Enzyme immunoassay (microtiter strips) for the quantitative determination of

Calcitonin in human serum and plasma

‘’For in-vitro diagnostic use only’’

Cat.-No. : RE 530 01

Size : 12 x 8

Storage : 2 - 8 °C

In USA imported and distributed by:

KMI Diagnostics, Inc.

818 51st Ave. NE, Minneapolis, MN 55 421-1746

Tel.: 1 (612) 572-9354 Fax: 1 (612) 586-0748

Email: postmaster@kmidiagnostics.com www.kmidiagnostics.com

Page

8. Method Comparison 15
1. Comparison of different specimen 16

The IBL Calcitonin Enzyme Immunoassay Kit provides materials for the quantitative determination of Calcitonin in serum and plasma. This assay is intended for in vitro diagnostic use only.

Calcitonin is synthesized primarily in the parafollicular C-cells of the thyroid gland as a prepro-hormone (MW = 17kDa). After cleavage of a short N-terminal signal peptide and the resultant N-terminal flanking peptide, the calcitonin molecule is packaged in secretory granules. At some point during the intracellular processing and/or shortly after secretion, the C-terminal flanking peptide, katacalcin, is cleaved. The mature calcitonin molecule (32 amino acids, MW 3.4 kDa in humans) is co-secreted in equimolar amounts with katacalcin (21 amino acids). Alternate splicing of the parent calcitonin gene results in production of the calcitonin gene related product (CGRP). CGRP is normally produced in lower quantities than calcitonin and secretory patterns of the two peptides are usually parallel. Other structurally-related proteins, such as amylin secreted by pancreatic islet cells and the apparently non-expressed human b -calcitonin gene, appear to represent examples of gene duplication.

The major physiological action of calcitonin is to lower plasma calcium concentrations. Relatively small increments in extracellular calcium concentrations have been demonstrated to stimulate calcitonin secretion. Calcitonin apparently acts by inhibiting osteoclast activity, resulting in decreased mobilization of calcium from bone. This effect is most evident in conditions associated with high levels of bone remodelling, e.g. Paget’s disease, following exogenous administration of calcitonin. Exogenously administered calcitonin has also been shown to reduce pain associated with Paget’s disease and malignancy, apparently by a direct effect on the central nervous system. Several other tissues may respond to calcitonin administration. However, the relative physiologic role of endogenous calcitonin in calcium homeostasis is not completely known. Calcitonin secretion is also regulated by gastrointestinal peptides, estrogens and vitamin D. Gastric peptide stimulation of calcitonin secretion following a meal may play a role in maintaining postprandial calcium homeostasis.

Serum calcitonin levels are relatively high in infants and may be higher in infants with very low weight at birth. Levels decline rapidly and are relatively stable from childhood to adult life. Calcitonin may also appear in the cerebrospinal fluid.

Abnormally elevated levels of calcitonin are characteristic of thyroid C-cell hyperplasia and medullary thyroid carcinoma. These related conditions may occur as isolated, idiopathic cases or as part of the familial conditions, multiple endocrine neoplasias type IIa and IIb. Early in the course of these conditions, basal calcitonin levels may be normal. However an abnormally high elevated serum calcitonin level following parenteral administration of calcium or pentagastrin is characteristic for C-cell disease, particularly if there is a positive family history and/or typical phenotypic characteristics of MEN IIb. Early diagnosis and treatment is thought to be essential in improving prognosis of medullary thyroid carcinoma. Elevated calcitonin levels may occur in a variety of other malignancies, possibly due to ectopic calcitonin production.

This assay is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. The wells of the microtiter strips are coated with donkey anti goat antibodies. The goat anti calcitonin antibodies are immobilized by binding to the donkey anti goat IgG coated wells

The antigen present in the standards or sample binds in a first incubation step to specific antibodies fixed on the wells. After a washing step and adding the biotinylated anti calcitonin antibodies a sandwich complex, fixed antibodies-antigen-labelled antibodies, is formed. The unbound biotinylated antibodies are washed off. Alkaline phosphatase (AP) labelled anti biotin antibodies bind in an additional incubation step to the biotinylated anti calcitonin antibodies. After washing off the unbound AP labelled antibodies an amplification system is used for detection.

