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A 64-year-old female presented with rapid onset of left ophthalmoplegia and truncal ataxia, after experiencing diplopia due to left abducens nerve palsy for a year. She had undergone surgery twice for left trigeminal neuralgia caused by a large intracranial epidermoid cyst at the age of 48 and 52 years. The intracranial epidermoid cyst grew and became predominantly enhanced by contrast medium on computed tomography (CT) and T(1)-weighted magnetic resonance (MR) imaging, which had not been observed earlier. The tumor was partially removed and the histological diagnosis was squamous cell carcinoma (SCC). Radiation therapy was administered, but she presented with paraplegia of the bilateral lower extremities and anesthesia due to spinal multiple metastases of SCC one year later. Radiation therapy was administered for the spinal lesions, but she died of multiple metastases to the cerebellum and medulla oblongata with hydrocephalus 2 years after the third surgery. Transformation of intracranial epidermoid cysts to SCC appears as predominant enhancement on CT or T(1)-weighted MR imaging with rapid deterioration of neurological features. All reported cases of malignant transformation of intracranial epithelial cysts to SCC with leptomeningeal carcinomatosis have occurred in intracranial epidermoid cysts.

Background: Adhesion molecules and eosinophils may play an important role in the pathogenesis of allergic inflammatory reactions. Objective: We attempted to clarify eosinophil activation, such as degranulation, by signaling through adhesion molecule and to determine whether degranulation is involved in adhesion molecule expression on endothelial cells. Methods: Eosinophils were cultured with or without recombinant soluble intercellular adhesion molecule-1 (ICAM-1), and the levels of eosinophil cationic protein and eosinophil-derived neurotoxin were determined. The influence of these eosinophil granule proteins or supernatant from eosinophil cultured with ICAM-1 on the expression of ICAM-1 or vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells was also examined by flow-cytometric analysis. Results: Supernatant levels of eosinophil granule protein were significantly increased by culture for 4 hourss or 16 hours with recombinant soluble ICAM-1, suggesting degranulation by adherence to ICAM-1. Both granule proteins and the supernatants of eosinophils cultured with recombinant soluble ICAM-1 induced expression of ICAM-1 and VCAM-1 on endothelial cells, with the latter showing a more prominant increase. Conclusion: Degranulation mediated through adherence to endothelial cells by ICAM-1 and its ligands may be involved in the expression of adhesion molecules, such as ICAM-1 or VCAM-1, on these cells. Our finding of the selective induction of VCAM-1 expression suggests that eosinophil adherence to endothelial cells, even if it is because of ICAM-1, may be involved in selective eosinophil recruitment and accumulation at sites of allergic inflammation. (J Allergy Clin Immunol 1999;103:S452-6.)

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

Sustained release of TAK-029 [4-(4-amidinobenzoylglycyl)-3-methoxycarbonyl-2-oxopiperazine-l-acetic acid], a glycoprotein (GP) IIb/IIIa antagonist, from injectable microspheres was achieved by a W/O/W emulsion solvent evaporation technique using copoly( d -lactic/glycolic)acid (PLGA). Entrapment of the drug into microspheres increased with addition of sodium chloride into an external aqueous polyvinyl alcohol solution. Addition of l -arginine to an internal water phase dose-dependently reduced initial burst of the drug from the microspheres in vitro and in vivo, probably by forming hydrophobic diffusion barriers with rigid inner structure and increased glass transition temperature (Tg). Microspheres obtained using sodium chloride and l -arginine demonstrated sustained plasma levels of TAK-029 for 3 weeks after subcutaneous injection in rats, while causing a slight increase of its plasma levels in 2–3 weeks.

For industrial production of human monoclonal antibodies (hmAb) against hepatitis B virus surface antigen (HBsAg), we scaled-up a short-term perfusion culture in serum-free medium, which was chosen as the most suitable culture method, to a 50-1 fermentor equipped with a rotating shear filter. Using hydrophobic chromatography as the initial step of hmAb purification, the mAb HBW4, HBW6 and W471 were isolated in good quality from the respective culture broths in yields of approximately 75%. Each of the three purified hmAb alone, and a cocktail of the three, protected chimpanzees against HB virus, when injected intravenously 3 h after viral challenge, as long as the serum antibody levels were significant. A pharmacokinetic study using cynomolgus monkeys demonstrated that the hmAb have a long plasma half-life and bioavailability of approximately 76% upon intramuscular injection in primates. Thus, anti-HBsAg hmAb produced by an industrial process are expected to be successfully used in clinical fields.

