the inter-assay precision profile (22% CV).

The value of functional sensitivity is: < 0.4 ng/ml.

Precision

5 control pool samples were assayed in 10 replicates to determine intra-assay precision. Values obtained are shown below.

Table 3

Reproducibility

To determine inter-assay precision 5 control pool samples were measured in duplicates in 15 independent assays by 3 operators using different kit batches. Values obtained are shown below.

Table 4

Linearity – dilution test

Individual human serum samples were diluted with the sample diluent of the kit. The diluted samples were measured according to kit protocol.

Table 5.

Expected Values

In 95% of healthy subjects, CEA levels are usually < 3.0 ng/ml.
For individuals who smoke normal CEA levels are usually < 5.0 ng/ml.

It is recommended that each laboratory determine a reference range for its own patient population.

The results obtained should only be interpreted in the context of the overall clinical picture. None of the in vitro diagnostic kits can be used as the one and only proof of any disease or disorder.

Recovery

Recovery was defined as the measured increase expressed as percent of expected increase upon spiking serum samples with known amount of carcinoembryonic antigen. The average per cent recover for 5 serum pooles spiked with hCEA at 3 levels was: 104.3 ± 5.01%, with a range of 95% to 115%.

Additional information

Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precaution

Radioactivity

This product contains radioactive material. It is the responsibility of the user to ensure that local regulations or code of practice related to the handling of radioactive materials are satisfied.

Biohazard

Human blood products used in the kit have been obtained from healthy human donors. They were tested individually by using approved methods (EIA, enzyme immunoassay), and were found to be negative, for the presence of both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B surface Antigen (HBsAg).

Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV-1), or other infectious agents are absent. Human blood samples should therefore be handled as potentially infectious materials.

Chemical hazard

Components contain sodium azide as an antimicrobial agent. Dispose of waste by flushing with copious amount of water to avoid build-up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 74 mg.

	Use by		In vitro diagnostic medical device		Control
	Batch code		Manufacturer		Standard
	Caution, consult accompanying documents		Radioactive material		Coated tube
	Biological risk		Temperature limitation Store between 2-8 °C		Tracer
	Consult instructions for use		Catalogue number		Wash buffer
					Serum diluent