Rabbit Globulin Research Articles

Comparison of Different Rabbit Anti-Thymocyte Globulin Formulations in Allogeneic Stem Cell Transplantation: Systematic Literature Review and Network Meta-Analysis

IntroductionSince 2000, phase III randomized controlled trials (RCT) investigated the efficacy of rabbit anti-thymocyte globulins (ATG) in patients following allogeneic stem cell transplantation (allo-SCT). However, comparisons of different ATG formulations do not exist. Our aim was to synthesize all efficacy evidence, enabling a comparison of all available formulations of rabbit ATG in the allo-SCT setting.MethodsWe performed a systematic literature review to identify all available phase III RCTs using EMBASE®, MEDLINE®, MEDLINE® in-process, the Cochrane Library and the website www.clinical-trials.gov. In addition, meeting abstracts (ASH, ASCO, EHA) were screened to include the most up-to-date evidence. Studies were included if they described a phase III RCT among adult patients who received rabbit globulins in the allo-SCT setting and if they reported results of the following end points of interest: chronic graft-versus-host-disease (GVHD, all grades), acute GVHD II-IV and III+IV, transplant-related mortality (TRM), overall survival (OS), incidence of relapse and CMV reactivation.The actual network meta-analysis (NMA) was performed by using a conventional Bayesian fixed-effect framework and hazard ratios (HR) for most end points while risk ratios (RR) were used for dichotomous outcomes. Analyses were done with the R package netmeta. The most common treatment, cyclosporine and methotrexate, was used as reference treatment.ResultsWe identified six phase III RCTs comprising five full publications and one conference abstract between 2001 and 2016, which investigated the following treatment options: 1) Grafalon® (Neovii Biotech, Graefelfing, Germany) consisting of polyclonal IgG obtained from hyperimmune sera of rabbits immunized with the human Jurkat leukemic T-cell line (ATLG), and 2) Thymoglobulin® (Genzyme-Sanofi, Lyon, France), in which immunization was done with human thymocytes recovered from patients undergoing cardiac surgery (Thymo).The overall population comprised 871 patients (ATLG: n = 610, and Thymo: n = 261). Population per trial ranged from 27 to 126 patients in the ATG arm and from 25 to 128 patients in the non-ATG arm. The median age of patients ranged from 28 to 49 years. More patients were male, except in the ATG arm of one ATLG trial.To include all trials within one framework, we had to assume: 1) the relative efficacy of ATG versus cyclosporine and methotrexate was identical to that of tacrolimus and methotrexate, which was used as reference treatment in one ATLG trial; 2) no difference in efficacy due to different graft sources or conditioning regimens; 3) no difference in efficacy due to dosage scheme. Figure 1a-f presents the NMA results of the main end points with HRs/RRs and 95% confidence intervals (CI) versus standard treatment (cyclosporine and methotrexate).Only ATLG was significantly better than standard treatment in preventing GVHD with a HR of 0.42 (95% CI, 0.31 to 0.56) regarding chronic GVHD, and HRs of 0.54 (95% CI, 0.39 to 0.73) and 0.50 (95% CI, 0.29 to 0.86) regarding acute GVHD II-IV and III+IV. The risk of chronic, acute GVHD II-IV and III+IV was reduced by ATLG with 58%, 46% and 50% versus standard treatment.Both ATLG and Thymo were at least similarly effective versus standard treatment and showed no difference in relative efficacy regarding TRM with HRs of 0.90 (95% CI, 0.61 to 1.32) and 0.90 (95% CI, 0.56 to 1.44). Considering OS, incidence of relapse and CMV reactivation, the NMA yielded at least similar results of both formulations versus standard treatment, with Thymo tending to be better regarding OS with a HR of 0.86 (95% CI, 0.59 to 1.26) while resulting less clearly in better relative efficacy regarding relapse with a HR of 0.92 (95% CI, 0.68 to 1.24) and CMV reactivation with a RR of 0.99 (95% CI, 0.84 to 1.16).ConclusionsOur NMA compared all available rabbit ATG formulations and identified ATLG being the best option to prevent chronic and acute GVHD. Both formulations show similar efficacy in TRM while Thymo tends to be better regarding OS. Since randomized head-to-head comparisons are missing, this NMA provides a complete overview of each formulation's relative efficacy. [Display omitted] DisclosuresKroeger:Neovii: Honoraria, Research Funding.