Nine murine monoclonal antibodies (MAbs) raised against recombinant soluble intercellular adhesion molecule-1 (sICAM-1) were evaluated by means of ELISAs, and their neutralizing activity was investigated in two bioassays employing Raji cells activated with phorbol myristate acetate. Three of the MAbs inhibited autoaggregation of the activated cells and adhesion to sICAM-1 fixed on a plastic plate. Non-neutralizing antibody SM1-255 is directed to an epitope that was not recognized by the other eight MAbs. This property enabled us to develop two ELISAs employing SM1-255 as a liquid-phase antibody for the quantitation of sICAM-1 circulating (cICAM-1) in human plasma. One assay, employing non-neutralizing antibody WIS2-11 as a solid-phase, has a sensitivity of two pg/well with coefficients of variation of 3.6-5.8% (within assay) and 5.5-9.5% (between assay). The other assay, employing neutralizing antibody WIS5-85 as a solid-phase, has a sensitivity of four pg/well with coefficients of variation of 2.7-7.2% (within assay) and 9.2-11.2% (between assay). There was a discrepancy between cICAM-1 levels of human plasma determined by these two assays. All samples showed 2- to 5-times higher levels in the assay using WIS2-11 than in that using WIS5-85. The result from the gel electrophoresis employing Western blotting suggests that ICAM-1 circulates in at least two molecular forms with molecular masses of about 85 and 130 kDa and with different reactivities to WIS2-11 and WIS5-85.

We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 μg ml−1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.

We have investigated the pharmacokinetic alteration in rats of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2. Serum rIL-2 levels after the intravenous administration of the immune complex at a dose of 100 micrograms/rat as rIL-2 were significantly higher than those after intravenous administration of rIL-2 alone at the same dose. Pharmacokinetic analysis indicated that the distribution volume of rIL-2 decreased from 74.0 to 10.3 ml/rat, while the elimination rate of rIL-2 was little changed by immunocomplexing with the antibody. On the other hand, serum rIL-2 levels after the subcutaneous administration of the immune complex at a dose of 100 micrograms/rat as rIL-2 were sustained longer than those after the subcutaneous administration of rIL-2 alone at the same dose, and Tmax shifted from 0.83 to 3.0 h by immunocomplexing with the antibody. Pharmacokinetic analysis also revealed that the mean-residence-time of rIL-2 increased from 1.98 to 6.52 h, and the area-under-the curve of rIL-2 decreased slightly, from 834 to 548 ng.h/ml, by immunocomplexing with the antibody.

We have investigated biological properties of an immune complex of recombinant interleukin-2 (rIL-2) and a monoclonal antibody against rIL-2 in mice for induction of killer cells and for anti-tumor activity. We have also examined the clearance of subcutaneously-injected immune complex in mice and compared it with that of rIL-2 alone. Plasma rIL-2 levels were sustained longer in mice given the immune complex than in mice given rIL-2 alone at a dose of 10 micrograms/mouse, and they were detectable even at 24 hours after the administration of the immune complex, while they fell to undetectable levels by 6 hours after the administration of rIL-2 alone. A more significant portion of rIL-2 was detected in lymph nodes after subcutaneous injection of the immune complex than that of rIL-2 alone. Splenic lymphocytes from mice given the immune complex demonstrated a higher killer cell activity against YAC-1 cells than those from mice given rIL-2 alone. The immune complex also exerted more significant anti-tumor effect in a dose-dependent manner in Meth-A fibrosarcoma-bearing mice than rIL-2 alone. Our results indicate that immunocomplexing of rIL-2 with an antibody against rIL-2 provides a useful tool as the drug delivery system for cancer therapy using rIL-2.