In this amplification system AP converts NADPH into NADH, which is an essential cofactor for the subsequent cycling step. Two enzymes catalyse the redox cycle in which NADH and NAD are interconverted. For each turn a coloured formazan is generated. The principle is shown below:

The intensity of colour developed is proportional to the antigen concentration in the sample. The measured ODs of the standards are used to construct a calibration curve against which the unknown samples are calculated.

Store all reagents at 2 - 8 °C and use before expiry date. When stored at 2 - 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

Once the foilbag of the coated microtiter strips has been broken, care should be taken to close it tightly again.

Allow all reagents and required number of strips to reach room temperature prior to use.

Do components contain < 250 µl solution, please care that all the solution is on the bottom of the vial.

5.1. Microtiter Strips 12 x 8 wells

break apart strips with anti calcitonin antiserum

(goat, polyclonal) attached to immobilized anti goat antiserum

(donkey, polyclonal) in foilbag with desiccant.

5.2. Standard A (Zero Standard) 1 vial

0.5 ml, lyophilized

human serum

and sodium azide (< 0.09 %) as preservative.

Reconstitute the standard with 0.5 ml distilled water prior to use.

5.3. Standards (B - G) 6 vials

0.25 ml each, lyophilized

containing the below mentioned

concentrations of calcitonin in human serum

and sodium azide (< 0.09 %) as preservative.

Reconstitute each standard with 0.25 ml distilled water prior to use.

The standards are calibrated against the WHO calcitonin International Standard (Second internal standard [2nd IS] 89/620) The concentration ranges of intact calcitonin are declared on the vial labels.

5.4. Control 1 vial

25. ml, lyophilized

human serum containing calcitonin

and sodium azide (< 0.09 %) as preservative.

The concentration is stated on the attached QC certificate

Reconstitute with 0.25 ml of distilled water prior to use.

5.5. Anti hCT Antibodies, concentrate (101x) 1 vial

150 µl, concentrate

containing anti hCT antibodies (mouse, monoclonal)

conjugated to biotin in tris buffer and sodium

azide (< 0.09 %) as preservative.

5.6. Enzyme Conjugate, concentrate (101x) 1 vial

150 µl, concentrate

containing anti biotin antibodies (mouse, monoclonal) conjugated

to the enzyme alkaline phosphatase supplemented

by enzyme stabilisers and sodium azide (< 0.09 %) as preservative.

5.7. Reagent 1 1 vial

4.5 ml, ready to use

containing alkoholdehydrogenase and diaphorase

in buffer supplemented by enzyme stabilisers.

5.8. Reagent 2 1 vial

4.5 ml, ready to use

containing iodonitrotetrazolium and ethanol

in buffer with stabilisers.

5.9. Reagent 3 1 vial

4.5 ml, ready to use

containing nictotinamide adenine dinucleotide phosphate

reduced form (NADPH) in buffer with stabilisers.

5.10. Stop Solution 1 vial

5 ml, ready to use

1 M phosphoric acid (H₃PO₄)

Avoid contact with stop solution it may cause

skin irritations and burns.

5.11. Wash Buffer, concentrate (10x) 1 vial

50 ml, concentrate

containing buffer with sodium azide (< 0.09 %) as preservative.

Dilute 1 to 10 with distilled water prior to use

5.12. Assaypuffer 1 vial

34 ml, ready to use

containing buffer with sodium azide (< 0.09 %) as preservative.

5.13. Sealing Tape 3 pieces

• Pipettes to dispense 20, 100 and 1000 µl (a multichannel pipetting device such as Titertek is suitable for adding reagents to the wells), 8-channel pipette
• Distilled water
• Microtiter plate spectrophotometer (ELISA reader) with 490 nm filter
• Microtiter plate shaker

6. Specimen Collection and Storage

7.1. Standards and control

Reconstitute standards and control with 0.25 ml (zero standard 0.5 ml) of distilled water allow to stand for 15 minutes. After reconstitution the standards and control should be stored frozen at - 20 °C.

Thawing and freezing of standards and control is possible for 3 times.

Dilute the required amount of anti hCT antibodies with assay buffer 1 to 101 (Example for 1/3 of the plate: 40 µl anti hCT antibodies + 4 ml assay buffer).