Desensitization Protocol Consisted of DFPP, Anti-CD20 Ab, Tacrolimus and Mycofenolate Mofetil, Improves Graft Function in Sensitized Patients of Living-Donor Kidney Transplantaion.

Objective) The aim of this study was to investigate the efficacy of desensitization protocol (DP) including double filtration plasmapheresis (DFPP), rituximab, tacrolimus (Tac), and mycofenolate mofetil (MMF), and to evaluate risk factors for graft function in sensitized patients of living-donor kidney transplantation (LDKT). Methods) Retrospectively, 35 recipients of LDKT, who had preformed donor-specific HLA antibodies (DSA), with average age of 51.0±13.0 (13 males and 22 females), were included in the study. Recipients were divided into 2 groups, Group 1 (n=9): DP consisted of DFPP and/or a rituximab (100mg) at four days before transplantation, and Group 2 (n=26): DP consisted of DFPP and/or a ritximab, and Tac (1mg/day) and MMF (250mg /day) for one month prior to transplantation. Occurrence of acute antibody mediated rejection (AMR), graft-survival rates (GSR) and eGFR at 3 years, were compared between them. And risk factors for developing AMR in patients with DSA were evaluated. Results) 3-year AMR free ratio was 33.3% in Group 1 and 76.7% in Group 2 (p=0.0190).Figure: No Caption available.All AMRs responded to treatment consisted of DFPP, intravenous immunoglobulin, and anti-thymocyte rabbit globulin. 3-year GSR in Group 1 and 2 were 100% and 90.2%, respectively (p=0.29). However, 3-year eGFR values in patients with AMR and without AMR were 23.6±8.3 and 50.0±15.8 mL/min (p<0.001). Interestingly, 11 of 12 recipients with AMR were females (p=0.01), and 10 of them (90.1%) were donated from husband. Other factors, such as age, PRA, value of MFI of Flow Cytmetric Crossmatch test were not significant. Conclusions)In patients with DSA, there was no significant difference in graft survival between two groups, however, occurrence of AMR in Group2 was significantly lower than that of Group 1. DFPP, rituximab plus prior prescription of Tac and MMF should be a useful DP. And female recipient donated from husband was significant risk factors for graft function after LDKT in patients with DSA.

Clinical and economic analysis of short-course versus standard-course antithymocyte globulin (rabbit) induction therapy in deceased-donor renal transplant recipients

The immunosuppressive effects of and costs associated with short-course antithymocyte globulin rabbit (ATG [rabbit]) therapy versus standard-course ATG (rabbit) therapy in deceased-donor renal transplant recipients were evaluated. The records of 84 consecutive patients who received a deceased-donor renal transplant at the Montefiore Einstein Center for Transplantation in 2008 were retrospectively reviewed. Donor and recipient characteristics, including rates of biopsy-confirmed acute rejection, serum creatinine (SCr) levels, and frequency of complications, and drug costs were collected. Patients were excluded if they had donor-specific antibodies identified before transplantation or hepatitis-C-positive serology or were under 18 years of age. A total of 60 patients were included in the study, with 28 receiving short-course ATG (rabbit) therapy and 32 receiving standard-course ATG (rabbit) therapy. Baseline patient demographic characteristics were similar between groups. Six months after transplantation, biopsy-confirmed acute rejection episodes did not significantly differ between the short-course ATG (rabbit) and standard-course ATG (rabbit) groups (17.8% versus 12.5%, respectively), nor did SCr concentrations (1.56 mg/dL versus 1.85 mg/dL). The frequency of therapy-related leukopenia was greater in patients receiving standard-course ATG (rabbit). Patients treated with short-course ATG (rabbit) received a total mean dose of 4.6 mg/kg, compared with 7.3 mg/kg for patients in the standard-course ATG (rabbit) group, resulting in a mean cost saving of $2548 per patient. After six months, there were no significant differences in biopsy- confirmed acute rejection episodes or SCr levels between deceased-donor renal transplant recipients receiving short-course versus standard-course ATG (rabbit) induction therapy. The mean cost saving associated with short-course therapy was $2548 per patient.