To detect in vivo activated platelets in humans as well as in animal models of thrombosis, we developed a new murine monoclonal antibody, 2T60, specific for activated human and rabbit platelets by immunizing with human thronibin-activated platelets. 2T60 (IgG(1) subclass) showed a great difference between binding to the thrombin-activated and resting human and rabbit platelets on ELISA and flow cytometer analysis. Immunoblotting analysis revealed that 2T60 reacted with a 130 or 106 kDa protein of human or rabbit platelets, respectively, only under non-reducing conditions. (125)I-labeled 2T60 inserted into the intermediate gel of CIE of solubilized human platelets was incorporated into a immunoprecipitation line. 2T60 immunoprecipitated a protein of 130 or 115 kDa from human or rabbit platelets, respectively, which had been activated and (125)I-surface-labeled. The N-terminal sequence of the affinity purified 2T60 antigen of human platelets was identical to that of GMP 140. There were differences in the carbohydrate chain content of GMP 140 between human and rabbit platelets. In experimental cerebral thrombosis of rabbits that had been injected with 2T60, the platelets adhering to the exposed subendothelium and contained in thrombi were found to bind 2T60 prominently. These results suggest that 2T60 may be a useful tool for clinical and experimental studies of thrombotic disease.

The utility of CEA and CA19-9 as colorectal carcinoma (CRC) markers is limited and development of additional reliable markers is under investigation. We previously showed that galectin-1 is overexpressed in CRC tissues. If such a protein leaks into the peripheral circulation, it might constitute a tumor marker candidate. Here, we test the hypothesis that the levels of circulating galectins could reflect the presence of CRC and/or its progression state. We constructed sandwich ELISAs for galectin-1/-2/-3/-4/-7 and determined their plasma concentrations in 105 CRC patients and 100 healthy volunteers (control). Matched pair samples of 56 patients pre- and post-surgery were also subjected to ELISA analysis. Circulating levels of galectin-1/-3/-4 in CRC patients were significantly higher compared to those in controls. Galectin-1 and galectin-4 levels significantly decreased after surgery (P

A 6-year-old boy had undergone ventriculoperitoneal (VP) shunt for acute hydrocephalus because of a brain tumor at the age of 11 months, and presented with vomiting and somnolence after the shunt malfunctioned 6 days after VP shunt reconstruction, during which the right external jugular vein was injured during the tunneling process and the peritoneal catheter was not fixed to the peritoneum with a purse string suture. Radiography revealed an abnormal route of the peritoneal catheter, suggesting that the distal VP shunt catheter had migrated into venous vasculature through the right external jugular vein. Computed tomography revealed that the peritoneal catheter had migrated into the internal jugular vein and the right atrium. At surgery, the peritoneal catheter was exposed through a small incision on the subclavicular region, was easily extracted from the internal jugular vein and the heart as there was no coiling or adhesion of the distal catheter to the vascular tissues, and was repositioned into the peritoneum with weak fixing between the subcutaneous tissues of the right subclavicular region and the right abdominal rectus muscle fascia as a temporary emergency measure. Peritoneal shunt migration into the internal jugular vein and the heart through the external jugular vein can be lethal because of pulmonary infarction or arrhythmia, and must be detected as soon as possible. Periodic follow-up radiography should be scheduled after VP shunt placement, even in the absence of symptoms.

We have constructed a murine hybrid hybridoma that secretes a bispecific monoclonal antibody (mAb) by fusing a hybridoma secreting an anti-ansamitocins mAb with a hybridoma secreting an anti-human transferrin receptor (TfR) mAb that binds to human A431 epidermoid carcinoma cells. The bispecific mAb, reactive to both ansamitocins and TfR, was purified by a combination of hydrophobic column chromatography and hydroxyapatite high-performance liquid chromatography, and evaluated in in vivo experiments using human tumor cell-implanted nude mice. Ansamitocin P-3 targeted through one of the antigen combining sites of the bispecific mAb was potentially more effective in suppressing the growth of established A431 tumor xenografts implanted on nude mice than unconjugated ansamitocin P-3 or the immunoconjugate of ansamitocin P-3 and monospecific anti-ansamitocins antibody, and the targeted ansamitocin P-3 finally eradicated the tumor mass. The bispecific mAb also played an important role in reducing such undesirable side-effects of ansamitocin P-3 as the loss of body weight, the damage to liver functions and the decrease in the number of white blood cells.

SummaryA hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent M r 150,000 on non-reduced SDS-PAGE and had an affinity for both human fibrin (K a = 2 × 107 M–1) and for u-PA (K a = 108 M–1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively.In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per εg/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FUl-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA.It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.

