First and Second Metatarsophalangeal Joint Open Dislocations: A Case Report.

Dislocation of multiple metatarsophalangeal joint is an uncommon injury. The mechanism of injury is a high energy force distal to proximal with foot in hyperextension at the metatarsophalangeal (MTP) joint. The acute hyperextension of the toe at the moment of injury causes avulsion of the plantar part of the capsule from the junction of head and neck of the metatarsal. If the collateral ligaments remain intact, they maintain the locked fibrocartilaginous plate over the dorsum of the head of the metatarsal, making closed reduction impossible. We report a case of simultaneous 1st and 2nd MTP joint open dislocation. In the present case, we chose the plantar approach utilizing the already present plantar wound. At 18 months post-operative follow-up, there was no instance of redislocations or signs of avascular necrosis of head of metatarsal.

Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin.

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg1-bradykinin and des-Arg9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.

The carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin.

The complete carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin was elucidated through chemical and enzymatic methods including gas chromatography-mass spectrometry (GC-MS) and lectin affinity chromatography. Pronase digestion of thiostatin yielded a major glycopeptide fraction with asparagine the most abundant amino acid present. Based on one mole of aspartic acid, the following molar ratios obtained for the four major amino acids: aspartic acid (1.0), threonine (0.53), glycine (0.48) and serine (0.30). Neutral sugar analysis yielded a 3:2 molar ratio for mannose to galactose based on an assigned value to mannose of 3. On this basis, the fraction also contained 3 residues of sialic acid and, on average, 0 to 1 residue of fucose. GC-MS of partially methylated alditol acetates from the glycopeptide fraction identified the presence of biantennary and triantennary structure. Analyses of the neutral sugar and amino-acid composition, together with methylation data, support a biantennary N-linked structure for this major glycopeptide fraction and a triantennary N-linked structure as a lesser component. Sequencing of the desialyated 14C-labelled glycopeptide fraction by sequential exoglycosidase digestion and lectin affinity chromatography uncovered the following saccharide order: terminal galactose, N-acetylglucosamine and pentasaccharide inner core. This sequence is consistent with the N-linked glycan structures demonstrated by methylation and compositional analyses.

Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins.

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.

Rodent kinin-forming enzyme systems--I. Purification and characterization of plasma kininogen.

Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 micrograms bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelectrophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).

Rodent kinin-forming enzyme systems--II. Purification and characterization of an acid protease from Murphy-Sturm lymphosarcoma.

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was purified 640-fold by extraction of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation, DEAE-Sephadex A-50 batch adsorption, QAE-Sephadex A-50 column chromatography, Sephadex G-200 gel filtration, and CM-32 cellulose chromatography. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. Two protease fractions obtained by Sephadex G-200 gel filtration had identical molecular weights of 39,500-41,000 and were similar in other physico-chemical and kinetic characteristics. The purified enzyme showed three major isozymic forms (alpha, beta and gamma) with isoelectric points (pI) of 5.2, 5.5 and 5.8, respectively, and nearly identical amino acid compositions. The enzyme had a high moles percent of both aspartic and glutamic acids. The carbohydrate moiety of the enzyme contained 2 moles of N-acetylglucosamine and 8 moles of mannose per mole of enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0 with a sharp decline of activity on either side of the pH optimum. The protease activity was very stable above pH 3.4. The Km values for the purified enzyme fractions A and B were 31.17 and 31.19 microM, respectively, and the corresponding Vmax values were 6.17 and 5.5 microM tyrosine per mg per min at 37 degrees and pH 3.0. The enzyme was inhibited strongly by pepstatin (Ki = 31 X 10(-9)M and alpha = 0.1). The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents/mg substrate after a 2-hr incubation at 37 degrees.

The effects of autonomic drugs on the concentration of kallikrein-like proteases and cysteine-proteinase inhibitor (cystatin) in rat whole saliva.

The influence of isoproterenol, phenylephrine, propranolol, and reserpine on the salivary concentration of kallikrein-like proteases and cysteine-proteinase inhibitor (cystatin) was investigated. The protease activities in saliva from treated rats were studied by means of five different chromogenic substrates. In the isoproterenol- and phenylephrine-treated groups, a significant decrease in protease activity was found, compared with the control group. The protease activity of saliva was found to be elevated by about 25-50% after chronic administration of reserpine (0.5 mg/kg). Specific polyclonal antibodies to rat glandular kallikrein and cystatin were utilized to determine the salivary concentrations of these proteins. Results from the use of anti-kallikrein antibodies in Western blot analysis and crossed immuno-electrophoresis indicated that differences observed in the kallikrein-like protease levels of saliva from treated animals were due to altered immunoreactive protein levels. The salivary concentrations of kallikrein and cystatin were measured by direct radio-immunoassays with specific antibodies. The concentration of cystatin in the saliva of normal animals or animals treated with reserpine or propranolol was very low, but was increased about 100-fold in phenylephrine-treated animals and more than 5000-fold in isoproterenol-treated animals. Western blot analysis with antibodies to submandibular gland mucin, glutamine/glutamic-acid-rich protein (GRP), and proline-rich proteins (PRP) were also utilized to compare the effects of autonomic drugs on these salivary proteins. The salivary mucin showed an increase in reactivity and increased mobility in saliva from both isoproterenol- and phenylephrine-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The association of basic proline-rich peptides from human parotid gland secretions with caries experience.