A Retrospective Evaluation of the Effects of Severe Aplastic Anemia Treatment in Children with Horse and Rabbit ATG.Polish Pediatric Hematology Group

Abstract 4375Antithymocyte horse globulin (hATG) or rabbit (rATG) is used to treat children with severe aplastic anemia (SAA) in the absence of a compatible bone marrow family donor's. We presenting (viewing) retrospective evaluation of the effects of SAA therapy using hATG and rATG.49 children (20 girls and 29 boys, aged 1.2–17) were treated between 1993 and 2000 - hATG (15mg/kg/day for 5 days with cyclosporine 5mg/kg/day for 12–24 mths). On day 180 day of treatment there was a complete response (CR) in 22 of 49 children (44.5%) and a partial response (PR) in 12 children (24.4%). 34 of 49 children (69,4%) responded to treatment. There was no remission (NR) in 15 of 49 children (30.6%). In the whole group there were 7 deaths during the first 6 months. There were no relapses within 5 years of observation. The 5-year probability of survival in this group was 84%.55 children (22 girls and 33 boys, aged 3,5–17,7 years) were treated in the years 1996–2009 using rATG (3.75 mg/kg/day for 5 days and cyclosporine 5mg/kg/day for 12–24 mths). On day 180 of treatment 5 patients (9%) achieved a CR, 23 children (41.8%) had a PR. 28 of 55 children (50.9%) responded to treatment. NR was found in 27 children (49%).In the rATG group 15 deaths (27%) were recorded, 4 patients (7%) had relapses. In one child paroxysmal nocturnal hemoglobinuria was noted.The 5-year survival in the rATG.group was 63.54%.Our results show the greater effectiveness of treatment with horse globulin than rabbit globulin in terms of hematological response and overall survival. Disclosures:No relevant conflicts of interest to declare.

Analysis of infusion-site reactions in renal transplant recipients receiving peripherally administered rabbit antithymocyte globulin as compared with basiliximab

Antithymocyte globulin rabbit (r-ATG) has been used for the treatment and prevention of acute rejection in renal transplant recipients (RTR). Current manufacturer recommendations for r-ATG dictate the need for administration through a high-flow vein (central line). Previous studies have shown peripheral administration of r-ATG to be safe; however, these studies suggest the co-administration of heparin and hydrocortisone and did not compare the infusion-site reaction rates to a control group. A retrospective analysis was conducted of adult RTR receiving r-ATG or basiliximab between January 2004 and October 2006. Each agent was administered through a dedicated peripheral line. The primary endpoint was the incidence of infusion-site reactions. Other endpoints included the need to replace the intravenous catheter and the incidence of systemic thrombosis within 1 month of transplantation. During the study period, 152 peripheral infusions of r-ATG and 92 peripheral infusions of basiliximab were administered. No difference in infusion-site reactions was noted between the groups. There was also no difference either in the need for peripheral line replacement or the rates of systemic thrombosis. Peripheral administration of r-ATG is safe and can be infused without concomitant heparin and hydrocortisone. This method of r-ATG infusion was shown to be as safe as peripherally administered basiliximab.