A 29-year-old woman presented with a severe headache. Computed tomography revealed a large cystic lesion with a mural nodule-like mass homogeneously enhanced with contrast medium in the right cerebellum. The tumor was removed, and pathological studies revealed a cerebellar astrocytoma corresponding to World Health Organization grade II. When she was 35 years old, or 6 years after the surgery, magnetic resonance imaging revealed a recurrence of the tumor in the right cerebellum, and subtotal removal of the recurrent tumor was performed. Pathological studies revealed a mixed glioblastoma multiforme and anaplastic ependymoma. Chemotherapy (Paraplatin and VePeside-S) and focal radiation therapy at 60 Gy were administered following surgery. Thereafter, at 39 years of age, or 4 years after radiation therapy, magnetic resonance imaging again revealed a recurrence of the tumor, which was heterogeneously enhanced with gadoliniumdiethylenetriamine pentaacetic acid in the right cerebellum. Subtotal removal of the tumor was performed; pathological studies revealed an anaplastic ependymoma with sarcomatous components. Immunohistochemical findings showed some parts of the sarcomatous components to stain positively for glial fibrillary acidic protein and, as a result, these sarcomatous components were diagnosed to be gliosarcoma.

The human thromboxane A2 receptor (TXA2-R)-coding gene was introduced into Chinese hamster ovary cells and a cell line (TCHO-25) stably expressing TXA2-R, at a level of 3 x 10(5)/cell, was obtained. An anti-asthmatic agent AA-2414 [(+-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7- phenylheptanoic acid] competitively inhibited the specific binding of a TXA2 mimic ([3H]U-46619) to the TCHO-25 cells, with an IC50 of 6.0 x 10(-8) M, indicating that the drug is an antagonist of human TXA2-R. The TCHO-25 cells offer a tool for the screening and characterization of human TXA2-R antagonists.

When sustained-release adhesive and non-adhesive microspheres which release the same drugs at similar rates are administered orally, drug absorption after administration of adhesive microspheres should, if the gastrointestinal residence of adhesive microspheres is prolonged as a result of mucoadhesion, be higher than that after administration of non-adhesive microspheres. The gastrointestinal transit of oral adhesive microspheres in man has been evaluated pharmacokinetically using furosemide and riboflavin, compounds with limited absorption sites in the upper small intestine. In a preliminary experiment with fasted rats it was confirmed that a higher percentage of the drug remained in the stomach and that plasma drug levels were higher when furosemide was administered in the form of adhesive rather than non-adhesive microspheres. Two kinds of sustained-release microsphere, adhesive and non-adhesive, containing furosemide and riboflavin in hard gelatin capsules were prepared and orally administered to 10 healthy fasted volunteers in a cross-over design. Areas under the plasma concentration-time curves (AUC) were 1.8 times larger for furosemide and urinary recovery was 2.4 times higher for riboflavin when adhesive microspheres rather than when non-adhesive microspheres were used. When adhesive microspheres containing riboflavin were administered to fed volunteers, urinary recovery was 2.1 times higher and mean residence time (MRT) was more prolonged than when the microspheres were administered to fasted volunteers. Adhesive microspheres were found to adhere to the gastric or intestinal mucosa with high affinity in man and rats, resulting in prolonged gastrointestinal residence.

An ex vivo antiviral assay was established which uses hepatocytes from mice given recombinant mouse interferon-beta (rmIFN-beta). Assay results were compared with results obtained with a 2',5'-oligoadenylate synthetase (2-5AS) assay. rmIFN-beta was intraperitoneally administered to C3H mice and the antiviral state of their liver parenchymal cells was evaluated in an in vitro cytopathic effect assay. In this assay, cells are infected with vesicular stomatitis virus (VSV) and surviving cells are determined colorimetrically. The antiviral state was measured as the resistance of hepatocytes to VSV infection with increasing doses of rmIFN-beta. The antiviral state correlated well with the dose-dependent increase in hepatic 2-5AS activity. This good correlation suggests that induction of 2-5AS mediates the antiviral action of interferon in liver tissue. This ex vivo assay could be a useful tool for estimating the ability of hepatocytes to resist hepatitis virus infection.

