Quantification of total IgM and IgG and specific IgM and IgG to a thymus-independent (LPS) and a thymus-dependent (tetanus toxoid) antigen in the rat by enzyme-linked immunosorbent assay (ELISA).

Rat IgM and IgG was determined by mechanized "sandwich" enzyme-linked immunosorbent assay (ELISA) using peroxidase labeled anti-rat-IgM and -IgG. Linear ranges in standard curves of a reference rat serum had a slope similar to the slopes found with sera of 25 rats of various age. IgM and IgG measurements by ELISA in these sera correlated well with results obtained by single radial immuno-diffusion (SRID). In addition, the precision of the enzyme immunoassay was the same as obtained with the SRID. Compared with SRID, ELISA is less time consuming and the amount of antiserum used in the macro-ELISA is one order of magnitude lower; and again 10 times lower in the mechanized micro-ELISA that is currently being developed. In conclusion, the ELISA is a specific, reliable, sensitive, and economic method for routine measurement of rat serum IgG and IgM e.g. in toxicity studies. In the second part of this study, ELISA and the passive hemagglutination test were compared to determine the primary and secondary antibody response to E. coli lipopolysaccharide (LPS) and tetanus toxoid in rats. In the ELISA, the antigens were bound to the wells of polystyrene microplates. Tetanus toxoid was coated directly, LPS after complexing with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled antiimmunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with the respective antigens. ELISA proved to be more sensitive than the hemagglution reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, ELISA is a convenient method for measuring both IgM and IgG antibodies. Finally, evidence is presented that in the rat, the humoral immune response to LPS is a thymus-independent phenomenon. Thus, by using the antibody response to LPS and tetanus toxoid in function studies of the immune system of the rat, insight can be obtained in the thymus-independent and thymus-dependent humoral immune response.

Adverse obstetric outcome in low- and high- risk pregnancies: predictive value of maternal serum screening.

OBJECTIVE: To determine whether the relationship between adverse pregnancy outcome and elevated maternal serum alpha-fetoprotein (MSAFP) and/or maternal serum hCG levels in women whose fetuses have no chromosomal abnormalities or neural tube defects is restricted to pregnancies with a priori elevated risk for pathology or also present in low-risk pregnancies. METHODS: The outcomes of pregnancy in two groups of patients with elevated MSAFP and/or maternal serum hCG values were compared with the outcomes of a reference group with normal serum values. The first study group consisted of 83 women without pre-existing risk for poor outcome as defined by the guidelines of the Dutch Society of Obstetrics and Gynecology. The second study group consisted of 62 women with a priori elevated risk according to these guidelines. RESULTS: Fetal or neonatal death, pregnancy-induced hypertension, placental abruption, placenta previa, preterm delivery, delivery of infants with birth weights in the 2.3rd percentile, and complications during the third stage of labor occurred significantly more often in patients with elevated values and low a priori risk than in women with normal values and without pre-existing risk factors. There was no significant increase in adverse pregnancy outcome in women with elevated values and high a priori risk compared with women with normal values and elevated a priori risk. CONCLUSION: In women at low risk, elevated MSAFP and/or maternal serum hCG values are predictive of adverse pregnancy outcome. In women with a priori elevated risk, abnormal serum values do not increase this risk.

A modified ELISA technique for the titration of antibodies to polio virus as an alternative to a virus neutralization test.

An ELISA based on inhibition of antibody binding for the determination of antibodies to polio virus type I is described. F(ab1)2-fragments of bovine antibodies to polio virus type I are used as the capture antibody thus lowering the background staining. A good correlation was found between the poliovirus neutralizing antibody level and the antibody titers as determined by ELISA.

Cord serum levels of IgM, IgA, and alpha-fetoprotein and their interrelationships.

IgM, IgA, and AFP concentrations were determined in a group of 30 umbilical cord sera chosen at random from the cord sera sampled over a period of one year. Because low levels were expected, use was made of sensitive techniques, which were standardized on the basis of international reference preparations. Geometric mean levels were 5.58 mg/l for IgA, 142 mg/l for IgM and 53.6 mg/l for AFP. Statistical analysis of the distinct IgM, IgA, and AFP levels showed no significant correlation with either birth weight or birth length. The difference between the sexes in the levels of these proteins was not significant either. The difference between the IgA concentration in sera from children of primipara and those of multipara mothers was significant. Moreover, a statistically significant negative correlation was found between the IgA and AFP levels. The results are discussed in relation to current knowledge about the ontogeny of humoral immunity in man and a possible biological function of AFP in ontogeny.

In vitro assays for the developmental toxicity of xenobiotic compounds using differentiating embryonal carcinoma cells in culture.