To address whether there are associations between the peptide composition of human parotid saliva and dental decay (caries) experience, we have characterized the peptides from parotid ductal saliva collected from nine adults who have remained free from dental caries (mean age = 59.2; Decayed Missing Filled Surfaces index [DMFS] = 0) and nine individuals who have experienced caries (mean age = 51.2; mean DMFS = 38.4). Ethanol-soluble peptides were size-fractionated on columns of Bio-Gel P-2; the salivary peptides derived from caries-susceptible subjects appeared larger than those found in the saliva of caries-free subjects. Peptides were then resolved into 19 species by cation exchange HPLC. Sequence analysis identified 18 peptides that appear to be proteolytic cleavage products of the basic proline-rich proteins IB-4, IB-5, IB-7, IB-8b, and P-B. The peptides that were more abundant in saliva obtained from the caries-free group differed from those isolated from the caries-susceptible group. The median peptide concentration of one possible precursor protein, IB-7, was found to be higher in saliva collected from caries-free individuals than in that from caries-susceptible individuals. Although differences were found in the phenotypes of proline-rich proteins expressed by these groups of caries-free and caries-susceptible subjects, no statistically significant associations were observed among proline-rich phenotypes and the level of any peptide. Collectively, our results indicate that proteolytic processing of parotid salivary proteins differs among individuals who have remained caries-free and those who have experienced dental decay.

The effect of adrenergic agonists and antagonists on cysteine-proteinase inhibitor (cystatin) in rat saliva.

The effect of a number of adrenergic agonists and antagonists on the induction of rat salivary cystatin was investigated. A highly sensitive and specific radioimmunoassay was used to determine cystatin in rat whole saliva. Treatment for 10 consecutive days with a non-specific beta-adrenergic agonist isoproterenol, or the beta 1-adrenergic agonists dobutamine or methoxyphenamine, resulted in the induction of the salivary cystatin. Induction was also found in rats treated for 10 days with arterenol. Only trace quantities of cystatin could be detected in saliva of rats treated with the beta 2-adrenergic agonists terbutaline or salbutamol. When isoproterenol was injected concomitantly with the mixed beta-antagonist propranolol or the beta 1-adrenergic antagonists metaprolol, proctocol or atenolol the production of cystatin was totally suppressed. However, the beta 2-antagonist, ICI 118551, produced only a partial reduction in salivary cystatin induction elicited by isoproterenol. The findings suggest that the induction of salivary cystatin is regulated, in part, by beta 1-adrenergic receptor stimulation.

Production of rat salivary cystatin S variant polypeptides in Escherichia coli.

Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.

Induction of type 2 cystatin in rat submandibular glands by systemically administered agents.

An inducible type 2 cystatin has earlier been characterized in submandibular glands and kidneys of rats treated with isoproterenol, as well as in kidneys of rats with experimental renal disease. The purpose now was to determine whether giving agents that have systemic toxicity could also be associated with induction of cystatin in rat salivary glands. Female Wistar rats (200-250 g) were given isoproterenol, cyclocytidine, potassium dichromate or turpentine oil. After autopsy, the organs were sectioned, fixed in 10% formalin, and processed routinely. Paraffin sections were processed for both the peroxidase-antiperoxidase and the avidin-biotin-alkaline phosphatase immunocytochemical methods. The submandibular glands of rats given cyclocytidine had generalized, strong staining of acinar cells, as well as occasional weak staining within granular convoluted tubules. Animals given either potassium dichromate or turpentine oil exhibited moderate staining for cystatin in submandibular acini. Rats given isoproterenol as a positive control exhibited strong acinar staining throughout the submandibular gland, while the glands of untreated rats were unreactive. Inducible type 2 cystatin could not be detected in the parotid or sublingual glands, or in trachea, lung, stomach, small intestine, large intestine, spleen, liver and pancreas, after treatment with any of the systemic agents evaluated. The results indicate that elaboration of type 2 cystatin can be induced by a variety of systemically administered agents other than isoproterenol, and suggest that elaboration of type 2 cystatin may represent a more generalized response to tissue injury.

