Study on the assay of proximal tubular antigen in urine and serum with an anti-human renal monoclonal antibody]

Monoclonal antibodies (Mabs) were produced by immunizing mice with human kidney microsomal antigen. Mab-B1 recognized brushborder (B1-Ag) in proximal tubules. Using Mab-B1, B1-Ag was assayed in the urine and serum of renal disease patients by sandwich ELISA. The subjects included normal control (Nor), minimal change nephrotic syndrome (MCNS), IgA nephropathy (IgA), membranous nephropathy (MN), membranoproliferative glomerulonephritis (MPGN), and chronic renal failure (CRF) (s-Cr greater than 2 mg/dl). Urinary B1-Ag demonstrated significant increases in the IgA (p less than 0.001), MN (p less than 0.001), MPGN (p less than 0.001) and CRF (p less than 0.01) groups as compared to the Nor group. There was no significant increase in the MCNS group. In the CRF group, B1-Ag in urine showed a significant increase in the progressive CRF group with delta s-Cr greater than 1.0 mg/dl/month as compared to the stationary CRF group with delta s-Cr less than 1.0 mg/dl/month. No correlation was observed between urinary B1-Ag and proteinuria, hematuria, s-Cr, s-BMG and u-NAG. The above findings suggested that the assay of urinary B1-Ag was useful as a new parameter in detecting the site and degree of proximal tubular damage.     

Prolongation of primate renal allograft survival by anti-Tac, an anti-human IL-2 receptor monoclonal antibody

In an effort to produce specific immunosuppression through the targeting of those lymphocytes expressing cell surface interleukin 2 receptors in response to an allograft, the anti-human IL-2 receptor monoclonal antibody anti-Tac was administered to cynomolgus monkeys receiving renal transplants. The data demonstrate that anti-Tac produces a significant delay in renal allograft rejection and prolongs host survival in cynomolgus monkeys. Though higher doses of anti-Tac produce modest delays in rejection, there was a surprising finding of greatly prolonged survival in three of five monkeys treated with much lower doses of anti-Tac. Anti-Tac was not shown to be synergistic with cyclosporine in this model. Animals treated with anti-Tac developed high titers of antibodies against the murine monoclonal antibody after 6-8 days of treatment, associated with the disappearance of plasma anti-Tac staining of activated lymphocytes as measured by flow cytometry. The data confirm the utility of the IL-2 receptor as a target for immunosuppressive therapy, and suggest that investigations of dosage and of methods to reduce the immunogenicity of anti-IL-2 receptor agents may be beneficial.     

A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats.

After immunization of mice with partially-purified heparan sulfate proteoglycan (HSPG) isolated from rat glomeruli, a monoclonal antibody (mAb JM-403) was obtained, which was directed against heparan sulfate (HS), the glycosaminoglycan side chain of HSPG. In ELISA it reacted with isolated human glomerular basement membrane (GBM) HSPG, HS and hyaluronic acid, but not with the core protein of human GBM HSPG, and not with chondroitin sulfate A and C, dermatan sulfate, keratan sulfate and heparin. Furthermore, it did not bind to laminin, collagen type IV or fibronectin. Specificity of JM-403 for HS was also suggested by results of inhibition studies, which found that intact HSPG and HS, but not the core protein, inhibited the binding of JM-403 to HS. In indirect immunofluorescence on cryostat sections of rat kidney, a fine granular to linear staining of the GBM was observed, along with a variable staining of the other renal basement membranes. Pretreatment of the sections with heparitinase completely prevented the binding of mAb JM-403, whereas pretreatment with chondroitinase ABC or hyaluronidase had no effect. The precise binding site of mAb JM-403 was investigated by indirect immunoelectron microscopy. It revealed a diffuse staining of the whole width of the GBM. One hour after intravenous injection of JM-403 into rats, the mAb was detected along the glomerular capillary wall in a fine granular pattern, which shifted towards a more mesangial localization after 24 hours. No binding was observed anymore by day 15. Intravenous injection induced a dose-dependent, transient and selective proteinuria that was maximal immediately after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of an acrosomal antigen recognized by a monoclonal antibody and antifertility effects of isoimmune serum