Murine embryonal carcinoma (EC) cells, which resemble the undifferentiated cells of the epiblast in the blastocyst, were used to establish two in vitro assays for developmental toxicants. The target processes in the assays are the differentiation of EC cells into endodermal and mesodermal derivatives, respectively. These processes were selected because they are crucial and specific for embryogenesis, and also because EC cell differentiation has been shown to be sensitive to various compounds that are teratogenic in vivo. The usefulness of the assays was studied with five pairs of xenobiotic compounds, with chemical analogy and different in vivo teratogenicity within each pair. Results for imidazoles and pyridines correlated well with known effectiveness in vivo; however, this was not the case for phthalates, sulfonamides and xanthines, which for the latter two is explicable in terms of mechanisms of action in vivo. Validation of the assays for classes of related compounds will determine their usefulness as a screen for each class, depending on the mechanism of action and physical properties. In this way in vitro assays will contribute to the refinement of testing, in addition to the more efficient use of laboratory animals.

[Maternal serum screening for Down syndrome and neural tube defects]. / Maternale serumscreening op Down-syndroom en neuralebuisdefecten.

OBJECTIVE: Evaluation of maternal serum screening for Down's syndrome (DS) and neural tube defects (NTDs). DESIGN: Longitudinal study. SETTING: Department of Obstetrics and Gynaecology, University Hospital Utrecht, the Netherlands. METHOD: 6362 pregnant women underwent serum screening for DS and (or) NTD between the 15th and 21st weeks of pregnancy between March 1991 and March 1996. Screening was performed using alpha-foetoprotein, unconjugated oestriol, human chorionic gonadotrophin and maternal age. The result of each individual test was a calculated risk for delivering a child with DS and (or) NTD. RESULTS: Nine out of 12 singleton pregnancies of a foetus with DS were detected. To this purpose, 573 women who, according to the serum screening had an increased risk of a child with the abnormality, were offered amniocentesis, which was performed in 471 of them. Two twin pregnancies with a total of 3 DS affected foetuses were also detected; one twin pregnancy of a DS foetus was screen-negative. The one case of spina bifida was screen-positive. The proportion of women eligible for invasive prenatal diagnosis because of maternal age increased from 9% to 25% in the course of the study. Of 1118 women aged > or = 36 years 913 (82%) declined invasive investigation compared with 40% in the general population. CONCLUSION: The results of the maternal serum screening program in Utrecht were comparable with other studies. Maternal serum screening is accepted as an alternative by women above 36 years, and allows to decrease the need for amniocentesis without a significant loss in detection rate.

Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin.

The enzyme-linked immunosorbent assay using different techniques has been applied to determine botulinum type B toxin. With the so-called "sandwich" technique, about 5,000 mouse ip LD50 of type B toxin can be detected. With the "double-sandwich" technique, about 400 mouse ip LD50 of toxin is detected and different commerical antisera are useful. For accurate quantification of botulinum toxins in culture filtrates, addition of EDTA to samples seems to be necessary. Cross-reactivity of the assay depends on the specificity of the antisera against botulinum type B toxin used and is almost eliminated with antiserum prepared against the toxic component of type B toxin.

Amniotic and maternal serum alpha-fetoprotein levels of rats with induced neural tube defects.

During the complete fetal period alpha-fetoprotein (AFP) was quantified in maternal sera and amniotic fluid from control, hypervitaminosis A and trypan blue treated normal rat fetuses, and from exencephalic and spina bifida aperta fetuses. The occurrence of histologic proven open neural tube defects was associated with amniotic fluid AFP levels that were much elevated over those of control and treated normal fetuses and those with closed neural tube defects at nearly the whole fetal period. In combining these results with the earlier reported morphologic data of the same rat fetuses as used in the present study, it is concluded that the elevation of amniotic AFP is caused by leakage of fetal serum through a disrupted and necrotic nervous tissue into the amniotic fluid. This experimental model of induced neural tube defects results in increase of amniotic fluid AFP levels similar to those found in human amniotic fluid in the presence of neural tube defects.

Comparison of ELISA and toxin neutralization for the determination of tetanus antibodies.

In a sandwich ELISA for tetanus antibodies, the influence of the tetanus toxoid concentration used for coating microtiter plates has been studied. The antibody levels by toxin neutralization bioassay and by ELISA were studied for a population with known immunization history. By decreasing the tetanus toxoid concentration in ELISA from 5 to 0.2 Lf/ml, a better correlation was found between the ELISA results and the bioassay titers, but sera from recently immunized people still showed high ELISA titers. This phenomenon cannot be ascribed to nonspecific reactions since sera from nonimmunized people are negative in both assays. All sera negative in ELISA had, however, a bioassay titer beneath 0.01 IU/ml.