Antithymocyte Rabbit Globulin Is Safe and Effective in Children with Severe Aplastic Anemia Report Polish Pediatric Hematology Group (Warszawa, Krakow, Wroclaw, Bydgoszcz, Poznan, Lodz, Bialystok, Szczecin, Gdansk).

Abstract 4220Bone marrow transplantation (BMT) is a therapy of choice in children with severe aplastic anemia (SAA), but it is possible only in about 15-20% patients who have family donors, so the majority of them received the immunosuppressive therapy (IST).We want to present the results of IST obtained in 116 children treated between 1993-2008 years at 11 pediatric hematology centers of the Polish Pediatric Hematology Group (PPHG), 55 patients (22 girls and 33 boys aged 0,6-17,5 years) received antithymocyte rabbit globulin (ATG) as a first course of therapy. ATG was administered according to PPHG and Working Party SAA Group of EBMT protocol: ATG 3.75mg/kg iv for 5 days, cyclosporin A (CSA) 5mg/kg orally from day 1 to day 180 and granulocyte-stimulating factor during deep neutropenia.Remission of the disease was assessed on days 112, 180 and 360 from start of therapy on the basis of blood and bone marrow examination. The results on 112 and 180 were similar. The remission was achieved in 28 of 55 children (51%), complete remission (CR) in 5 children (9%), and partial remission (PR) in 23 children (42%). On day 360 remission was obtained in 30 of 55 patients (42 %), CR in 10 children (18%) and PR in 20 children (36%) There was no response to treatment (NR) in 25 patients (45%).In the group of 55 patients, 15 died (27%). 5 of them died early (day 52 and day 72 of therapy) due to septicemia and central nervous system (CNS) hemorrhage. The other 10 died late (day 99 and day 360 of therapy) due to CNS hemorrhage. Observation time of patients ranged from 1 to 14 years. During this time we noted four relapse. PNH was observed in one patient. The probability of 14 year survival in our group of patients treated initially with rabbit ATG is 72,7%. The results in the last follow up (Jun 2009) are better then earlier. We observed CR in 23/55 children (42%), PR in 10/55 (18%), NR 18/55 (33%). o information about 4 patients.We conclude that initial treatment with rabbit ATG is safe and effective in children. Further studies are needed, to assess long-term effectiveness of rabbit ATG in IST. Disclosures:No relevant conflicts of interest to declare.

Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis.

In immune-induced glomerulonephritis (gn), glomeruli (gl) synthesize nitric oxide (NO), and urinary nitrite (NO2-) excretion is increased. In mammals on a low nitrate (NO3-) diet, urinary NO3- is a measure of endogenous NO3- synthesis. Excretion is increased after administration of macrophage activators, reflecting induction of NO production. To determine whether increased urinary NO2- gn is due to glomerular synthesis we studied urinary NO2-/NO3- in accelerated nephrotoxic nephritis induced by preimmunization with rabbit immunoglobulin G (IgG), followed by rabbit anti-rat nephrotoxic globulin, and in control rats similarly preimmunized with rabbit IgG, but followed by normal rabbit serum. Both urinary NO2- and NO3- were increased by i.p. preimmunization with rabbit IgG (peak 463 +/- 171 nmol NO2-/60.3 +/- 9.4 nmol NO3-/24 h, P < 0.001 for both NO2- and NO3- compared with preimmunization levels). Repeat immunization with i.v. rabbit anti-rat nephrotoxic globulin (nephritic rats) or normal rabbit globulin (control rats) again increased urinary NO2- and NO3-. There was no statistically significant difference in urinary NO2- and NO3- levels between nephritic rats where globulin had nephrotoxic activity and the control rats injected with normal rabbit globulin, despite increased NO2- synthesis in ex vivo nephritic glomeruli after nephrotoxic globulin (7.9 +/- 1.9 nmol/2000 gl/48 h; controls 3.2 +/- 1.0 nmol/2000 gl/48 h). Thus neither urinary NO2- nor NO3- levels reflect local activation of the NO pathway in glomeruli. As reported for other stimulants, we show here that systemic stimulation with foreign antigen increased NO synthesis.