A highly conserved acrosomal antigen reactive to a monoclonal antibody (HS-63), generated against human sperm, was purified to homogeneity with a combination of conventional procedures and immunoaffinity chromatography using a soluble extract of mouse and rabbit testes. The molecular weight of the purified antigen was 42-50 kD when analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis. The high specificity of the purified antigen to monoclonal antibody HS-63 was shown by indirect immunofluorescent inhibition assay, enzyme-linked immunosorbent assay, Western blot analysis and radioimmunosorbent assay. The purified antigen was used for isoimmunization of mice and rabbits. Following successive immunizations, antisera of high titres were raised and reacted specifically with antigen on the sperm acrosome and in testes of several mammalian species, but not with somatic tissues. These isoimmune sera exhibited strong inhibition on mouse in-vitro fertilization and human sperm penetration of zona-free hamster eggs. The results of this study suggest that the sperm-specific acrosomal antigen reacting with HS-63 could be a good candidate for the development of immunocontraceptive vaccines in humans and in other animals.     

Intracerebral granuloma with serum anti-human ascaris antibody: case report

A 1.5-year-old girl with a convulsion attack due to intracerebral granuloma in the right frontal lobe is reported. Her serum was positive with anti-human ascaris antibody, although no ova of the parasites were detected in the feces. She had grown up intimately with several cats in the home. These findings suggested that the granuloma was due to larva migrans of toxocara, which cross-reacts immunologically with human ascaris. Histological examination of the granuloma revealed no eosinophilic infiltration. No systemic reactions such as eosinophilia and hepatomegaly were found except for elevation of protein in cerebrospinal fluid. These were similar to those of ocular type of toxocara larva migrans.     

Study of the changes in glomerular antigenicity in various renal diseases with anti-human renal monoclonal antibodies

We produced 22 different kinds of monoclonal antibody (Mab) by immunizing mice with human GBM antigens. In these Mabs, Mab-G1 to G5 recognized only GBM in the glomerulus, Mab-E1 and E2 recognized only glomerular epithelial cells, and Mab-M1 to M4 recognized mainly mesangium. The reactions of these Mabs with known GBM antigens such as type IV collagen, fibronectin and laminin were negative by immunoblotting. Using Mab-G1, Mab-E1 and Mab-M1, changes in the antigenicity of antigens recognized by Mabs were examined on kidney sections from the patients with various renal diseases by the indirect immunofluorescence test. When Mab-G1 recognizing GBM was used, there was no particular change of antigenicity in minimal change nephrotic syndrome (MCNS) and IgA nephropathy (IgA), whereas in membranous nephropathy (MN) thickened GBM was found to maintain antigenicity and the region of deposits was observed as negative punched-out region. In type I and III of membranoproliferative glomerulonephritis (MPGN), GBM was observed only outside of subendothelial deposits without showing double contour. In type II MPGN, GBM showed a double linear pattern and antigenicity of GBM in regions of dense deposits was not detected. When Mab-E1 recognizing glomerular epithelial cells was used, there was no change of antigenicity in the renal diseases. Further, in crescentic glomerulonephritis, the region of the cellular crescents was not stained. When Mab-M1 recognizing mesangium was used, extensive staining was observed in the increased mesangium in IgA, MPGN, and diabetic nephropathy. We feel that it is of significance in elucidating the pathogenesis of renal diseases to study the changes of glomerular antigenicity in diseased kidneys by using anti-human renal monoclonal antibodies.     

Differential immunostaining of oncocytic renal tumors with an anti-renal cell carcinoma monoclonal antibody

Formalin-fixed, paraffin embedded tissue sections from twenty three oncocytic renal neoplasms were stained with hematoxylin and eosin, retrospectively examined, and graded according to the criteria reported by Lieber and associates. Additional sections were stained by the avidin-biotin immunoperoxidase technique with anti-renal cell carcinoma monoclonal antibody 5F4. The results showed that cytological heterogeneity was the most prominent feature of the tumors. Four cases were composed predominantly of grade 1 cells, but also had foci of grade 2 cells. Seventeen cases were composed predominantly of grade 2 cells. Two cases were composed predominantly of grade 3 cells. Immunostaining with 5F4 showed differential reactivity between grade 1 and grades 2 and 3 cells. The antibody highlighted the foci of atypical cells which were difficult to detect by routine hematoxylin and eosin staining and thus could be useful in the differential diagnosis of oncocytic renal neoplasms.     

