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Food & Feed Analysis

With the exclusive distribution in Belgium of the product lines of :

R - Biopharm AG www.r-biopharm.de

Also Fapas www.fapas.com

And DSM - Rotronic

AFP Coated Tubes

Immunoradiometric quantitative measurement of Alpha-Fetoprotein (AFP) in serum and plasma.

BL-26-CT

for in vitro diagnostic use only

1.Summary and background of the test:

Α-Fetoprotein (AFP) is a 70.000 MW oncofetal protein synthesized by liverparenchymal cells, yolk sac and gastrointestinal tract of human fetus. The peak of AFP concentration occurs between weeks 12 and 15 of gestation. After birth AFP concentration in plasma rapidly decrease to less than 5 IU/ml.

2.Principle of the test :

The Bio-Line AFP-Irma is a two-step immunoradiometric assay based on coated-tube separation. Mabs1, the capture antibodies, are attached to the lower and inner surface of the plastic tube. Standards or samples added to the tubes. After incubation, washing remove the occasional excess of antigen. Mab2, the ¹²⁵I-labelled-antibody is added. After incubatin and washing, the remaining radioactivity bound to the tube reflects the antigen concentration. Because of the occasional extremely high concentration of AFP in serum (>1 mg/ml) in case of cancer, the present Bio-Line AFP-Irma has been developed on a two steps procedure to avoid high dose hook effects.

3.Precautions:

1. Radioactive material: Radioactive material may be received, acquired, possessed and used only by physicians, clinical laboratories, or hospitals for "In-Vitro" clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals.

Compliance with these basic rules of radiation safety should provide adequate protection:

1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive material is used.

2. Do not pipet by mouth reagents containing radioactive materials.

3. Wear protective clothing; i.e., lab coats and disposable gloves, in order to avoid direct contact with radioactive reagents.

4. Work with radioactive materials should be performed in a designed area.

5. Radioactive materials should be stored in an acceptable location.

6. A log should be kept for receipt and disposal of radioactive materials.

7. Radioactive spills or accidents should be taken care of immediately according to established procedures.

8. Disposal of radioactive materials must comply with prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory.

2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in acid medium. In addition, it may form potentially explosive lead or copper azides. To avoid dangerous deposits, waste solutions should be flushed away with large volumes of water.

3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line reagents included in this kit have been tested and found to be non reactive for hepatitis B surface antigen. They have also been screened and determined to be non-reactive for HTLV-III antibody. However, human serum products should be handled as if potentially capable of transmitting hepatitis, Acquired Immune Deficiency Syndrome, or other infectious agents.

4.Materials provided:

Kit contains sufficient reagents for 100 determinations.

1. Bovine serum with sodium azide based standards & control: 8 vials containing each 0.5 ml except zero 10.0 ml.

Standards: 0-1.0-3.0-10.0-30.0-100.0-150.0 mIU/ml.

2. ¹²⁵I-AFP tracer: 1 vial (red solution) containing 10.5 ml. Activity < 9.5µCi or 350 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-AFP monoclonal antibodies.

4. Wash solution concentrate: 1 vial of 10 ml of concentrate, to be diluted into 700 ml distilled water and stored at 4° C.

Reagents provided should be stored at 2^o - 8^o C.

Refer to the expiration date on the kit label for stability.

5.Materials required but not provided:

The following material is required but not provided in the kit:

1. Distilled water

2. Pipettes for delivery of:, 100 µl,- 200 µl, 500 µl, and 1 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3. 5 ml automatic syringe (Cornwall type) for washing

4. Aspiration system (optional)

5. Gamma counter, set for ¹²⁵I counting and Vortex mixer and magnetic stirrer.

6.Specimen collection and preparation:

- Serum, plasma or amniotic fluid samples must be kept at 2-8°C.

- If test is not run within 24 hours, storage at –20°C is recommended and will not result in loss of immuno-reactivity for at least 6 months.

- Serum, heparinized or EDTA plasma provide similar results:

y(serum) = 1.05 x (hep. plasma) - 0.2 r=0.98 n = 31

y(serum) = 1.05 x (EDTA plasma) - 0.5 r = 0.99 n= 32

7.Assay procedure:

1. Label coated tubes in duplicate for each standard, sample, control. For determination of total counts, label 2 normal tubes.

2. Vortex mix briefly standards, samples, controls and dispense 50 µl of each into the respective tubes.

3 Shake the tube rack gently and manually to remove any bubbles.

4. Incubate for 1 hour at room temperature.

5. Aspirate (or decant) the content of each tube. Be sure that the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.

6. Wash the tubes with 2 ml Wash Solution and aspirate (or decant) Avoid foaming during the addition of the Wash Solution.

7. In order to increase the reproducibility of the assay, leave the tubes on the tableI for two minutes and aspirate the remaining drop of liquid.

8. Dispense 100 ~l tracer in each tube, including total counts.

9. Shake gently and manually tube rack.

10. Incubate for 1 hour at room temperature.

11. Aspirate (or dceant) the content of each tube (except total counts). Be sure that the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.