We have investigated the biological properties of an immune complex composed of recombinant interleukin-2 (rIL-2) and an F(ab')2 fragment of a monoclonal antibody against rIL-2 in mice for the induction of killer cells and anti-tumor activity, as well as the pharmacokinetic properties of the immune complex injected subcutaneously into mice. The immune complex demonstrated sustained serum rIL-2 levels, with a 2.4-times longer "mean-residence-time" than free rIL-2. A more significant portion of rIL-2 was detected in lymph nodes after the subcutaneous injection of the immune complex than with rIL-2 alone. Splenic lymphocytes from mice given the immune complex showed higher killer cell activity against YAC-1 and P815 cells than those from mice given rIL-2 alone. The immune complex also exerted a more significant anti-tumor effect in a dose-dependent manner in Meth-A fibrosarcoma-bearing mice than dit rIL-2 alone.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.

Two hybridoma systems, mouse.human-human (m.h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m.h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semi-continuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.

By fusing a human hybridoma producing an IgG2 kappa antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2 kappa antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reactive cells, A431, for attack by the exotoxin in vitro.

A hybridoma secreting human monoclonal antibody (MAB) against Pseudomonas aeruginosa exotoxin A (PEA) was constructed by fusing Epstein-Barr virus-transformed peripheral blood lymphocytes with human B lymphoblastoid cell line TAW-925. The human-human hybridoma stably produced human IgG2 MAB at the rate of 0.4-0.5 microgram/ml per 10(6) cells per day for more than six months, and the MAB was capable of neutralizing the in vitro cytotoxic and in vivo lethal effects of PEA with approximately 100- and 70-fold, respectively, higher activity than serum polyclonal antibody preparations.

Murine hybrid hybridomas secreting bispecific monoclonal antibodies (bs mAbs) were constructed by fusing hybridomas secreting an anti-tissue plasminogen activator (tPA) or anti-urokinase (UK) mAb with hybridomas secreting a mAb which binds to human fibrin but not to fibrinogen. The bs mAbs reactive to both fibrin and PA (tPA or UK) were purified by affinity chromatography employing the respective antigen-coupled columns and characterized by fibrin-binding, amidolytic and fibrinolytic assays. Immunochemical conjugation of PAs and the bs mAbs did not impair the catalytic activity of PAs at all and made it possible to concentrate PAs on fibrin clot. Pretreatment of fibrin with the bs mAbs enhanced the fibrin-binding of PAs and the subsequent fibrinolysis.

An enzyme immunoassay of pancreatic glucagon was established by using E. coli beta-D-galactosidease [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immunoassay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1 : 100,000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).

Lymph-node lymphocytes of a patient with stomach cancer were fused with the mouse-human heterohybridoma, HM-5. A clone (2F9) was isolated that showed stable production of an IgM antibody reactive with NUGC-4 stomach cancer cell line. This antibody reacted predominantly with a cell surface antigen on cell lines originating from gastro-intestinal cancer and adenocarcinoma of lung, whereas it was not generally reactive with other types of cancers, or with normal kidney cells or fibroblasts. Biotin-labeled 2F9 antibody clearly stained cell smears and the nude mouse tumor of NUGC-4, but it did not show a positive reaction with stomach cancer tissues obtained from more than 10 patients, indicating that the antigen detected is very weakly expressed on tumor cells or on a limited number of stomach cancers. The antigen shed from NUGC-4 cell line was detected in the culture supernatant. 2F9 antibody precipitated a glycoprotein with a molecular weight of over 200 kilodaltons as well as a possible glycolipid, from NUGC-4 cells labeled with [3H]glucosamine or [35S]-H2SO4. Periodic acid treatment of the tissue section decreased reactivity with 2F9 antibody, but heat, neuraminidase or protease treatment did not. These results suggested that the epitope is present on a carbohydrate moiety not containing sialic acid, and that a part of the antigen molecule is sulfated.