抗前列腺分化抗原单克隆抗体的研究

目的 为利用单克隆抗体（ＭＡｂ）结合放射性核素对前列腺癌及其转移灶提供诊断和治疗手段。 方法 以前列腺癌细胞株ＰＣ－３Ｍ作为免疫原,亮氨酸甲基酯处理经免疫后的脾细胞作融合,产生抗前列腺癌ＭＡｂ（ＹＤＰＣ）。 结果 ＹＤＰＣ在正常前列腺腺管上皮、良性前列腺增生和所有前列腺癌组织表达,其它正常组织除肾小管上皮、肾上腺和汗腺腺管上皮外均不表达。另外,３例前列腺癌淋巴结转移和１例前列腺癌膀胱转移染色均呈阳     

Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation

BACKGROUND: Anti-CD154 monoclonal antibodies (mAbs) cause long-term graft survival in preclinical allotransplantation experiments. This is the first report on the efficacy and safety of ABI793, a novel human anti-human CD154 mAb, in Cynomolgus renal transplant recipients. METHODS: ABI793 (human immunoglobulin-G1:kappa) was derived from a hybridoma generated after immunization of human immunoglobulin transgenic mice (HuMAb-Mouse, Medarex Inc., Annandale, NJ). Cynomolgus monkey recipients of major histocompatibility complex-mismatched, life-supporting renal allografts were treated repeatedly with intravenous ABI793 for a 3-month period posttransplantation. Graft function was monitored by serum creatinine, and rejection was confirmed histologically. RESULTS: ABI793 binds to human, Cynomolgus and Rhesus monkey CD154; it inhibits dose dependently in vitro CD154:CD40 binding and human mixed lymphocyte reaction. ABI793 is comparable to the mouse anti-human CD154 mAbs 5c8 and 24-31 with respect to affinity, inhibitory capacity, and species specificity; however, ABI793 binds to a different CD154 epitope. With 20 mg/kg of ABI793, five of nine recipients showed substantially prolonged graft survival after cessation of treatment, whereas four of nine recipients were killed because of high serum creatinine while still receiving treatment. ABI793 treatment was associated with episodes of severe acute tubular necrosis (which was unrelated to rejection and responded to fluid and diuretic treatment) and a decrease in platelet numbers. Chronic and acute thromboembolic vascular lesions with hemorrhages were observed in the lung and brain of two allograft recipients. None of these side effects were observed in animals that underwent autotransplantation, thus excluding direct toxicity of ABI793. CONCLUSIONS: ABI793 treatment effectively prevents graft rejection in Cynomolgus monkeys. Evidence for rare thromboembolic events, as also previously observed with different anti-human CD154 mAbs, suggests that thromboembolic complications may be a class effect of anti-CD154 mAbs, unrelated to their epitope specificity.     

A monoclonal antibody to an antigen present in cells from a patient with Hodgkin's disease

A monoclonal antibody-secreting hybridoma cell line, VCD-1, was derived from the fusion of murine myeloma cells with splenocytes from a BALB/c mouse that had been immunised with chronic B-lymphocytic leukaemia cells. The cells came from a patient who had developed the leukaemia approximately 10 years after a course of radiotherapy for nodular sclerosing Hodgkin's disease. The antibody bound to a 30,000-dalton protein that was present in normal and malignant B cells, in monocytes, neutrophils, and interdigitating reticulum cells, and in malignant cells present in Hodgkin's disease lymph nodes. The reactive epitope was not accessible to antibody in viable intact cells; binding to peripheral blood cells could only be seen if the cells were fixed. The antibody recognises a determinant that probably resides on the alpha-chain of HLA class II molecules.     

Antitumor effect of adriamycin entrapped in liposomes conjugated with anti-human alpha-fetoprotein or anti-carcinoembryonic antigen monoclonal antibodies

