On rat alpha-fetoprotein as a fatty acid carrier. Influence of the structure of fatty acids.

Fatty acids interfere with rat alpha-fetoprotein-estrogen interaction. We present here a quantitative study of the association constants for the binding to alpha-fetoprotein of different fatty acids. It can be concluded that fatty acids bind more strongly if the number of carbon atoms increases with the number of double bonds. The maximum binding ability occurred with the C20 fatty acids having three or four double bonds. As these fatty acids are the precursors in the synthesis of prostaglandins, we tested some compounds in this series and showed that prostaglandins presented no particular affinity for rat alpha-fetoprotein.

Alpha-foetoprotein and oestrogen metabolism. I. -Influence of alpha-foetoprotein on the metabolism of steroids by rat liver microsomes in vitro.

Rat alpha-foetoprotein (AFP) was shown to inhibit the formation of water soluble metabolities of oestrone and oestradiol by incubation with microsomes from rat liver in the presence of NADPH. The results support the proposal that in young animals the low activity of enzymes responsible of oestradiol metabolism may be due in part to the presence of AFP and not only to the low level of these enzymes.

Purification and characterization of an estrogen-binding peroxidase from human fetuses.

A peroxidase found under two forms with a molecular weight of 220,000 and 170,000 respectively, was purified from human fetuses. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, gel filtration and hydrophobic interaction chromatography. The purification factor approximated 400. These two forms of peroxidase were found to be immunologically identical as shown when utilizing immunodiffusion. They were able to bind estradiol in the presence of H2O2. This bond resisted to denaturation and solvent extraction therefore suggesting a covalent binding of estradiol to the enzyme.

A sandwich method of enzymoimmunoassay. I. Application to rat and human alpha-fetoprotein.

An enzymoimmunoassay (EIA) for the quantitation of soluble antigens having at least two antibody-combining sites is descirbed. This non-competitive sandwich method comprises three steps: 1) the antigen to be assayed is reacted with the antibody-coated cellulose immunosorbent, 2) the enzyme-labelled antibody is then incubated with the antigen bound to the solid phase, 3) the enzymatic activity of the immunosorbent is then measured. This activity increases with the quantity of antigen to be assayed. Examples of the application of this method to the assay of rat and human alpha-fetoprotein (AFP) are given. When applied to rat and human AFP, this assay gives reproducible results in the range of 10--1000 ng/ml and 3-1000 ng/ml respectively. AFP sera concentrations of normal and pregnant rats were assayed by EIA, radioimmunoassay RIA and rocket-immunoelectrophoresis (RIE). In all the cases good agreement was noted among these three techniques. Rheumatoid factor, whenever present, may interfere with the assay. However, the effect of this interaction can be eliminated. The advantages of the EIA method can be listed as follows: a) no pure antigen is required, b) the final color reaction is developed from the solid phase. This feature eliminates most non-specifically interfering factors, c) the range of the assay covers a 2 log-scale, d) only inexpensive equipment is used, e) results are obtained within 24 hr, f) sensitivity and reproducibility lie within a range comparable to that of RIA.

Effect of pH on the recovery of human alpha-fetoprotein from immunosorbent columns. An improved elution procedure.

A slow decrease in pH was shown to be more efficient in eluting human alpha-fetoprotein bound to Sepharose-immobilized antibodies than a pH shock. With this method, nearly 50% of the antigen absorbed onto the column was recovered in a single peak whereas the yield was 3 times lower in the case of a single elution at pH 2.6. Our result suggests that dissociation of the antigen-antibody complex occurred only within a narrow pH range.

Coupling of gamma-globulin to microcrytstalline cellulose by periodate oxidation.

Anti-rabbit IgG sheep gamma-globulins were covalently coupled to periodate oxidized microcrystalline cellulose using the Schiff reaction. Optimal conditions (sodium m-periodate concentration and gamma-globulin amount) were studied measuring the ability of this solid-phase antibody to bind glucose oxidase-labeled rabbit IgG.

Enzymoimmunoassay of human alpha-fetoprotein.

A non-competitive method for the determination of human alpha-fetoprotein (AFP) in serum, using a pure specific antibody linked to glucose oxidase is described. When applied to human AFP, this method gives reproducible results in the range 0.7 to 15 ng/ml, in a relatively short time (6 hr). AFP sera levels of healthy human adults, pregnant women and adults with liver diseases, were tested both by enzymoimmunoassay and radioimmunoassay. In all cases, good agreement was noted between the two methods.