Phenotypic characterization of resident macrophages in submandibular salivary glands of normal and isoproterenol-treated rats.

Macrophages exert a major effect in the stimulation of lymphocytes and the modulation of immunological responses. To determine the presence and phenotypic distribution of the resident cells of the mononuclear phagocyte system in submandibular glands, frozen sections were prepared from five normal rats, and from six rats treated with 20 mg/kg isoproterenol/day for 10 days. A panel of six monoclonal antibodies was used to identify membrane markers associated primarily with circulatory monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), Ia antigen (OX6), CD5-positive T lymphocytes (OX19) and rat B lymphocytes (OX33). Cells identified by each monoclonal antibody were quantified by averaging the number of positive cells in 10 consecutive random high-power fields. ED2 cells (165 cells/field) were predominant in normal rat submandibular gland, followed by lower numbers of OX6-positive cells (18 cells/field). Cells positive for the remaining markers were also present in smaller amounts. In submandibular glands, treatment of rats with isoproterenol resulted in an increase in ED1-positive cells (from 2 to 39 cells/field), but also in substantial decreases in the number of cells positive for the remaining cell markers. B cells were not detected in any of the submandibular glands examined. These data suggest that isoproterenol induces a mild inflammatory response within rat submandibular glands that is not observed in normal glands. This results in an increase in the relative number of infiltrating monocytes compared to the number of more mature tissue macrophages.

Expression of a functional rat salivary cystatin S polypeptide in Escherichia coli.

Cystatin S is a cysteine proteinase inhibitor that is transiently expressed during rat submandibular gland development and can be induced by isoproterenol in the adult. A cDNA for rat cystatin S which included the entire coding sequence of the secreted cystatin was cloned. A coding region of the cystatin gene was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector. The chimeric plasmid was transformed into Escherichia coli, and protein expression was induced by isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and fast protein liquid chromatography on a MonoQ column. The purified recombinant cystatin reacted with antibodies to cystatin S purified from rat submandibular glands and showed an amino-terminal amino acid sequence identical to that of rat cystatin S. The recombinant protein exhibited papain inhibition activity comparable to natural cystatin. This was a successful expression and purification of a functionally and immunologically reactive recombinant cystatin from E. coli, an approach which will be used later towards generating recombinant variants to study the binding and functional domains of this cysteine protease inhibitor.

Further characterization of monoclonal antibodies against rat plasma kallikrein, rat low molecular weight kininogen and synthetic bradykinin.

Monoclonal antibodies (MAbs) to rat plasma kallikrein, rat plasma low molecular weight kininogen and synthetic bradykinin were characterized further. The MAbs were obtained after the immunization of BALB/c mice with the purified reagents and synthetic bradykinin coupled to ovalbumin. The MAbs were very useful in characterizing components of the kallikrein-kininogen-kinin system. Plasma kallikrein MAbs bound specifically to both rat and human plasma kallikrein but did not interact immunologically with either rat or human glandular kallikrein. The MAbs immunoprecipitated about 75% of the kallikrein enzyme activity. Five stable hybridomas were obtained that produced MAbs against bradykinin. These MAbs cross-reacted with bradykinin, bradykinin analogues, purified rat plasma kininogen and tryptic digests of rat plasma kininogen. The MAbs also neutralized the smooth muscle contractile activity of bradykinin. The specificity of MAbs against rat plasma kininogens was confirmed by immuno-precipitation of 125I-kininogen with MAbs followed by SDS-PAGE. These MAbs were used successfully to develop immunoassays (ELISA) and immunoadsorbents to purify components of the kallikrein-kininogen-kinin system.

Monoclonal antibodies to rat plasma kininogen.

Spleen cells from Balb/c mice immunized with purified rat plasma kininogen were fused to P-3 mouse myeloma cells. Positive clones were identified by enzyme linked immunosorbent assay (ELISA), cloned successively two times with limiting dilution and expanded as ascites tumors. Five hybridomas were developed that produced monoclonal antibodies against plasma kininogen. Two of the secreted antibodies were of the IgG1 (k) isotype and the remaining three were of the IgG1(lambda), IgG2A(k) and IgM(k) isotypes respectively. The specificity of the monoclonal antibodies was confirmed by the immunoprecipitation of kininogen with the antibodies coupled to Sepharose-4B followed by SDS-polyacrylamide gel electrophoresis. These monoclonal antibodies recognize at least two distinct epitopes on rat plasma kininogen.

A rapid radioimmunoassay for rat plasma kininogen.