Are Down syndrome fetuses detected through maternal serum screening similar to those remaining undetected?

This study was designed to examine whether fetuses with Down syndrome (DS) identified through serum screening are different from those whose mothers have normal serum screening results. It was a retrospective follow-up study of pregnancies where maternal serum alpha-fetoprotein (MSAFP) concentrations were measured to identify women at increased risk of having a baby with a neural tube defect (NTD). An enhanced risk for NTD was the only reason for intervention in the screened population. Clinical features of fetuses or children with DS were related to the screening results. A retrospectively calculated term risk of 1/250 classified a pregnancy as having been at an elevated risk of DS. The outcome measures were fetal or neonatal death and severe somatic disease. Human chorionic gonadotrophin (hCG) and unconjugated oestriol (uE3) were measured retrospectively in frozen samples of the DS pregnancies and the same cut-off level was used for classification (so-called 'triple test'). Ten thousand women were included in the study. Pregnancy outcome was known in 93.5 per cent of the cases. Children with and without anatomic defects were found in all subgroups of test results combinations. All mothers of children with a congenital heart defect (CHD) had a DS risk of > or = 1/250 according to the triple test.

The influence of malaria and gestation on the immune response to one and two doses of adsorbed tetanus toxoid in pregnancy.

The effect of Plasmodium falciparum infection on the response to immunization with tetanus toxoid in pregnancy is of importance because malaria is more frequent and severe in pregnant women. This paper presents the results of a study in west Kenya of the antibody response to an adsorbed tetanus toxoid in primigravidae and multigravidae living under holoendemic conditions. There was no apparent influence of either P. falciparum infection or gestational age on the immune response to one and two doses of adsorbed toxoid. The antibody response in pregnant women with and without malaria was comparable to that in non-pregnant healthy adults. Previous studies of responses to primary immunization schedules in pregnant and non-pregnant women are reviewed.

Maternal serum markers in second-trimester oligohydramnios.

The levels of the maternal serum markers alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE3) in 35 pregnant women with early second-trimester oligohydramnios differed from those in a reference population of 1699 singleton pregnancies. Maternal serum AFP levels above the 95th centile of the population distribution were observed in 80 per cent (16/20) of oligohydramnios cases with a normal fetus and in only 20 per cent (3/15) of the cases with a fetus displaying urogenital tract malformations. Elevated levels of hCG (above the 95th centile) and decreased levels of uE3 (below the fifth centile) were encountered in 26 per cent (9/35) and 17 per cent (6/35) of the women, irrespective of the fetal condition. The abnormal profile of the serum markers in early second-trimester oligohydramnios resulted in 57 per cent (20 out of 35) of screen-positive cases for either fetal Down's syndrome or neural tube defects, compared with 8.4 per cent (143 out of 1699) in the reference population.

Maternal serum alpha-fetoprotein in fetal anal atresia and other gastro-intestinal obstructions.

The fetal gastro-intestinal (GI) tract contributes to alpha-fetoprotein (AFP) levels in amniotic fluid and hence to those in maternal serum (MS). This study retrospectively analysed results of second trimester MSAFP screening in cases of fetal GI tract obstruction. 18 cases of fetal GI obstruction were diagnosed amongst 17,036 women who underwent MSAFP screening between 1979 and 1997: seven had oesophageal atresia, four had duodenal atresia, six had anal atresia, and one had both anal and oesophageal atresia. MSAFP in pregnancies of a fetus with anal atresia was significantly lower than the population median. MSAFP in cases of fetal oesophageal or duodenal atresia was low but these differences did not reach significance.

Comparison of enzyme-linked immunosorbent assay and passive hemagglutination method for quantification of antibodies to lipopolysaccharide and tetanus toxoid in rats.

In a comparative study, the enzyme-linked immunosorbent assay, using peroxidase labeled anti-rat immunoglobulin M and immunoglobulin G, and the passive hemagglutination test were applied to determine the primary and secondary antibody response to lipopolysaccharide and tetanus toxoid in rats. In the enzyme-linked immunosorbent assay, the antigens were bound to the wells of polystyrene microplates, tetanus toxoid directly, and lipopolysaccharide after complexing it with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled anti-immunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with their respective antigens. The enzyme-linked immunosorbent assay proved to be more sensitive than the hemagglutination reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, the enzyme-linked immunosorbent assay is a convenient method for measuring both immunoglobulin M and immunoglobulin G antibodies. At low serum dilutions of lipopolysaccharide antisera, inhibition of the reaction in the enzyme-linked immunosorbent assay occurred. This phenomenon could be prevented by heating the sera at 56 degrees C for 30 min. Lipopolysaccharide was immunogenic in rats over an extremely wide dose range (from 10 pg to 1 mg); the optimal immunogenic dose of lipopolysaccharide for young adult rats was 0.1 to 1,000 mug when administered intravenously, and that of tetanus toxoid was 5 to 10 lines of flocculation, as determined by the Ramon flocculation test.