An enzyme immunoassay design using labelled antibodies for the determination of haptens. Application to methotrexate assay.

A competitive enzyme immunoassay with labelled antibodies has been developed for methotrexate (MTX). Methotrexate in the sample and a constant quantity of this hapten physically absorbed to polystyrene spheres through a methylated bovine albumin carrier were allowed to compete for a limiting amount of peroxidase labelled antibody. After washing, the residual enzyme activity bound to the solid phase was measured. This test was able to detect 10 fm of MTX per sample. A comparative study of this test with a commercial radioimmunoassay kit using the same antiserum and a high pressure liquid chromatography method showed that the sensitivity, specificity and precision of this test were as good as of the radioimmunoassay. The high pressure liquid chromatography method was 500 times less sensitive. Good agreement was found among the 3 methods on 83 serum samples from patients receiving methotrexate therapy.

A sandwich method of enzyme immunoassay. III. Assay for human beta-2 microglobulin compared with radioimmunoassay.

A sandwich enzyme immunoassay has been developed to measure human beta-2 microglobulin (beta 2m) in bilogical fluids. beta 2m is first bound by specific antibody covalently coupled to microcrystalline cellulose. The solid phase is washed and reincubated with glucose oxidase-labeled anti-beta 2m antibody. After washing, the enzymic activity of the solid phase is measured by incubation with the appropriate chromogenic substrate. The OD is directly related to the quantity of beta 2m to be measured. A second sandwich assay has also been developed which uses plastic microplates coated with the IgG fraction of rabbit anti-beta 2m serum. Reproducible results are obtained in the range 2-150 and 0.75-48 microgram/l respectively. These tests detect 17 fmoles of beta 2m. Assays of 27 sera showed good agreement between these two enzyme immunoassay methods and two radioimmunoassays.

A novel enzyme immunoassay for total thyroxine using immobilized antibodies and hydrophobic chromatography purified thyroxine-peroxidase conjugate.

A new and simple competitive enzyme immunoassay method for the measurement of total thyroxine in serum is described. Anti-thyroxine antibodies, raised in sheep with a bovine serum albumin-thyroxine conjugate prepared with carbodiimide as coupling initiator, were physically adsorbed onto a large surface area polypropylene support. Competition occurred between thyroxine in the sample and a thyroxine-peroxidase conjugate prepared with a glutaraldehyde spacer and further purified by octyl Sepharose hydrophobic chromatography. The entire assay was performed in 2 h with a useful range of 10-300 micrograms/l. The coefficients of variation ranged from 6.9 to 12% and good agreement (r = 0.93-0.97) was found between this method and 2 radioimmunoassays on 71 serum samples having well distributed thyroxine values.

Transfer, tissue localization and proliferation of fetal cells in pregnant mice.

Matings between Rb7-Rb7 male mice, possessing the Rb7 chromosomal marker, with CBA, C57/B1, Swiss or F1 BALB/SJL females were carried out to determine whether fetal cells transferred into maternal tissues. Rb7 cells undergoing spontaneous mitosis were sought in maternal blood, bone marrow, thymus, para-aortic lymph nodes, Peyer's patches and liver. Proliferation fetal cells were unequivocally demonstrated in maternal spleen and bone marrow, 12-21 days after mating.

Evolution of alpha-fetoprotein serum levels throughout life in humans and rats, and during pregnancy in the rat.

PIP: A radioimmunoassay method (RIA) with specific antisera for human and rat alpha fetoprotein (AFP) was used to study the evolution of AFP levels in humans and rats, respectively, throughout the life span. Sera of 66 rats, aged 3-70 weeks, as well as sera of 201 clinically well children, 192 healthy blood donors, and 16 persons aged 70-98 were assayed. In humans, AFP levels decreased steeply during the 1st year of life, reaching a low basal range by the end of the 2nd year which is maintained throughout adulthood. Rats showed a similar pattern, but concentrations were higher at every stage of life. Puberty occurs in rats while AFP is still decreasing, whereas in humans the adult basal level is stabilized long before puberty. Maternal serum AFP was studied in 29 rats, and the levels were found to begin a sharp increase on the 10th-13th day of gestation. From Day 13-19, a deceleration of AFP increase was observed which led to a decrease between Day 17 and 19. From Day 19-21, AFP levels increased sharply again. A significant correlation was observed on Day 21 of pregnancy between the number of rat fetuses and the maternal AFP level. Hemihysterectomy led to a reduction of AFP level that was grossly proportional to the number of remaining fetuses. The stable level of AFP that persists throughout adult life is attributed to the stability of this protein's synthesis. Conversely, in pregnancy the evolution of serum AFP curve is dependent on the fetal synthesis, maternal and fetal catabolism, and permeability of the placental barrier.^ieng

The impact of clinical biochemistry on university education in France.