Rabbit Antithymocyte Globulin in Treatment of Children with Severe Aplastic Anemia.

In children with severe aplastic anemia (SAA) bone marrow transplantation is the chosen therapy. However, it is only possible in about 15–20% patients who have family donors. Immunosuppressive therapy (IST) is administered in the remaining cases. 105 children in 11 hematology centers of the Polish Pediatric Hematology Group (PPHG) were given IST treatment. 27 patients (12 girls and 15 boys aged 6–16 years) received antithymocyte rabbit globulin (ATG) in the first course of therapy. ATG was administered according to PPHG and Working Party SAA Group of EBMT protocol: ATG 3.75mg/kg iv for 5 days, cyclosporin A (CSA) 5mg/kg orally from day 1 to day 180 and granulocyte-stimulating factor during deep neutropenia. Remission of the disease was assessed on days 84, 112 and 180 from start of therapy on the basis of blood and bone marrow examination. On day 180 remission was obtained in 17 of 27 patients (62.9%), complete remission (CR) in 11 children (40.7%) and partial remission in 6 children (22.2%) There was no response to treatment (NR) in 10 patients (37%). In the no response group, 1 child died and 4 had bone marrow transplants from unrelated donors. The remaining 5 patients received a second course of IST therapy, and 2 of them had CR. In the group of 27 patients, 4 died (14.8%). 2 of them died early (day 52 and day 72 of therapy) due to septicemia and central nervous system (CNS) hemorrhage. The other 2 died late (day 99 and day 365of therapy) due to CNS hemorrhage. Observation time of patients ranged from 1 to 13 years. During this time we noted one relapse. MDS/AML or PNH was not observed in any of the patients. The probability of 13 year survival is 85.1% in our group. Our data suggest that initial SAA treatment with ATG is safe and effective in children. Further studies are needed, however, to assess long-term effectiveness of ATG in IST.

Development of Coated Tubes RIA for Serum T3 (Tri‐Iodothyronine) for Production Scale

A coating procedure that could provide immobilization of antibodies, with increased binding capacity, that is cost effective, simple, robust, and appropriate for production scale application, is described. This coating approach of T3 antibodies to the polystyrene tubes has been systematically investigated to determine its utility for the development of coated tube Radioimmunoassay (RIA) for T3 in human serum. Further, the results obtained by the developed coating procedure are found to be comparable with those obtained by the “gold standard,” the liquid phase RIA for T3. The coating procedure is completed in three major steps, each step involving an overnight incubation. The normal rabbit γ‐globulins are physically adsorbed onto the polystyrene tubes and incubated. After washing, a second antibody (goat anti‐rabbit antiserum) is added and incubated. To this antigen specific antibody is added (T3 antibody produced in rabbit) and further incubated. Finally, the non‐specific sites on the tubes are saturated by the blocking solution. The concentration of normal rabbit globulin, titers of second antibody and T3 antibody, and time required for coating are optimized to arrive at a suitable coating protocol. The coated tubes were evaluated for precision, reproducibility, and stability. Various parameters such as total reaction volume, incubation time and temperature, total number and volume of washings, concentration of 8‐anilino‐1‐naphthalene sulfonic acid (ANS), and quantity of tracer per tube are optimized to arrive at a suitable standard curve. The optimized assay is validated for the quality control parameters such as intra‐ and inter‐assay variations, recovery, and parallelism. The developed coated tubes assay had an assay range of 0.3–4.8 ng/mL with a sensitivity of 0.3 ng/mL at 90% B/B 0. Batch to batch variation in coating was <10%. The coated tubes were stable up to 1 year, which is adequate for production scale.

