Studies of carcino‐fetal proteins. III. Development of a radioimmunoassay for α‐fetoprotein. Demonstration of α‐fetoprotein in serum of healthy human adults

Ruoslahti, Erkki; Seppälä, Markku

doi:10.1002/ijc.2910080304

Cited by 244 publications

(81 citation statements)

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“…We purified immunochemically human AFP [14] and developed a radioimmunoassay (RIA) for its determination [15]. It became evident thät small amounts (2-16 ng per ml) of AFP are present in normal human serum [15,16]. This finding has now been confirmed by other groups [6,13].…”

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Alpha fetoprotein: Physiology and pathology during pregnancy and application to antenatal diagnosis

Seppälä¹,

Ruoslahti²

1973

Journal of Perinatal Medicine

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Alpha fetoprotein (AFP) is synthesized by the x human fetal liver and yolk sac [8,9]. During the 12 th -14 th intrauterine weeks the serum concentration of AFP is at its highest, reaching a few milligrams per ml [7]. However, the total amount of AFP in the fetus increases until the 22 nd week of gestation, remaining constant until about the 32 nd week, and thereafter decreasing [7]. In the newborn at term, the serum AFP concentration is 50-ISO^g per ml [19]. AFP remains detectable by immunodiffusion or related methods for about 5 weeks after birth [24]. Greatly increased serum AFP levels cften reappear in adults with primary hepatocellular cancer [25] or teratocarcinomas [1]. Until recently, the occurrence of AFP in maternal serum had been contested [3]. We purified immunochemically human AFP [14] and developed a radioimmunoassay (RIA) for its determination [15]. It became evident thät small amounts (2-16 ng per ml) of AFP are present in normal human serum [15,16]. This finding has now been confirmed by other groups [6,13]. AFP levels in maternal serum increase during pregnancy and the highest AFP concentrations occur during the third trimester. The half life of maternal serum AFP after delivery is about 5 days [19]. In amniotic fluid, the AFP concentrations decrease from the 15 th week of gestation, and at term the concentrations are similar to those in maternal serum [19,21]. Recently, we found that the maternal serum AFP concentration often increases in asso- 1963-1965. Resident at Dept. of Obstetrics and Gynecology., University Central Hospital, Helsinki 1966-1969. Senior lecturer in Obstetrics and Gynecology, 1970. Assistant professor at Dept. II of Obstetrics and Gynecology (Head: Professor PAAVO VARAJ, University Central Hospital, Helsinki, 1970.'* ciation with fetal death [20], and that this increase may occur even before the fetal death [23]. In the present paper we describe results on the AFP concentrations in normal and pathological pregnancies, and evaluate the clinical significance of the serum AFP levels in the antenatal diagnosis of high risk pregnancies.1. Materials 1.1 Serum samples from pregnant women Sera from 204 pregnant women between the 8 th and the 42 nd week of gestation were studied for the normal ränge. The following types of high risk pregnancies were investigated: 67 sera from 20 diabetic women, 116 specimens from 74 pregnant women with toxemia or hypertension, and 48 samples from 27 pregnant women with a liver disorder. Intrauterine fetal death occurred in 22 patients.

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Alpha fetoprotein: Physiology and pathology during pregnancy and application to antenatal diagnosis

Seppälä¹,

Ruoslahti²

1973

Journal of Perinatal Medicine

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“…In adults the concentration of AFP is extremely low (4) except in patients with primary liver cancer or teratoma (5,6). AFP has a molecular weight of 70,000, consists of a single polypeptide chain, and contains about 4% carbohydrate (7,8).…”

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“…Immunodiffusion was performed on plates cast in a conventional buffer and soaked overnight in PBS-GTX prior to use. Radioimmunoassays were performed using the double antibody technique as described (4). The first and second antibody were added in the form of an IgG fraction in assays where native albumin was tested for crossreactivity with AFP.…”

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Immunological crossreaction between alpha-fetoprotein and albumin.

Ruoslahti¹,

Engvall²

1976

Proc. Natl. Acad. Sci. U.S.A.

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(16). Bovine serum albumin (BSA) was from Pentex, bovine hemoglobin from Sigma, and bovine fetuin from Gibco.The samples to be reduced and alkylated (17) were dissolved at a concentration of about 10 mg/ml in 0.1 M Tris-HCI buffer (pH 8.2) containing 8 M urea. After deaeration with nitrogen, dithiothreitol was added to a final concentration of 10 mM. After 3 hr at room temperature under nitrogen, the reaction mixture was cooled on ice and recrystallized iodoacetamide was added to make the final concentration 20 mM. After another 15 min the sample was dialyzed against distilled water and lyophilized. Phosphate-buffered saline containing 0.05% gelatin, 0.02% sodium azide, and 0.1% Triton X-100 (PBS-GTX) with urea added to it to give a 2 M solution was used to dissolve the dry proteins, some of which were poorly soluble in ordinary buffers. In some cases the reduced and carboxamidomethylated (RC-) protein was diluted directly into this solution for testing without dialysis and lyophilization. HAFP labeled with 1251 was reduced and alkylated similarly and freed of reactants by gel filtration on Sephadex G-15 equilibriated with PBS-GTX.Antisera. Anti-HAFP sera have been described (6,8). Using a similar schedule, we produced one antiserum each to RC-HSA and RC-BSA and two to RC-HAFP in rabbits. The dose used per injection was 5 mg (RC-HSA, RC-BSA) or 0.5-2 mg (RC-HAFP) of protein suspended in PBS and given in complete Freund's adjuvant. A precipitating antiserum to RC-CEA was obtained similarly, using 0.25 mg of RC-CEA per injection.Immunological Tests. Immunodiffusion was performed on plates cast in a conventional buffer and soaked overnight in PBS-GTX prior to use. Radioimmunoassays were performed using the double antibody technique as described (4). The first and second antibody were added in the form of an IgG fraction in assays where native albumin was tested for crossreactivity with AFP. The diluent buffer used throughout was PBS-GTX. RESULTSLack of Crossreaction between Native AFP and Albumin. Antisera produced against highly purified HAFP do not react with albumin in immunodiffusion (ref. 15, our unpublished results). Radioimmunoassay also failed to reveal any crossreactivity between native HAFP and native HSA or RC-HSA. RC-HAFP was also almost completely devoid of activity in the usual AFP assay (Fig. 1).

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“…Radioimmunoassays (RIA) have been applied to the quantitation of human AFP [62,80,90,102,125,139,169,1971 and AFP's of various animal species [85,90,123, 1511 since 1971. The literature has recently been reviewed [ …”

Section: Q U a N T I T A T I O N O F A F Pmentioning

confidence: 99%

Alpha‐fetoprotein

1978

Scand J Immunol

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Studies of carcino‐fetal proteins. III. Development of a radioimmunoassay for α‐fetoprotein. Demonstration of α‐fetoprotein in serum of healthy human adults

Cited by 244 publications

References 23 publications

Alpha fetoprotein: Physiology and pathology during pregnancy and application to antenatal diagnosis

Alpha fetoprotein: Physiology and pathology during pregnancy and application to antenatal diagnosis

Immunological crossreaction between alpha-fetoprotein and albumin.

Alpha‐fetoprotein

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