In France, clinical biochemistry, similar to other disciplines of laboratory medicine, is taught in both the regular medical and pharmacy curricula, but medical teaching is oriented more towards the interpretation of laboratory findings than test performance. At present, there is no compulsory program of lifelong continuing education, but it is planned to introduce such an obligation in the near future. The practice of laboratory medicine is regulated strictly by the national Health Administration. Clinical laboratories are multidisciplinary, covering simultaneously clinical biochemistry, microbiology, parasitology, hematology and immunology. The only officially recognized laboratory profession is that of 'Director of a Laboratory for Medical Analysis'. The practice of this profession is only open to physicians and pharmacists, provided they graduated in 'Medical Biology' after 4 years of specialized training through a particular type of residency called the 'internat'. The 'interns' are selected by competitive examination. After completing their curriculum, specialized physicians or pharmacists can without further examination or certification either enter a career in a hospital, a university, or both, or direct or co-direct a private laboratory. In this scheme, clinical biochemistry exists as a separate academic discipline, but barely as a distinct profession.

Distribution of Trop 3 and 4 antigens as defined by monoclonal antibodies raised against a human choriocarcinoma cell line.

The reactivities of Trop 3 and 4 monoclonal antibodies (MAbs) were studied on human term and 7-week extraembryonic membranes, adult tissues, and cell lines. Trop 3 MAb reacted with cells of chorionic villi, decidua, amniotic epithelium, and basal plate trophoblast. Trop 4 MAb reacted only with syncytiotrophoblast. On epithelium, Trop 3 MAb bound to stratified and glandular epithelium, but Trop 4 MAb reactivity was limited to basal keratinocytes. On peripheral blood mononuclear cells, Trop 3 MAb reacted with the majority of cells and Trop 4 MAb with the totality. Most cell lines were positive with both MAbs. However, one chemically induced MHC mutant was negative and another decreased its expression of Trop 3 antigen. Our results suggest Trop 3 MAb might recognize a monomorphic determinant of TLX antigens and Trop 4 is involved in cell proliferation or in cell-to-cell interaction.

Alpha-fetoprotein favours accumulation of estrone but not arachidonic acid into the fetal and new-born rat brain.

Tritriated estrone or arachidonic acid, two high affinity ligands for rat alpha-fetoprotein (AFP), were injected into adult, pregnant or newborn Sprague Dawley rats in order to evaluate their possible transfer into the brain. This study shows that the developing brain accumulates the estrogen but not the fatty acid, suggesting that the uptake of AFP by the developing brain is a mechanism for transporting estrogens, but not fatty acids.

In vivo regulation of ovarian activity by alpha-fetoprotein.

Adult female rats, each injected with 760 microgram alpha-fetoprotein (AFP) weekly, presented after the third week a decrease of ovarian activity evidenced by a decreased number of maturing follicles, corpora lutea, and a drop in ovarian weight. The level of progesterone was low and agreed with the decreased number of corpora lutea. Conversely, total and free estradiol-17 beta levels remained normal despite the known ability of AFP to bind estrogenic hormones. A regulatory role of AFP during the postnatal stage of sexual maturation is proposed.

Antibody quantitation in human and veterinary medicine.

The determination of antibodies is a matter of clinical importance since it may prove or disprove the existence of an immune reaction against a specific foreign or autologous antigen. It is useful to monitor the immune response against a vaccine or the disappearance of injected therapeutic antibodies and in fundamental immunological research or in industrial R & D. However, this determination of this particular analyte cannot end up with a significant mass measurement, as a result of the heterogeneity of the antibodies present in most samples (with the exception of monoclonal antibody research). This heterogeneity has multiple facets concerning the constant region (isotypes) and the variable region (degeneracy of the antibody response). What is measured is a binding activity of the antibodies for the corresponding antigen. Calibration is a challenge impossible to carry out. In all cases, the selection of an antibody calibrator results from a compromise. The expression of the results can be done in a variety of ways, and presently is far from being standardized. The present state of the art is characterized by the absence of reference method, the scarcity of reference preparation both for antibody and antigen and the lack of comparability of the methods. Improvement can be expected from the better knowledge of antigens and of their epitopes, the selection and the preparation of pure antigen molecule or synthetic molecules. There are a few technical possibilities to approach a better standardization. Finally, the results of such standardized methods should be interpreted operationally, without reference to ponderal estimations, for a specific clinical or epidemiological purpose. Each method should be validated by epidemiological studies.