Urgent Allogeneic Bone Marrow Transplantation Using a Preparative Regimen of Cyclophosphamide Anti-Human Thymocytes Rabbit Globulin in a Patient with Severe aplastic Anemia with Pneumonia

Urgent Allogeneic Bone Marrow Transplantation Using a Preparative Regimen of Cyclophosphamide Anti-Human Thymocytes Rabbit Globulin in a Patient with Severe aplastic Anemia with Pneumonia

New cause for false-positive results with the Pastorex Aspergillus antigen latex agglutination test

The Pastorex Aspergillus antigen test for detection of Aspergillus galactomannan antigen in the sera of patients with invasive aspergillosis is used in many clinical laboratories. A serum sample contaminated with Penicillium chrysogenum gave a strongly positive reaction (1:128) which was heat stable, was not eliminated by pronase treatment, and was not detected by a normal rabbit globulin control. This observation was shown to be due to cross-reactions of the monoclonal antibody EB-A2 used by the kit with several airborne fungi likely to contaminate serum samples, including Penicillium chrysogenum, Cladosporium herbarum, Acremonium species, Alternaria alternata, Fusarium oxysporum, Wangiella dermatitidis, and Rhodotorula rubra.

Recombinant Tumor Necrosis Factor Stimulates Interleukin-1 Production in Glomerular Cultures from Rats with Nephrotoxic Serum Nephritis

We have investigated the effect of recombinant tumor necrosis factor (r-TNF) on the release of interleukin-1 (IL-1) in glomerular cultures from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN). Freshly isolated glomeruli from the NTSN rats were incubated for 24 h in the presence of r-TNF. When the glomeruli were activated by r-TNF substantial amounts of IL-1 could be detected in the supernatants as measured by the mouse thymocyte assay. The r-TNF-induced IL-1 activity was significantly higher in rats with NTSN than those in normal controls and the other control group, consisting of preimmunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. To avoid the effect of prostaglandins on the IL-1 assay, we cultured the glomeruli with addition of indomethacin and assayed IL-1 activity in the culture supernatants. This cyclooxygenase inhibitor augmented r-TNF-induced IL-1 production. Our data suggest that r-TNF can serve as a potent stimulator of IL-1 synthesis in glomerular cultures from rats with NTSN.

Interleukin-1 production by lipopolysaccharide-stimulated glomeruli from rats with nephrotoxic serum nephritis.

The objective of the present work was to characterize some aspects of interleukin-1 (IL-1) production by nephritic glomeruli after stimulation with bacterial lipopolysaccharide (LPS). Freshly isolated glomeruli from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN) were incubated for 24 h in the presence of LPS. The modified NTSN was produced by an intravenous injection of nephrotoxic serum (NTS) into the rats which had been previously immunized with rabbit IgG and Freund's complete adjuvant. The glomerular cultures of the NTSN rats were found to release significantly increased amounts of IL-1 after LPS stimulation when compared to the values obtained with normal controls and the other control group, consisting of preimmunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. To block the effect of prostaglandins on the IL-1 assay, we cultured the glomeruli with the addition of indomethacin and assayed IL-1 activity in the culture supernatants. The use of indomethacin resulted in a further increase in IL-1 production. The administration of a rabbit anti-rat macrophage serum reduced the production of IL-1 activity in the NTSN rats. Our findings support the notion that at least in NTSN rats activated macrophages are present and probably account for their increased IL-1 activity. This description of IL-1 activity produced by LPS-stimulated nephritic glomeruli may introduce a new element to the early events leading to glomerular inflammation.

Characterization of the effector mechanism of help for antigen-presenting and bystander resting B cell growth mediated by Ia-restricted Th2 helper T cell lines.

