Identification of lung major GTP-binding protein as Gi2 and its distribution in various rat tissues determined by immunoassay

Antisera were raised in rabbits against the 40-kDa alpha subunit of bovine lung GTP-binding protein, which were identified as the alpha subunit of Gi2 (Gi2 alpha) by the analysis of the partial amino acid sequence. Antibodies were purified with a Gi2 alpha-coupled Sepharose column and then were passed through a Gi1 alpha-coupled Sepharose column to remove antibodies reactive also with 41-kDa alpha. Purified antibodies reacted with Gi2 alpha, but not with Gi1 alpha, Gi3 alpha, or Go alpha in an immunoblot assay. A sensitive enzyme immunoassay method for the quantification of Gi2 alpha was developed by using these purified antibodies. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 1 fmol, or 40 pg. Samples from various tissues were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Gi2 alpha were determined. Gi2 alpha was detected in all the tissues examined in the rat. The highest concentration was found in platelets and leukocytes when the data were expressed as picomoles per milligram of protein. The spleen, lung, and cerebral cortex contained relatively high levels of Gi2 alpha. In the bovine brain, Gi2 alpha was distributed almost uniformly among the various regions. The concentrations of Gi2 alpha were constant in the rat brain throughout ontogenic development, in contrast with those of Go alpha which were markedly increased with age.     

Highly Sensitive Immunoassay for Rat Brain-Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.     

Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain

Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha.     

Sensitive immunoassay for rat parvalbumin: tissue distribution and developmental changes

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.     

Developmental and Age-Dependent Changes of 28-kDa Calbindin-D in the Central Nervous Tissue Determined with a Sensitive Immunoassay Method

For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin-D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4-5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human Brain-Type Glycogen Phosphorylase: Quantitative Localization in Human Tissues Determined with an Immunoassay System

Abstract: Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity. Antisera were developed in rabbits with purified phosphorylase as the immunogen. Antibodies were first affinity-purified with a column of brain phosphorylase-coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase-coupled Sepharose to remove antibodies reactive also with muscle phosphorylase. By using the specific antibodies, a sandwich-type immunoassay system for measurement of brain phosphorylase was prepared. The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab')₂ fragments and the same antibody Fab' fragments labeled with β-D-galactosidase from Escherichia coli. The assay was sensitive and specific to the brain phosphorylase. The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross-reactivity of the assay with muscle phosphorylase was <1%. Tissue concentrations of immunoreactive brain-type phosphorylase were estimated. The phosphorylase was present in the heart at as high a level as in the brain. The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis. Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex.     

Monoclonal antibodies to thromboxane synthase from porcine lung. Production and application to development of a tandem immunoradiometric assay

Two hybridoma cell lines secreting antibodies against thromboxane synthase of porcine lung were produced. Clone TS1 secretes IgG2a antibody of lower affinity, while clone TS2 secretes IgG1 antibody of higher affinity. Both antibodies (when bound to rabbit anti-mouse IgG-Staphylococcus aureus complex) can immunoprecipitate thromboxane synthase from crude enzyme preparations in an active form suggesting that binding was not directed at the active site. Each antibody showed a distinctive pattern of cross-reactivity with thromboxane synthase from different porcine tissues. Neither of the antibodies cross-reacted with the enzyme from tissues of other species tested, indicating the heterogeneous nature of the enzyme among species. Competitive binding assay revealed that TS1 and TS2 recognized different determinants on the enzyme. The fact that two antibodies bind to separate epitopes on the same enzyme allows the development of a sensitive tandem immunoradiometric assay. The assay, based on binding of 125I-TS2 to thromboxane synthase immobilized on TS1-S. aureus complex, was linear with 7.5 approximately 75 ng of purified lung thromboxane synthase as standards and applicable to enzyme preparations regardless of their purity. The concentration of immunoreactive thromboxane synthase in porcine tissues as determined by this assay followed the order of platelet greater than colon greater than duodenum greater than lung greater than kidney greater than stomach. The fact that gastrointestinal tract is enriched with thromboxane synthase suggests that thromboxane may have significant physiological roles to be recognized in these organs.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain

Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column. Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined. The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.     

Immunochemical and immunohistochemical localization of the G protein Gi1 in rat central nervous tissues

Antisera were raised in rabbits against purified alpha subunit of G protein Gi1 (Gi1 alpha) and also against a synthetic decapeptide corresponding to a sequence of Gi1 alpha. Antibodies in both antisera were purified with a Gi1-coupled Sepharose column, but purified anti-Gi1 alpha protein antibodies still reacted equally with both Gi1 alpha and Gi3 alpha, while anti-Gi1 alpha peptide antibodies reacted principally with Gi1 alpha. Using these antibodies, an enzyme immunoassay method for the quantification of Gi1 alpha was developed. The assay system consisted of polystyrene balls with immobilized anti-Gi1 alpha protein antibody F(ab')2 fragments and the anti-Gi1 alpha peptide antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 25 fmol of Gi1 alpha, and it did not cross-react with Gi2 alpha, Go alpha, or beta gamma. Samples from various regions of the rat central nervous system were homogenized in a 2% sodium cholate solution, and the concentration of Gi1 alpha in each extract was determined. Gi1 alpha was detected in all the regions, and the highest concentration was found in the olfactory bulb. Immunohistochemical study showed that Gi1 was mainly localized in the neuropil.     

Sex-Dependent and Sex-Independent Distribution of the β-Subunit of Nerve Growth Factor in the Central Nervous and Peripheral Tissues of Mice

Levels of the beta-subunit of nerve growth factor (beta-NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich-type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. beta-NGF-specific antibody isolated on a column of Sepharose CL-4B coupled with purified beta-NGF reacted only with beta-NGF. The assay for beta-NGF was performed by incubation of F(ab')2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab' fragments labeled with beta-D-galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of beta-NGF in almost all organs of mice. The amount of beta-NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3-5 mg/g of tissue). In serum, some of the 2-month-old males, but none of the females, exhibited a fairly high level of beta-NGF (greater than 100 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.     

Monoclonal Antibodies to Mammalian Carnosine Synthetase

A set of mouse monoclonal antibodies has been generated against rabbit muscle carnosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross-reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low-abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.     

Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles

The tissue distribution of muscle-type alpha beta and beta beta enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the alpha and to the beta subunit of enolase, and beta-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both alpha beta and beta beta enolases. The beta beta enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The alpha beta enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The beta beta enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the alpha alpha and alpha beta enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of beta beta enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum longus (about 800 pmol/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of alpha alpha (2 pmol/mg) and alpha beta (80 pmol/mg) usually showed a slight increase by the treatment (alpha alpha, 7 pmol/mg; alpha beta, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of beta beta enolase is closely correlated with the functional state of the muscle tissue.     

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay

Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme‐linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity‐purified anti‐C‐terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin‐linked horseradish peroxidase. Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2‐ to 5‐fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or β‐estradiol, in cultured adipocytes reduces resistin protein levels in a dose‐dependent manner. Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.     

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.     

The effect of the neonatal Fc receptor on human IgG biodistribution in mice

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an ¹²⁵I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0–96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.     

Tissue distribution and kininogen gene expression after acute-phase inflammation

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykinin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.     

The effect of the neonatal Fc receptor on human IgG biodistribution in mice

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an ¹²⁵I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0–96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.