Fetal ductus venosus flow velocity waveforms and maternal serum AFP before and after first-trimester transabdominal chorionic villus sampling.

Doppler flow velocity waveform recording in the fetal ductus venosus and umbilical artery as well as maternal blood sampling for serum alpha-fetoprotein (MSAFP) was performed before and after transabdominal chorion villus sampling (TACVS) in 36 women of advanced maternal age (> or = 36 years). Gestational age ranged between 11 and 13 weeks. No chromosomal anomaly was detected. No statistically significant difference was observed in ductus venosus velocity parameters or in the umbilical artery pulsatility index (PI) before and after CVS in 35 women with a normal pregnancy outcome. One case resulted in fetal loss. Post-CVS median MSAFP levels at 12 weeks (25 kIU/l) and 13 weeks (35 kIU/l) were significantly higher than pre-CVS levels. In three cases, post-CVS MSAFP levels were higher than 600 kIU/l, correlating with feto-maternal transfusions of approximately 1.0-1.4 ml, i.e., of around 40 per cent of feto-placental blood volume. One of these cases displayed absence of fetal peripheral blood flow velocities and fetal bradycardia following TACVS, resulting in fetal loss 1 week later. The remaining two cases had a normal pregnancy outcome, but showed a more than 50 per cent reduction in ductus venosus velocity after TACVS, whereas umbilical artery PI remained unchanged. However, similar velocity changes were associated with only small feto-maternal transfusions. Umbilical artery PI values remained unchanged.

Prenatal diagnosis of spina bifida aperta after first-trimester valproate exposure.

In the context of a prospective study on the adverse effects of anti-epileptic drugs on fetal outcome, we evaluated our experience with prenatal diagnosis by ultrasonography and alpha-fetoprotein (AFP) determination in amniotic fluid. We compared these results with AFP values in maternal serum obtained prior to amniocentesis. From November 1985 to July 1990, amniocentesis at 16-18 weeks of gestation was performed in 267 pregnancies of 237 different women using anti-epileptic drugs. Among 92 pregnancies with maternal valproic acid use, five (including one concordantly affected monozygotic twin-pair) were terminated because of a spina bifida aperta, all prenatally diagnosed by AFP determination and acetylcholinesterase electrophoresis in amniotic fluid. The maternal serum AFP level was raised (> or = 2.5 multiples of the median (MOM) for singleton pregnancies and > or = 4.5 MOM for twin pregnancies) in only two of these five affected pregnancies. We emphasize that maternal serum AFP levels may be unreliable for prenatal screening for fetal neural tube defects in women taking valproate and recommend that amniocentesis and fetal ultrasound examination should be offered directly.

Maternal serum alpha-fetoprotein levels and fetal outcome in early second-trimester oligohydramnios.

Early second-trimester oligohydramnios was associated with normal maternal serum alpha-fetoprotein (MSAFP) levels in nine out of 26 cases (35 per cent). Congenital malformations of the fetal urinary tract resulting in fetal anuria were present in nine cases; in seven of them, normal MSAFP levels were measured. In contrast, normal MSAFP levels were established in only 2 out of the 17 cases without fetal malformations. These data suggest that fetal urine is the major source of elevated AFP in the maternal compartment in early second-trimester oligohydramnios. This is further supported by the lack of any relationship between concentrations of MSAFP non-reactive with Concanavalin A, originating mainly from the yolk sac-derived amniotic fluid AFP pool, and the presence of fetal diuresis. Three out of 26 women had experienced early second-trimester oligohydramnios in a previous pregnancy, suggesting the existence of a recurrence risk for this condition.

Transabdominal chorionic villus sampling in the second trimester of pregnancy: feto-maternal transfusions in relation to pregnancy outcome.

Volumes of feto-maternal transfusions (FMTs) in transabdominal chorionic villus sampling (TACVS) in the second trimester of pregnancy were calculated from the difference between maternal alpha-fetoprotein (AFP) concentrations before and 1 h after TACVS. In 50 pregnancies, there existed no correlation between FMT volume and the amount of villi collected or the number of TACVS attempts. The expected risk of fetal exsanguination due to very voluminous FMT could not be substantiated. In one case, immunization could have been the cause of hydrops fetalis, although only a volume of 0.15 ml could be calculated.