Kazuhiko Kuroda

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.

[27]Cell culture assay of biological activity of lactoferrin and transferrin

Publisher Summary This chapter describes the methods for cell culture in serum free medium supplemented with lactoferrin, and compares the biological activities of lactoferrin with those of transferrin. Lactoferrin shares with transferrin the property of reversibly binding two atoms of iron, and presents structural and functional homology with transferrin. Both are monomeric glycoproteins, having two similar oligosaccharide chains, with molecular weights of about 8 × 10 4 . Both molecules possess two independent metal binding sites, each of which can bind a ferric ion together with a bicarbonate anion. The complete amino acid sequence of human serum transferrin ∼6 and partial amino acid sequence of lactoferrin have been reported. It has been reported that lactoferrin may play a role in iron transport in the intestine, and transferrin may not, though lactoferrin and transferrin are similar as described above. It has also been reported that lactoferrin binds to a specific macrophage receptor without competition with transferrin.

Histone H2B as an antigen recognized by lung cancer–specific human monoclonal antibody HB4C5

Histone H2B was demonstrated to be an immunoreactive material recognized by the human monoclonal antibody HB4C5, which had been already established to be specific for lung cancers. The inhibitory effect of histone H2B on the activity of HB4C5 antibody to immunostain the cytoplasmic antigen in lung adenocarcinoma tissue indicated that histone H2B at least had antigenic determinant comparable to the cytoplasmic antigen. A mouse anti-histone H2B monoclonal antibody could immunostain the cytoplasm of lung adenocarcinoma cells in sliced tissue sections in the same manner as the human monoclonal antibody HB4C5. Enzyme-linked immunosorbent assay of HB4C5 antibody on plastic immunoplates coated with histone H2B also showed specific reactivity of this antibody with histone H2B, and the reaction was effectively inhibited when extra histone H2B or mouse anti-histone H2B monoclonal antibody was added to the reaction mixture. These results consistently lead us to a conclusion that histone H2B possesses antigenicity to the human monoclonal antibody HB4C5.

Serodiagnosis of cancer by using Candida cytochrome c recognized by human monoclonal antibody HB4C5

Cytochrome c from various sources, such as Candida krusei, yeast, horse, and cattle, was found to be recognized by human monoclonal antibody HB4C5 specific to lung cancer. Therefore, the cytochrome c was applied to the measurement of antibody amount in patient sera with a similar reactivity to the antibody HB4C5 for serodiagnosis of cancer. The cytochrome c from Candida krusei was most valuable for the serodiagnosis of various cancers, and the yeast cytochrome c was also useful. However, horse and bovine cytochrome c did not react with antibody of the cancer patients. By using Candida cytochrome c lung, bile duct, esophagus, and liver cancers were detected at high rates of more than 50%. In the case of lung cancer, the detection rates of small-cell, squamous, large-cell and adenocarcinoma were 78%, 63%, 100%, and 34%, respectively. The rate for small-cell carcinoma was higher than that with the currently used NSE assay system, and the rate for squamous carcinoma was comparable to that with the SCC assay system, although the system using cytochrome c did not show similar reactivity to that with the SCC system. Furthermore, lung cancer was detected at early stages by using cytochrome c, and even in the case of adenocarcinoma, the rate at early stages with the cytochrome c system was higher than that with the CEA assay system. On the other hand, false positive rates of benign diseases and normal were low--8% and 2%, respectively.

Serodiagnosis of cancer using porcine carboxypeptidase A as an animal antigen recognized by human monoclonal antibody HB4C5

Protein from hog which is recognized by human monoclonal antibody (HB4C5), generated from a patient with large cell lung carcinoma, was identified as carboxypeptidase A by comparison of the protein with carboxypeptidase A in enzymatic activity, immunologic reactivity, and amino acid sequence. Carboxypeptidase A activity was also found in human cancer tissue, and purified antigen from cancer tissue recognized by the antibody HB4C5 was reacted with rabbit anti-carboxypeptidase A serum, indicating that carboxypeptidase A is an antigen of HB4C5. Since large amounts of carboxypeptidase A can be obtained from porcine sources, a simple method for its purification was established. The fraction which was most reactive with HB4C5 was obtained from acetone powder of porcine pancreas by successive applications of water extraction, ammonium sulfate precipitation, trypsin treatment, and Mono Q column chromatography. Its apparent molecular weight was 40,000, according to SDS polyacrylamide gel electrophoresis. When the reactivity of IgG in sera with the purified carboxypeptidase A was measured, the detection rates for lung, ovary, larynx, uterus, and liver cancer were more than 50%, while the rates for stomach and breast cancer were around 30%, and pancreatic cancer, benign diseases, and normal controls were minimally detected.

A human monoclonal antibody to carbohydrate moiety of carcinoembryonic antigen

Hybridoma BSRF-S-97, secreting a human monoclonal antibody of IgG1 subclass reactive to the carcinoembryonic antigen, was generated by fusing the regional lymph node lymphocytes from a cervical cancer patient with RF-S1 human-mouse heteromyeloma fusion line. This monoclonal antibody was found specifically reactive to carinoembryonic antigen-producing cell lines, including those of cervical cancer (SKG-II), mucinous type ovarian cancer (RMUG-L), stomach cancer (MKN-45), and lung cancer (PC-10). The monoclonal antibody reactivity with pepsin- and periodate-treated carcinoembryonic antigen demonstrated that this monoclonal antibody recognizes the carbohydrate moiety of carcinoembryonic antigen specifically. Possibilities of the monoclonal antibody reaction with mucin and blood-group antigens were excluded by the comparative studies with a placental mucin-containing protein which reacted with carcinoembryonic antigen-specific rabbit polyclonal antibody. The monoclonal antibody conjugated with Pseudomonas exotoxin showed potent regression effects on the growth of the MKN-45 cell line in both the dish culture and xenografted nude mice, indicating potential usefulness of this human monoclonal antibody as a promising tumor targeting vehicle.

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination

AbstractFood allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an “amino acid sequence immunoassay” approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%–106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstractThe ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.