The assay of plasma kininogen most often relies on bioassay techniques that measure biological effects of vasopeptides formed by proteolytic action on the substrate. These assays are tedious, time consuming, subject to the variations in tissue response and expensive. In the present report a sensitive and rapid radioimmunoassay for rat plasma kininogen is described. Polyclonal antibodies of high avidity and specificity were produced in rabbits immunized with purified rat plasma kininogen. Optimal conditions for each step of the assay were established from a series of initial experiments. Fifty percent (50%) precipitation of 125I-kininogen was obtained with a 1:100,000 dilution of antirat plasma kininogen antibody. Precipitation of the antigen-antibody complex was achieved with 10% polyethylene glycol in 0.05 M phosphate buffer, pH 7.5. Since precipitation with polyethylene glycol requires no incubation, this procedure eliminates lengthy waiting periods and the need for a second antibody. The assay can be completed within one day. A standard curve constructed by plotting the fraction of bound 125I-kininogen against the plasma kininogen in a logit-log mode yielded a straight line for kininogen concentrations between 0.1 and 30 ng. The lowest limit of detection of the rat plasma kininogen was 0.1 ng.

Purification and characterization of an acid protease and vasopeptide kinins from Murphy-Sturm lymphosarcoma.

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was isolated, purified and characterized. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. The purification was carried out in seven steps involving homogenization of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation to 60% saturation, DEAE-Sephadex batch treatment, chromatography on a QAE-Sephadex column, gel filtration on Sephadex G-200 and finally chromatography on CM-32 cellulose. The purification scheme resulted in a 640-fold purification and a homogeneous preparation as confirmed by disc gel electrophoresis and Ouchterlony immunodifussion. Sephadex G-200 elution profiles indicated two different protease fractions containing protease activity which showed a single band with identical relative mobility on polyacrylamide gel electrophoresis in the presence and absence of SDS, and also showed immunological cross-reactivity against anti-acid protease antiserum raised in the rabbit against one of the fractions. The acid protease is presumed to be a single protease which has a tendency to aggregate. An apparent molecular weight of 39,500 was estimated by gel filtration on Sephadex G-200, and 41,000 by polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis showed that the acid protease was comprised of a major band with a molecular weight of 4,000 and two fainter bands with molecular weights of 27,000 and 12,000. The purified enzyme showed three major isozymic forms (alpha, beta, gamma) which had isoelectric points (pI) of 5.2, 5.5 and 5.8 respectively, and had near identical amino acid compositions. The carbohydrate moiety contained 2 mol of N-acetylglucosamine and 8 mol of mannose per mol enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0. The protease activity was very stable above pH 3.4. The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents per mg substrate after 2 hr incubation at 37 degrees. The vasopeptide was purified to electrophoretic homogeneity by gel filtration on Sephadex G-50 and chromatography on a column of CM-Sephadex. The amino acid composition of vasopeptide was Ser2, Gly1, Pro4, Ile1, Leu1, Phe2, Arg2.

Production and characterization of monoclonal antibodies to rat plasma kininogen.

Five hybridoma cell lines were established that secrete monoclonal antibodies (MAbs) directed against rat plasma low molecular weight plasma kininogen. Two of the secreted antibodies were of the IgG1k isotype. The remaining three were of the IgG1 lambda, IgG2ak and IgMk isotypes, respectively. The dissociation constants (Kd) of these MAbs ranged from 0.58 X 10(-9) M to 5.4 X 10(-9) M. At least two distinct epitopes on the rat plasma kininogen were recognized by these MAbs. Further characterization of the MAbs showed that four MAbs cross-reacted strongly with kininogen from the Murphy-Sturm lymphosarcoma (MSLS) tumor while one of the MAbs cross-reacted weakly with MSLS tumor kininogen.

Monoclonal antibody to rat plasma kallikrein.

Monoclonal antibody (MAb) was obtained to rat plasma kallikrein after immunization of BALB/c mice with the purified enzyme. Spleen cells from immunized mice were fused to P-3 mouse myeloma cells. Four antibody secreting hybridomas were identified by enzyme-linked immunosorbant assay (ELISA) for production of antibody to rat plasma kallikrein. One hybrid was cloned by limiting dilution and expanded as ascites tumor by injection of 5 X 10(6) cells into the peritoneal cavity of male BALB/c mice primed with 2,6,10,14-tetramethylpentadecane. Antibodies at a dilution as great as 1:20,000 were detected in ascites fluid and sera of immunized animals. The lower limit of detection of kallikrein was 0.1 microgram/ml. Secreted antibodies bound specifically to rat and human plasma kallikrein, but did not show any immunological interaction with human urinary or porcine pancreatic kallikrein. The MAb provides the necessary component for an enzyme-linked plasma kallikrein immunoassay.