Standardization of immunoassays.

The aim of standardization is to ensure that assays of the same analyte in the same samples, done at different places or at different times or both, can be readily compared. Standardization is especially desirable for immunoassays because all external quality assessment surveys have shown that this type of method involves a much greater variability than traditional assays. A major problem in immunoassays is that the recognition of the analyte is determined by the reagent (i.e. an antibody or conversely, if antibodies are to be determined, an antigen) using a limited portion of the molecule, the epitope or the paratope, respectively. Therefore, the specificity of whole process is questionable, even if the specificity of the epitope/paratope reaction is fair. Biological variability is such that many molecules possessing the same recognition site coexist in biological fluids. A first example is the different secretion forms of hormones, their fragments, and other related hormones which occur simultaneously in the blood. A second example is the high variety of antibodies possessing the same paratope. Many other examples can be cited. Thus, a precise definition of the analyte should be given, dependent on the aim of the assay. This is a major conceptual difficulty because several different molecules having the same recognition site may cooperate in a physiological function. In this context, it may be useful to assay them simultaneously, but it is impossible to express their concentrations in common mass units. Another major difficulty in immunoassay standardization is due to the fact that such methods operate at very low concentrations and are sensitive to the environmental conditions created by ambient molecules existing in much higher concentrations. These "non-specific" effects create problems of non-comparability due to the prevalent conditions in different samples. From the different causes mentioned above, it follows that the major difficulty is to ensure perfect identity of the conditions in the calibrator and in the sample (analyte and matrix). The current procedures of assay standardization are reviewed and their deficiencies are stressed. Recent proposals for rationalization of standardization of immunoassays are described.

[Principles and methods of immunological diagnosis in infectious and parasitic pathology]. / Principes et méthodes du diagnostic immunologique en pathologie infectieuse et parasitaire.

Immunological methods have always represented a major contribution to the diagnosis of infectious diseases. Recent technical advances in immunochemistry make this contribution even more essential than in the past. The author reviews the classic principles of immunological diagnosis in infectious pathology. In the best cases, this diagnosis is based on parasite identification (in the broad sense), or identification of specific products from the parasite. It is also possible to consider shortening culture time, when necessary, by performing an early immunological identification. The most recent developments in this field are presented. When the identification of the parasite or its secretion products is not feasible, one must be content to demonstrate the occurrence of a specific immune response, essentially of humoral nature. The author reminds of the basic theoretical difficulties preventing a real analytic antibody titration. The importance of the expression of the results as well as the predictive value of the tests, is stressed. Finally, the author analyzes some of the recent advances such as the development of immuno-enzymatic techniques, rapid testing which can be performed by non-biologists, the contribution of monoclonal antibodies and the possibility of using them in a new detection diagram of antibodies. The author reminds of the difficulty in the standardization of immunological methods, especially the determination of antibodies.

[Enzymo-immunological estimations. Principles and applications (author's transl)]. / Dosages enzymo-immunologiques. Principes et applications.

Diagrams of the reactions of enzymo-immunological estimations are similar to those of radio-immunoassay. The only difference is that an enzymatic label is substituted on the antigen or the antibody for the isotopic label and that consequently a measurement of enzyme activity replaces isotopic counting. One must therefore add a further stage of substrate addition and a stage of enzyme reaction. There is however a technic which has no equivalent in radio-immunoassay, the EMIT system, where one measures the change in activity of a haptenised enzyme under the influence of the corresponding anti-haptene antibody. The author reviews the main problems raised by the first group of technics and gives a few examples of the results of estimation of alpha-foeto-protein obtained with 3 different reactions with fairly similar performances. The advantages and disadvantages of enzymo-immunologic technics are reviewed. The two major advantages seem to be the stability of the reagents and the fact that they avoid the administrative controls which restrict the use of isotopes.