A hybrid hybridoma cell line secreting a bispecific monoclonal antibody (MAb) was constructed by fusing horseradish peroxidase (HRPO)-immunized mouse splenocytes with previously established mouse hybridomas secreting anti-human lymphotoxin (hLT). This cell line was grown in ascitic fluid in mice to obtain large quantities of hybrid MAbs and a bispecific antibody, reacting with both HRPO and hLT, was separated from the monospecific antibody or other inactive immunoglobulin populations by hydroxylapatite chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the bispecific antibody molecule contained two different types of heavy and light chains of both anti-HRPO and anti-hLT origin. The bispecific antibody was used to establish one-step enzyme immunoassays (EIAs) employing competitive and sandwich systems. The simple sandwich EIA was able to detect 1-100 U/ml of hLT and there was good correlation (r = 0.96) between hLT concentrations measured by the one-step EIA and a bioassay using L929 cell-lysis.

The level of human chorionic gonadotropin (hCG)-like substance in normal serum was measured using 50 μl of serum specimen by a highly specific and sensitive sandwich enzyme immunoassay for hCG which did not significantly cross-react with hLH. The level was calculated using a standard hCG and expressed in mIU/ml as that of hCG. In normal male (20–59 years of age) and normal, non-pregnant female (20–42 years of age) adults, the level was below 0.6 mIU/ml with mean levels of 0.16 mIU/ml (n=209) and 0.14 mIU/ml (n=36), respectively. In older subjects, however, the level tended to rise. In normal male adults, aged 60–91 years, the level was mostly below 0.8 mIU/ml (mean=0.56 mIU/ml, n=99), but levels of 15 specimens out of 99 were 1.1–5.5 mIU/ml. In normal, non-pregnant female adults, aged 45–88 years, the level was widely distributed between 0.1 and 11.7 mIU/ml (mean=1.6 mIU/ml, n=141). In these older female adults, levels of hCG-like substance were not well correlated to either levels of hLH and hFS...

Antitumor agents including mitomycin C (MMC) and methotrexate (MTX) were coupled to monoclonal antibodies against murine bladder tumor cell line MBT-2 for the purpose of enhancing their drug activity. MMC was conjugated with antibody through two carriers, periodate-oxidized dextran and dithiopyridylated serum albumin, and MTX was conjugated with antibody either directly or through dithiopyridylated serum albumin or poly-L-lysine via an amide bond. These conjugates were assayed for growth inhibitory effect on MBT-2 bladder tumor cells, MCA clone 15 embryo cells and P388 leukemic cells. The cytotoxicity tests demonstrated that drug-antibody immune conjugates were 10 to 100 times more cytotoxic against antibody-reactive MBT-2 cells than nonimmune drug conjugates and showed a similar level of cytotoxicity against antibody-nonreactive cells to that of the nonimmune conjugates. This selective cytotoxicity was also confirmed by competitive inhibition of unconjugated antibody, showing a dependency on antibody binding to the target cell surface antigens. The targeting effect was further assessed by evaluating the suppression of tumor growth subsequent to in vitro treatment of MBT-2 cells with immune conjugates. Half of the mice receiving cells treated with immune conjugates survived more than 40 d after inoculation, while none or one of 7 mice receiving cells treated with unconjugated drug, antibody alone or nonimmune conjugates survived at 40 d after inoculation.

We have constructed a murine hybrid hybridoma that secretes a bispecific monoclonal antibody (bs mAb) by fusing a hybridoma secreting an anti-tissue plasminogen activator (tPA) mAb with a hybridoma secreting a mAb that binds to human fibrin but not to fibrinogen. The bs mAb, reactive to both fibrin and tPA, was purified by affinity chromatography employing antigen-coupled columns and characterized by SDS-PAGE, a fibrin-binding assay, an amidolytic assay, a fibrinolytic assay and a thrombolytic assay. The immunochemical conjugation of tPA and the bs mAb did not impair the catalytic activity of tPA and made it possible to concentrate tPA at fibrin clots. Pretreatment of fibrin with the bs mAb enhanced the fibrin-binding of tPA and subsequent fIbrinolysis. The tPA-bs mAb immunoconjugate exhibited more thrombolytic activity than tPA alone in the rabbit jugular vein model.

In order to investigate the validity of an enzyme immunoassay for glucagon, the glucagon levels of human plasma were determined by both enzyme immunoassay (EIA) and radioimmunoassay (RIA). After a glucose load, plasma glucagon measured by both EIA and RIA fell in 12 normal subjects. The glucagon levels measured by both assays during glucose tolerance test showed good agreement in a group of 10 patients. After arginine infusion, plasma glucagon increased in 6 normal subjects and 3 patients and glucagon values measured by EIA correlated well with those by RIA. The present study demonstrates correlation between glucagon levels measured by RIA and EIA and indicates the usefulness of EIA for determining glucagon in human plasma.

