Influence of the antibody-peroxidase coupling methods on the conjugate stability and on the methodologies for the preservation of the activity in time.

We studied the influence of conjugation methods and storage conditions on the stability of immunoconjugates with peroxidase. We demonstrate here that conjugates formed by the maleimide-sulfhydryl method and by the periodate oxidation method lose activity when maintained in diluted solutions. However, while the loss of activity of MSM conjugates is due exclusively to hydrolysis of the thioether bond, the loss of activity of periodate complexes is caused by a reduction of both the enzymatic and antibody immunochemical activities. Based on these observations, we developed a buffer that stabilizes the thioether bond, thus permitting long time storage of these immunoconjugates at low concentration and at above freezing temperatures.

The impact of reagent standardization on the quality of diagnostic devices.

Global quality is becoming a major concern for the medical device and diagnostics industry. Good Manufacturing Practice (GMP) regulations aim to guarantee that effective and safe products are produced. However, the overall quality of a device depends on the quality of its components, which are frequently not manufactured by the manufacturer of the final product. The quality of components, such as reagents can be controlled by setting acceptance criteria, but the standardization of reagents would result in improved quality of the final product. This article briefly reviews the role of GMP regulations with regard to product quality and provides examples of how the application of modern technologies can, and in some cases has, contributed to the realization of standardizable reagents.

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.

Autologous peritoneal mesothelial cell implant in rabbits and peritoneal dialysis patients.

Human and rabbit peritoneal mesothelial cells have been successfully cultured and autoimplanted. An original biopsy technique was used to take samples of peritoneal mesothelial cells and after culture they were characterized from the structural and caryological points of view. Staphylococcal peritonitis was induced in 12 rabbits with indwelling peritoneal catheters and after 4 days of antibiotics 6 of them were autoimplanted with cultured autologous mesothelial cells previously marked in 3 cases with thymidine (H3TdR). Direct morphological observation and autoradiography were used to compare the mesothelium of control rabbits and implanted rabbits sacrificed on days 3 and 6 and showed that the cell implants had taken. Four uremic CAPD patients recovering from severe peritonitis were implanted with about 300 million autologous peritoneal mesothelial cells, previously cultured and frozen. Morphological signs of taking were evident from peritoneal laparoscopy biopsies performed 3 and 6 days after implant. The aim of the study was merely to demonstrate that such implants are possible; however, the techniques may have interesting applications not only in peritoneal dialysis, but also in the vaster fields of medicine and surgery.

Unique structure of glycopeptide from alpha-fetoprotein produced in human hepatoma cell line, as determined by 1H-nuclear magnetic resonance spectroscopy.

Determination of alpha-fetoprotein is used in diagnosis of tumors and neural tube defects. A good reliable source of alpha-fetoprotein would be an obvious advantage to the preparation of diagnostic reagents and their standardization. We have recently developed a method for the production of alpha-fetoprotein from a human hepatoma cell line. This method, which is suitable for scaling up, allowed us to produce 40 g of alpha-fetoprotein from culture supernatant liquid through a simple purification procedure. We have previously shown this protein to be identical to alpha-fetoprotein produced from other sources. However, because the presence of different glycoforms has been reported in alpha-fetoprotein preparations, both from human sources and from other species, it was important to establish the type and extent of glycosylation of alpha-fetoprotein prepared by our method. By using 1H-NMR spectroscopy we were able to establish that our product contains a single N-linked biantennary, fully sialylated complex-type oligosaccharide, typical of human hepatomas.

Sialyglycoconjugates release during in vitro capacitation of human spermatozoa.

Modifications of the human sperm surface during incubation in capacitating conditions were studied by radiolabeling terminal sialic acid residues of cell surface glycoconjugates using the sodium metaperiodate/sodium tritiated borohydride method. During in vitro capacitation, sialyglycoconjugates were released from the human sperm surface according to well reproducible kinetics. This release could be inhibited by the presence of seminal plasma in the capacitation buffer. Two principal size classes of sialyglycoconjugates were detected in the capacitation medium and analyzed by gel filtration chromatography and SDS-PAGE. The smaller class was characterized by glycopeptides less than 5,000 Da, whereas the larger class was characterized by two sialyglycoproteins of approximately 15,000-16,000 and 22,000-23,000 Mr. The role of human albumin, a key component of the capacitation buffer, in the removal of these molecules from the sperm surface was studied in light of its constant association with large amounts of released material.

Synthesis and characterization of a recombinant fragment of human alpha-fetoprotein with antigenic selectivity versus albumin.