The mechanism of help for resting B cell growth in MHC-restricted T-B collaboration was investigated using an in vitro polyclonal model for these T cell-B cell interactions. In the presence of rabbit anti-mouse Ig, small, size-selected B cells elicit help from syngeneic Ia-restricted Th2 cell lines specific for F(ab')2 rabbit globulin. Both Ag-presenting and bystander B cells receive signals from stimulated Th cells that lead to B cell proliferation. The results suggest that the direct activation of resting Ag-presenting and bystander B cells by Th2 cells is mediated by a similar effector mechanism. Although proliferative responses by Ag-presenting B cells are of greater magnitude, help for both Ag-presenting and bystander B cell populations is characterized by the lack of a requirement for membrane Ig cross-linking, by identical kinetics, and by the necessity for direct cell contact or close proximity with Th cells. B cell proliferation is not induced by exposure to the sequence of diffusable mediators released from a synchronized Ag-specific T-B interaction. The T cell-dependent proliferation by both B cell populations can be inhibited by excess mitomycin C-treated syngeneic "cold target" B cells, demonstrating a requirement for a short-range T cell-B cell interaction. mAb inhibition experiments fail to identify a role for class II, LFA-1, or CD4 membrane molecules in the delivery of help to bystander B cells. Antibody against H2d bystander class II molecules has no effect on bystander B cell proliferation at concentrations that completely block Ag presentation by H2d B cells to an H2d-restricted Th cell line. Antibodies against the cell adhesion molecule LFA-1 or the Th cell molecule CD4 do inhibit bystander B cell proliferation, but only to the extent that they block T cell activation and the induction of help. The inductive stimulus leading to resting B cell growth results from an early, short-range interaction with Th cells. B cell proliferation is supported by T cell soluble mediators as a consequence of this interaction, which is required for at least 8 hr after T cell recognition of Ag/Ia on the surface of Ag-presenting B cells.

Monoclonal Antibodies to Rat Androgen-Binding Protein Recognize Both of Its Subunits and Cross-React with Rabbit and Human Testosterone-Binding Globulin*

A series of four monoclonal antibodies to rat androgen-binding protein (ABP) has been developed. These antibodies recognize both the heavy (48,400-dalton) and light (43,000-dalton) subunits of the native ABP molecule. In addition, they recognize the subunits from which Asn-linked oligosaccharides have been removed by treatment with N-glycanase, indicating that these moieties do not form the immunological determinants recognized by the antibodies. Two of these antibodies are capable of recognizing both nondenatured ABP, as assessed by dot blot analysis, and denatured ABP, as determined by Western blot analysis of ABP after electrophoresis under denaturing conditions. The immunoreactivity of denatured ABP is decreased with two of the antibodies, suggesting that they more readily recognize the antigenic epitopes when the protein is in its native configuration. The antibodies were capable of immunoprecipitating covalently labeled ABP from solution. All four monoclonal antibodies produced were determined to be immunoglobulins M by both enzyme-linked immunosorbent assay and Ouchterlony immunodiffusion even though the initial serum response of the immunized animals indicated the presence of immunoglobulins G. All of the monoclonal antibodies raised against rat ABP cross-reacted with rabbit and human testosterone-binding globulin (TeBG). They were able to detect two subunits when Western blots of intact rabbit [mol wt (Mr, 43,000 and 40,500] or human (Mr, 47,600 and 44,500) TeBG were probed, but only a single subunit (Mr, 39,300) when deglycosylated samples of rabbit TeBG were analyzed.

Production of Interleukin-1 in Macrophage Cultures from Rats with Nephrotoxic Serum Nephritis

To investigate the role of cell-mediated immunity in the pathogenesis of experimental glomerulonephritis, we examined interleukin-1 (IL-1) activity in the culture supernatant of lipopolysaccharide-stimulated peritoneal macrophages from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN). Modified NTSN was produced by an intravenous injection of nephrotoxic serum (NTS) into the rats which had previously been immunized with rabbit IgG and Freund's complete adjuvant. It was found that the NTSN rats had significantly increased levels of IL-1 activity when compared to the values obtained with normal controls and the other control group, consisting of pre-immunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. The administration of a rabbit anti-rat macrophage serum reduced the production of IL-1 activity in the NTSN rats. In this experimental model IL-1 synthesis by peritoneal macrophages is present early in the disease process and may be an important mediator of glomerular immune injury.