12. Wash the tubes with 2 ml Wash Solution and aspirate (or decant). Avoid foaming during the addition of the Wash Solution.

13. In order to increase the reproducibility of the assay, leave the tubes on the table for two minutes and aspirate the remaining drop of liquid.

14. Count tubes in gamma counter for 60 seconds.

AFP Coated Tubes Flow chart

Total Counts (ml)

Standards (ml)

Sample(s), controls (ml)

Standards (0-5)

Samples, controls

-

-

0.05

-

-

0.05

Incubation

1 hour at room temperature

Separation

Wash solution

Separation

Wash solution

Separation

-

-

-

-

-

Aspirate (or decant)

2.0

Aspirate (or decant)

2.0

Aspirate carefully

Tracer

0.1

0.1

0.1

Incubation

1 hour at room temperature

Separation

Wash solution

Separation

-

-

-

Aspirate

2.0

aspirate carefully

Counting

Count tubes for 60 seconds

Data table (example)

AFP-IRMA		Cpm	B/T (%)
Total count Standard	0IU/ml 5IU/ml 14.5IU/ml 46 IU/ml 143IU/ml 475 IU/ml	154183 158 2600 7058 19667 46377 78169	0.1 1.7 4.6 12.8 30.1 50.7

8.Calculation of results:

The standard curve is prepared by plotting cpm or B/T (%) on the ordinate against the standard concentration on the abscissa using either linear-linear or semi-log paper. Draw the best curve rejecting obvious outlyers. Determine AFP concentrations of samples by interpolation of cpm or B/T (%) values. If a computer assisted method is used, a polynomial function usually gives the best results.

B/BO x 100 = Counts (Std or sample) / Counts (Zero Standard) x 100

9.Specific performance characteristics:

A. Sensitivity

The sensitivity of 0.5 IU/ml was calculated by measuring 20 times the zero standard and equals the mean cpm value +2 SD.

B. Specificity

The AFP-IRMA does not cross-react with structurally related substances Human serum albumin (HSA) at a concentration of 60 ng/ml was added to a low (5 lU/ml) and a high (50 lU/mI) AFP standard, the apparent AFP response was measured.

Observed AFP value (IU/ml)

Std 5 IU/ml + HAS (60ng/ml)

Std 50 IU/ml + HAS (60ng/ml)

4.8

49.0

C. Precision

INTRA ASSAY INTER ASSAY

Serum	Replicate	X±S.D. (pg/ml)	CV (%)	Serum	Replicate	X±S.D. (pg/ml)	CV (%)
A B	20 20	8.9±0.3 29.2±1.3	3.4 4.5	A B	20 20	9.1±0.6 28.2±1.3	6.6 4.6

D. Accuracy

RECOVERY TEST

Sample	Added AFP (IU/ml)	Recovered AFP (IU/ml)	Recovery (%)	Sample	Dilution	Theor. Conc. (IU/ml)	Recovery (%)
Serum	7.8 15.6 31.3 62.5 125.0 250.0	7.2 14.8 29.8 59.6 110.0 255.0	92 95 95 95 88 102	Serum	1/1 ½ 1/4 1/8 1/16 1/32 1/64	200.4 100.2 50.1 25.1 12.5 6.3 3.1	200.4 104.2 47.9 24.7 13.0 6.3 3.4
Plasma	7.8 19.6 31.3 62.5 125.0 250.0	7.2 14.6 28.3 54.0 106.0 251.0	92 94 90 86 85 100	Amniotie fluid	1/50 1/100 1/500 1/1000 1/2000	128 64 13 6.4 3.2	128 66 12 6.2 3.2

10.Bibliography

1. CUCKLE, H.S., WALD, N.J. 1984.Maternal serum alpha-fetoprotein measurement : a screening test for Down Syndrome.Lancet, I:926

2. HUNTER, W.M. BUDD, P.S. 1981.Immunoradiometric versusradioimmunoassay a comparison using alpha-fetoprotein sa the model analyte.

J. of Immunol. Meth.45:255

3. KOHN, J. WEAVER, P.C. 1974.Serum alpha-fetoprotein in hepatocellullar carcinoma.Lancet, i:334

4. KOHN, J., ORR, A.H. MC EL WAIN, T.J. et al.Serum alpha-fetoprotein in patients with testicular tumours.Lancet,II:334

5. NORGAARD-PENDERSEN, B.1976Human alpha-fetoprotein – A review of recent methodological and clinical studies.Scand. J. Immunol., Suppl. 4:7.

6. U.K. Collaborative study, First report. 1977Maternal serum alpha-fetoprotein measurement in antenatal screening for anencephaly and spina bifidein early pregnancy.Lancet, i:1323.

Very important notice for export

Since 1rst of January 2012

The production and the export of the clinical laboratory diagnostic kits RIA and ELISA

are transferred to the company : Asbach Medical Products GmbH

Tel: + 49 62 62 91 74 02 - Fax: + 49 62 62 91 76 99 - Email: info@amp-asbach.com

If any problem please do not hesitate to contact us.

Last updating 14/12/11