A highly sensitive and specific enzyme immunoassay (EIA) for human lymphotoxin (hLT) has been developed. The assay is based upon a sandwich system employing two kinds of anti-hLT antibodies with neutralizing activity. One of them was mouse monoclonal antibody raised against Escherichia coli-derived recombinant hLT with a deletion of 20 aminoterminal amino acids and used as labelled antibody. The other was rabbit antibody raised against the carboxyl-terminal portion of hLT and used as solid-phase antibody. The EIA employing such a combination was able to detect less than 50 pg/ml of hLT, showing that this method was approximately 5–10 times higher sensitivity than the conventional bioassay employing L929 cell-lysis. The mean recovery of hLT added to serum specimens was 101% and the coefficients of variation were 3.3–7.8% (intra-assay) and 2.9–17.2% (interassay). There was a good correlation between the present EIA and the bioassay (r=0.93).

A simple and sensitive radioimmunoassay procedure is described for the screening and detection of specific antibodies in hybridoma cell lines. The specific procedure was developed to screen for antibodies against human thyrotropin (hTSH), but the procedure is applicable to screening for any desired antibodies. The immunoglobulin G (IgG) fraction of goat anti-mouse IgG is used to coat wells of microtiter plates. Anti-hTSH antibodies are measured by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated hTSH. Unlabeled hTSH may also be detected by its ability to inhibit binding of125I-hTSH to the coated wells. This assay technique meets the demands of simplicity, sensitivity, reproducibility, and rapidity as a screening assay of hybridoma cell lines capable of secreting anti h-TSH.

A highly specific enzyme immunoassay for determining hCG was established by using beta-D-galactosidase as label. In order to increase the specificity of the assay, an antiserum against whole hCG was purified on a column of hCG beta carboxyl-terminal peptide (residues 123-145) covalently linked to Sepharose 4B. The antibody (N101S) thus prepared showed a weak cross-reactivity with human LH in an assay using hCG-enzyme conjugate, but the slight cross-reactivity was virtually avoided when an hCG beta carboxyl-terminal peptide was used as a peptide in the enzyme conjugate. N101S antibody was compared with antiserum (B1B) directed against a carboxyl-terminal peptide (123-145). In hCG measurement N101S gave about 30 times higher sensitivity than B1B, although the former antibody was less sensitive to carboxyl-terminal peptides of hCG beta. The enzyme immunoassay using a combination of N101S antibody and a carboxyl-terminal peptide (130-145)-enzyme conjugate was able to detect as little as 0.25 mIU of hCG without the interference of LH. The performance and validity of this assay were comparable to those of conventional radioimmunoassay.

Antisera against tetrahydronaphthalenols, which are conformationally rigid derivatives of adrenergic catecholamine, were produced in rabbits immunized with trans-5-amino-6-hydroxy-2-isopropylamino-1,2,3,4-tetrahydronaphthalene-1-ol (I) conjugated to succinylated bovine serum albumin at the C5 position on the tetralin ring. Antisera were screened by immunodiffusion and further characterized by passive hemagglutination assay using erythrocytes sensitized with trans-I-ovalbumin conjugate and by enzyme immunoassay using trans-I-β-galactosidase conjugate. Cross-reactivity studies indicated that the antiserum was highly specific for the tetralin structure and for substitution at the C2 position. The antiserum also selectively discriminated the stereoisomers about the C1C2 bond. The anti-trans-I serum was used to develop EIA for trans-5-hydroxymethyl-6-hydroxy-2-isopropylamino-1,2,3,4-tetrahydronaphthalene-1-ol (IIb), which exhibited strong β-stimulating activity fairly selective to tracheal muscle, since it recognized trans-IIb to the same degree as trans-I. The assay could detect as little as 100 pg of this compound. The mean recovery of trans-IIb added to plasma was 105%, and values for plasma trans-IIb determined by this immunoassay correlated well with those determined by gas chromatography-mass spectrometry.