Monoclonal antibodies against human alpha-fetoprotein (AFP) or carcinoembryonic antigen (CEA) were conjugated to liposomes containing adriamycin (ADM), and the therapeutic effects of the conjugates were experimentally studied in vitro and in vivo. The liposomes were prepared from a lipid mixture of egg phosphatidyl choline, cholesterol and dipalmitoylphosphatidyl ethanolamine, and were covalently coupled with anti-AFP monoclonal antibody (19-F-12) or anti-CEA monoclonal antibody (1-C-11) after activation of antibody with the N-hydroxysuccinimidyl 8-(2-pyridyldithio) propionate and dithiothreitol. The selective binding of the 19-F-12 conjugated liposomes to AFP-positive human hepatoma cell line PLC and the 1-C-11 conjugated liposomes to CEA-positive colon cancer cell line C-1 as demonstrated using fluorescent liposomes. In vitro studies with PLC and C-1 clearly indicated that monoclonal antibody-conjugated liposomes containing ADM exerted much more effects than unconjugated liposomes containing ADM on target cells in the inhibition assay of [3H]-thymidine incorporation. The therapeutic effects of the conjugates were tested in vivo on AFP-positive human hepatoma xenograft, Li-7, and CEA-positive human colon cancer xenograft, Co-4, maintained in BALB/c nu/nu mice. The antitumor effect of the antibody-conjugated liposomes containing ADM was far greater than that of unconjugated liposomes containing ADM or that of ADM alone as assessed by tumor weight and histological findings.     

Enhancement of rat renal allografts with monoclonal antibody.

Treatment of rat allograft recipients before grafting with donor spleen cells and whole, pooled antidonor alloimmune serum results in indefinite renal allograft survival. The enhancement is immunologically specific. In these experiments a monoclonal, homogenous anti-BN antibody was produced by a hybridoma clone created by fusing the mouse-P3 myeloma with spleen cells from Lewis rats immunized with BN lymphoid cells. The hybridoma supernatent enhanced survival of LBN renal allografts in Lewis recipients as effectively as whole Lewis anti-BN antiserum. Dilution of the hybridoma supernatent by tenfold or a hundredfold abrogated the enhancement effect.     

The effect of antithymocyte globulin on anti-human leukocyte antigen antibody detection assays

BACKGROUND: We evaluated the effect of antithymocyte globulin (ATG) on anti-human leukocyte antigen (HLA) antibody assays. METHODS: We tested sera from six in vivo ATG-treated kidney transplant patients after measuring serum concentrations, as well as six nonsensitized sera with ATG added in vitro. T- and B-cell complement-dependent cytotoxicity (CDC), flow cytometric (FXM), and solid-phase HLA class I and II assays based on antigen-coated microspheres and enzyme-linked immunosorbent assay (ELISA) were studied. Sera were then retested after treatment to remove ATG. RESULTS: We found that ATG affects test results differently depending on whether sera is obtained from in vivo treated patients or added in vitro. In vitro treated sera produced ATG concentration-dependent positive results for T/B CDC, FXM, and flow bead testing for HLA I/II, while the ELISA-based assay was unaffected. In vivo treated sera from ATG-treated patients produced positive test results for T CDC and T/B FXM, while the B-cell CDC crossmatch remained negative. Solid phase assays were minimally affected using in vivo treated sera. After ATG extraction, all tests became negative. CONCLUSION: We conclude that ATG produces positive results in anti-HLA antibody testing, and treatment to remove ATG abolishes this effect. This treatment allows ATG-treated patients to be monitored for anti-HLA antibodies.     

抗人C5单克隆抗体对补体介导的超急性排斥反应的抑制作用

目的 研究抗人C5单克隆抗体对补体介导的超急性排斥反应的抑制作用.方法 获取8例临床心脏移植供者的主动脉血20ml和主动脉血管,分别用于提取淋巴细胞和主动脉内皮细胞.同时,采集相应8例心脏移植受者移植前的中心静脉血20 ml和外周静脉血5 ml,分别用于提取淋巴细胞和制备补体.供者主动脉内皮细胞经培养后,依次加入供、受者淋巴细胞混合培养上清50μl和受者静脉血浆(含补体)20 μl,建立主动脉内皮细胞超急性排斥反应模型.模型建立后,将供者主动脉内皮细胞平均分为2组,C5组:加入20 mg/L的抗人C5单克隆抗体20μl;对照组:加入M200培养液20 μl.经台盼蓝染色,倒置显微镜下观察主动脉内皮细胞的染色情况及活性,并测定主动脉内皮细胞刺激指数(SI).结果 经观察,对照组大部分主动脉内皮细胞蓝染、肿胀死亡,而C5组大部分主动脉内皮细胞未见蓝染,存活良好;对照组和C5组吸光度值分别为1.528±0.046和1.644±0.058,两组比较,差异有统计学意义(P<0.001),SI为1.076±0.038.结论 在离体主动脉内皮细胞超急性排斥反应模型中,抗人C5单克隆抗体可以抑制补体介导的主动脉内皮细胞溶解.     