A DNA sequence coding for human alpha-fetoprotein amino acid sequence 38-119 was synthesized and cloned in a bacterial expression vector. The alpha-fetoprotein sequence was selected as the least homologous to albumin, since the two proteins have an overall amino acid identity of approximately 38%. A chimeric protein was obtained which was purified by preparative electrophoresis and characterized in its primary structure by fast atom bombardment mass spectometry. About 70% of the alpha-fetoprotein sequence was physically mapped and found to correspond to the amino acids encoded in the synthetic gene. The use of this recombinant protein allowed the selection of monoclonal antibodies recognizing both the recombinant fragment and native alpha-fetoprotein. These antibodies should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.

Monoclonal antibodies recognizing a recombinant portion of human alpha-fetoprotein with antigenic selectivity versus albumin.

Determination of serum alpha-fetoprotein is useful in the clinical management of liver cancer, but it has not been particularly helpful in the early diagnosis of this disease, since also non-neoplastic liver diseases may result in small increases of its serum concentration. To improve the clinical performance of this assay, we have previously developed an in vitro culture system, in which the expression of alpha-fetoprotein and albumin could be coordinately modulated by thyroid hormone. This system allowed large scale production and purification of native alpha-fetoprotein to be used as reference material. In addition, we synthesized and cloned in a bacterial expression vector a DNA sequence coding of human alpha-fetoprotein amino acid sequence 38-119. This alpha-fetoprotein sequence was chosen since it is the least homologous to albumin, being the amino acid sequence of the two proteins extremely similar with an overall identity of about 38%. Now we have obtained three hybridomas recognizing with high affinity and specificity both the recombinant fragment and native alpha-fetoprotein. These antibodies, which therefore recognize the native protein in the amino acid sequence 38-119, should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.

Concomitant purification of prostatic carcinoma tumor markers from human seminal fluid under nondenaturing conditions.

We report a protocol for concomitant purification to homogeneity of both prostatic acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and prostate-specific antigen, from human seminal fluid. The method requires only two chromatographic steps: passage through an Affigel-Blue column and gel filtration HPLC. This is a fast, efficient procedure for purification of these two important tumor markers, which are specific for prostatic cancer.

One-step, high-yield purification of human prostatic acid phosphatase from seminal fluid by gel-filtration HPLC under nondenaturing conditions.

This is a fast, efficient method for purification to homogeneity of human prostatic acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2], from seminal fluid. Use of a "high-pressure" liquid-chromatographic gel-filtration column permits high-yield recovery of the purified enzyme with most of its enzymatic and immunological activity retained.

Correlation between differentiation and lung colonization by retinoic acid-treated F9 cells as revealed by the expression pattern of extracellular matrix and cell surface antigens.

For study of the correlation between differentiation and organ colonization properties of tumor cells, F9 embryonal carcinoma (EC) cells were treated with retinoic acid, an inducer of differentiation; and their organ colonization pattern was assessed by the experimental metastasis assay. Untreated cells were found to colonize the liver, whereas treated cells colonized the lungs. This pattern held true when metastases were scored after spontaneous death or after a careful microscopic search for micrometastases. Histologic examination revealed that both the tumor nodules produced by the untreated and the treated cells had the characteristics of EC devoid of any evidence of differentiation. The immunohistochemical study of the expression of markers typical of embryonal carcinoma cells or of the extracellular matrix components laminin and collagen type IV, typical of differentiated cells, confirmed these results. However, the lack of expression of stage-specific embryonal antigen 1 (SSEA-1), a marker generally associated with the undifferentiated state, observed only in the tumors obtained after injection of treated cells, indicates that the lung nodules probably derive from cells that have responded to the induction in vitro but have dedifferentiated in vivo.

High-yield and high-degree purification of human alpha-fetoprotein produced by adaptation of the human hepatoma cell line Hep G2 in a serum-free medium.

The human hepatoma cell line Hep G2 secretes both albumin and alpha-fetoprotein when grown in the presence of serum. The present report describes how adaptation to growth in serum-free medium results in a progressive switch in the expression of the two proteins; i.e., alpha-fetoprotein becomes the main protein secreted while albumin production is greatly reduced. The culture supernatant obtained, being very enriched in the protein, allows the development of a purification procedure by preparative electrophoresis. By this procedure it is possible to easily obtain large amounts of alpha-fetoprotein from a constant and unlimited source. The availability of these protein preparations should improve the reproducibility and the quality of standardization in clinical immunoassays for alpha-fetoprotein and should permit a more accurate study of the structure and biological functions of the protein.

Analysis of F9 embryonal carcinoma cell lactosaminoglycans in relation to their differential expression during induction of differentiation.