7.3. Enzyme Conjugate (Example for 1/3 of the plate)

Dilute the required amount of enzyme conjugate with assay buffer 1 to 101 (Example for 1/3 of the plate: 40 µl enzyme conjugate + 4 ml assay buffer). Prepare the diluted enzyme conjugate freshly.

4. Substrat Solution

Mix the solutions of reagent 1, reagent 2 and reagent 3 in the ratio 1 + 1 + 1 (Example for 1/3 of the plate: 1.5 ml of each) to obtain the substrate solution. Prepare the required amount only immediately before use.

7.5. Wash Buffer

Dilute the wash buffer concentrate with distilled water 1 to 10 (e.g. 10 ml concentrate + 90 ml distilled water). Ready to use wash buffer is stable for 6 weeks when stored at 2 - 8 °C.

All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.

Once the test has been started, all steps should be completed without interruption.

Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination.

Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents be ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.

1. Leave sufficient microtiter strips in the strip holder to enable the running of standards, controls, and samples in duplicate. Secure the desired number of microtiter strips in the holder.
2. Pipet 100 µl of assay buffer into the appropriate wells of the strips.

4. Incubate for 3 - 4 hours at room temperature (18 - 24 °C) on a microtiter plate shaker (500 rpm).
5. Washing: discard the incubation solution, rinse the wells 4x with 250 µl wash buffer (dilute concentrate 1 to 10 with distilled water) and remove any residual.
1. Add 100 µl of anti hCT biotin to each well in sequence, cover plate with sealing tape.
7. Incubate overnight (10 - 16 hours) at 2 - 8 °C.

12. Promptly pipet 100 µl of the substrate solution into the rinsed wells

without air bubbles.

Any ELISA reader capable of determining the absorbance at 490 ± 10 nm may be used. The antigen concentration of each sample is obtained as follows:

Using linear-linear or semi log graph paper, construct an standard curve by plotting the average absorbance (Y) of each reference standard against its corresponding concentration (X) in pg/ml.

Use the average absorbance of each sample to determine the corresponding antigen value by simple interpolation from this standard curve, multiplying by the initial sample dilution, if necessary.

Alternatively the use of electronic device is possible. The results can also be calculated with normal programs for automatic data processing, i.e.
4 parameter, spline, logit-log.

Any sample reading greater than the highest standard should be diluted appropriately with the zero standard and reassayed.

Do not use this calibration curve. In the laboratory the standard curve should be established in each assay run.

The specificity of the calcitonin ELISA was assessed according to Abraham's method. The percentage indicate crossreactivity at 50 % displacement compared to hCT.

The below mentioned compounds do not show any crossreactivity up to the stated concentrations.

Within run variation was determined by replicate determination of 4 different control sera in one assay. The within assay variability is shown below:

Between run variation was determined by replicate measurements of 4 different control sera in several different assay. The between assay variability is shown below:

The IBL Calcitonin ELISA was compared to another commercially available Calcitonin IRMA. Serum samples of 45 obviously healthy blood donors and 15 patients with MTC were analysed according in both test systems. A correlation coefficient of r = 0.99 was found between the two test systems. The linear regression curve was calculated from y = mx + b where m is the slope and b the y - intercept. The resulting equation is:

y = 1.03 IBL-ELISA +3.2

19 serum were compared to their plasma samples using the IBL Calcitonin ELISA. A correlation coefficient of r = 0.99 was found between the two kinds of samples. The linear regression curve was calculated from y = mx + b where m is the slope and b the y - intercept. The resulting equation is:

Plasma = 0.94 Serum - 0.67

Reliable and reproducible results will be obtained when the assay procedure is carried out exactly with a complete understanding of the package insert instructions and with adherence to good laboratory practice.

The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbances.

The amplification system works with three enzymes, which leads to high sensitivity to temperature differences. Temperature has to be taken in consideration when substrate reaction has to be stopped. The results of this effect are demonstrated in the schedule below.

This test has been developed at the laboratories of IBL Hamburg. Any modification of this test as well as exchange or mixture of any components from different lots might influence the results. In such cases there is no claim for a replacement.

8. Motte P et al. J. of Immunology, 10: 3332 (1987).