Imaging of human colorectal adenocarcinoma with indium-labeled anticarcinoembryonic antigen monoclonal antibody

Twenty patients with 21 primary colorectal adenocarcinomas were studied with 2 mCl (7.6 X 10(7) becquerels) of indium-labeled monoclonal antibody (200 micrograms) specific for carcinoembryonic antigen (CEA). Fifteen lesions (71%) were visualized by gamma camera scintigraphy at 48 hours postinjection. Tumors that were identified by immunoscintigraphy were large (38.10 +/- 17.76 cm3 vs 6.00 +/- 1.65 cm3), had a grossly fungating component, had a high content of CEA by enzyme immunoassay (12.9 +/- 3.6 micrograms/g vs 3.3 +/- 1.7 micrograms/g), and had an apical and/or intraluminal staining pattern on immunohistologic section. Patients whose tumors were visualized had a low serum CEA level (1.9 +/- 0.4 ng/mL vs 14.6 +/- 8.0 ng/mL). Prospective selection of patients for follow-up imaging or therapy with radiolabeled monoclonal antibodies may be feasible using these measurements.     

The Goodpasture antigen in Alport's syndrome: studies with a monoclonal antibody

A mouse monoclonal antibody (MCA-P1), which recognizes an antigenic determinant in human glomerular basement membrane against which autoantibodies are directed in Goodpasture's syndrome, was used in indirect immunofluorescence studies to investigate glomerular basement membrane structure in Alport's syndrome. We found reduced or absent binding of MCA-P1 to glomerular and distal tubular basement membranes in renal biopsy tissue from ten patients with Alport's syndrome. Antiglomerular basement membrane antibody eluted from the kidneys of a patient who had died from Goodpasture's syndrome was used to confirm these findings. In contrast, there was bright linear fluorescence of MCA-P1 on glomerular and tubular basement membranes of normal renal material and renal biopsy tissue obtained from patients with a variety of glomerulonephritides. These results suggest an abnormality or a variable quantity of the immunoreactive autoantigen in the glomerular basement membrane of patients with Alport's syndrome. Furthermore, MCA-P1 may be of value in the diagnostic interpretation of renal biopsies from patients with familial nephritis.     

Experimental studies on targeting chemotherapy using adriamycin entrapped in liposomes conjugated with anti-human alpha-fetoprotein monoclonal antibody]

The in vivo tissue distribution of adriamycin (ADM) entrapped in liposomes conjugated with anti-human alpha-fetoprotein (AFP) monoclonal antibody (Lip-ADM = Ab) and the therapeutic effects of the conjugates on the AFP producing human hepatoma xenograft, Li-7, transplanted in BALB/c nu/nu murine liver were studied. The tissue distribution studies of Lip-ADM = Ab revealed that ADM levels were augmented in tumors, when Lip-ADM = Ab was administered intravenously as compared with free ADM or ADM entrapped in liposomes (Lip-ADM) alone. In addition, cardiac distribution of ADM was decreased in mice using ADM entrapped in liposomes. The therapeutic effects of Lip-ADM = Ab were tested in vivo on the experimentally transplanted Li-7 in liver. The anti-tumor effects of Lip-ADM = Ab were greater than those of Lip-ADM or free ADM as assessed by tumor weight. This experiment also indicated that the toxicity of ADM was reduced when the drug was injected using liposomes entrapped form by the body-weight losses and spleen weights.     

Carcinoembryonic antigen in serum,urine and cells of patients with bladder carcinoma

Summary A raised level of CEA-like substance has been demonstrated by radioimmunoassay in the urine of patients with bladder carcinoma, in concentrations which increase with a more advanced stage, and in serum of patients with advanced disease. In a 2-year follow-up of patients receiving chemotherapy, a correlation of raised urinary CEA to local recurrence was seen, as well as rising and high serum values with metastases. In the patients who responded to treatment, CEA values became normal. CEA was also located in carcinoma cells from bladder washings in 24–61 % of the cases. Combined studies of CEA in serum, urine and cells may be used to study the biology of the tumor and perhaps also in the monitoring of patients with urothelial carcinoma.