Teratocarcinoma stem cells can be used to study the events related to early differentiation, and many cell surface changes have been described which correlate with the different stages of early embryogenesis. In this work we analyze the [3H]galactose-labeled glycopeptides derived from the mouse embryonal carcinoma cell line F9. We show that the high-molecular-weight glycopeptides typical of embryonal carcinoma cells are composed of two distinct molecular weight classes, namely H1 and H3, and that retinoic acid-induced differentiation determines a relative increase of the larger peak (H1) which is mainly due to a decrease in the expression of H3 species. We also show that, beside this decrease, there is a greater increase in the expression of lower-molecular-weight species. Furthermore, we present evidence that H1 and H3 species are polylactosaminoglycans N-linked to the peptidic backbone, and that induction of differentiation determines slight modifications in the structure of such species.

Organ colonization pattern of retinoic acid-treated and -untreated mouse embryonal carcinoma F9 cells.

The mouse embryonal carcinoma cell line F9 differentiates into parietal endoderm cells after a 3-day exposure to retinoic acid and dibutyryl cyclic AMP. Using the experimental metastases assay, we investigated the organ colonization properties of RA-treated and -untreated populations of F9 cells. The results show that untreated F9 cells colonize the liver with a high degree of specificity while the treated populations colonize the lungs. Cells derived from a lung colony colonized only the liver unless they were treated with RA. However, removal of the inducer from culture of differentiated cells did not cause reversal of the lung colonization potential. Our observations also indicate that it is unlikely that lung colonization is due to cell aggregation or to interaction between differentiated and undifferentiated cells. Taken together, these results suggest that RA induces the observed changes of organ colonization properties of F9 cells.

Identification of a novel glycopeptide species that correlates with differentiation of F9 cells.

The high-molecular-weight fucosyl glycopeptides of differentiated F9 cells have been analyzed. We found that these high-molecular-weight surface structures contain two components with different molecular weights, the largest of which, peak I, has never before been reported. The material eluting in this peak seems to contain only acidic species. Removal of sialic acid from both the peak I and the peak II species does not eliminate the differences in molecular weight, indicating that the two species have more profound structural differences than can be accounted for by sialic acid. Since peak I glycopeptides were found both in differentiated F9 cells and in two parietal endoderm cell lines, we suggest that its presence is related to parietal endoderm differentiation.

[Observations on the diffusion of hepatitis A and B virus during an epidemic in a Sicilian town with a high rate of infection]. / Osservazioni sulla diffusione di virus epatitici A e B durante una epidemia in un comune Siciliano con alta prevalenza di infezioni.

Epidemiological investigations were carried out during a viral hepatitis outbreak occurring in a Sicilian town of 30,000 inhabitants with poor sanitary standards, with the aim to study the mode of spread of HAV and HBV in conditions of high incidence of infections. HBsAg, anti HBs, anti HAV (RIA), HBeAg, anti HBe and anti HBc were investigated in serum samples from patients, their family contacts and from healthy individuals of different age groups. Morbidity was inferred from case notifications; search of unreported cases among school children, through the study of absenteeism, did not reveal further cases. In all 148 cases, occurred from August 1976 through July 1977 with a peak in January, 44% were under 5 and 93% under 10 years of age. All but 8 of 59 cases in which laboratory data were available were due to HAV. Anti HAV antibodies were highly prevalent in serum samples obtained in February through April 1977: 62% were positive in the 1 to 3 years age group, and more than 90% among school children. Prevalence of HBsAg was age and sex dependent, ranging from 4% to 15%; anti HBs was present in 8% of the children 1-10 years and in 30% or more in age groups 30-41 and over. It is suggested that direct contact between very young children was the main mode of spread of HAV, and inapparent cases the main source of infection, although ambient diffusion through water contamination could not ruled out. HBV was probably propagated mostly by intrafamilial spread with little overt pathology.

Requirement of a cell division step for stalk formation in Caulobacter crescentus.

Penicillin G at low concentrations blocked cell division in Caulobacter crescentus without inhibiting cell growth. The long filamentous cells formed after two to three generations under these conditions had a stalk at one pole and usually one or more flagella at the opposite pole. The failure of the filaments to form a second stalk at the flagellated pole indicates that stalk formation was dependent upon completion of a step that was also required for cell division. Two observations support this conclusion. (i) Penicillin did not stop the normal development of synchronous swarmer cells into stalked initiation and stalk elongation. (ii) When the action of penicillin was reversed by the addition of penicillinase to cultures of filaments, stalks were not formed at the nonstalked pole until after cell division had occurred; thus the normal order of development events was maintained: cell division leads to stalk formation. These results are consistent with a model in which the organization of the developmental program for stalk formation occurs before cell division as a consequence of steps that branch from the cell division pathway.