Competitive solid-phase enzyme immunoassay for measuring digoxin in serum.

In this clinically useful enzyme immunoassay of digoxin in serum, we mix sample, beta-galactosidase-labeled digoxin, and anti-digoxin Fab fragments for 30 min at room temperature, then use Sepharose-bound second antibody for phase separation, and measure the unbound enzyme activity directly in the supernate of the equilibrium reaction mixture. The immunoassay buffer--phosphate-buffered isotonic saline with added rabbit globulin (4 g/L), hydrolyzed gelatin (2 g/L), Brij 96 detergent (5 g/L), glycerol (0.25 mol/L), and N-acetyl-8-anilinonaphthalene-1-sulfonic acid (2 mmol/L)--minimizes serum matrix effects for convenient measuring of unbound enzyme--digoxin conjugate. The immunoassay developed with Fab fragments has better displacement characteristics than that with intact antibody. Performance of the assay compares favorably with that of other manual digoxin immunoassays; in comparison studies with EMIT involving 110 clinical specimens, the coefficient of correlation was 0.97.

Role of membrane immunoglobulin (Ig) crosslinking in membrane Ig-mediated, major histocompatibility-restricted T cell-B cell cooperation.

Resting murine B lymphocytes can present rabbit anti-Ig to T cell lines specific for normal rabbit globulin. The T cell-B cell interaction is major histocompatibility complex (MHC)-restricted, and leads to activation, proliferation, and differentiation of the resting B cell into an antibody-secreting cell. Efficient antigen presentation and B cell activation depends upon binding of rabbit globulin to (membrane) mIg. To investigate the role of mIg in this polyclonal model for a T cell-dependent primary antibody response, we determined whether crosslinking of mIg is required either for efficient antigen presentation, as measured by helper T cell activation, or for the B cell response to T cell help, since all the direct effects of anti-Ig on B cells require crosslinking of mIg. We found that monovalent Fab' fragments of anti-IgM or anti-IgD work as efficiently as their divalent counterparts. Therefore, a signal transduced through the antigen receptor seems not to be required when T cell help is provided by an MHC-restricted T helper cell recognizing antigen on the B cell surface. Moreover, rabbit globulin bound to class I MHC molecules in the form of anti-H-2K also results in efficient antigen presentation and T cell-dependent B cell activation. However, mIg still appears to be specialized for antigen presentation, since anti-Ig is presented about three- to fivefold more efficiently than anti-H-2K.

Allergen-specific ELISA for horse IgE

An enzyme-linked immuno sorbent assay (ELISA) for measuring horse IgE specific to ovalbumin, bencylpenicilloic acid and odinitrocarboxyphenol is described. We used a sandwich type of ELISA by which horse serum was incubated in antigen-coated tubes containing one additional polystyrene ball, followed by rabbit anti horse IgE serum. The tubes were then incubated with biotinylated goat anti rabbit globulin followed by avidin coupled to phosphatase. Endpoint titrations were compared. The ELISA is highly reproducible due to the pretreatment of the polystyrene with glutaraldehyde. The increased antigen surface area led to a higher sensitivity of the test. The assay can be easily modified for the measurement of other Ig classes and of IgE of other antigen specificities.

Inhibition of a mouse hepatoma by the alkylating agent Trenimon linked to immunoglobulins.

Trenimon was conjugated in active alkylating form to rabbit anti-mouse H6 hepatoma globulin (AHG) with retention of antibody activity. H6 hepatoma-inoculated mice were given various combinations of conjugates, free Trenimon, and unconjugated immunoglobulins in daily injections for 5 days. Linkage of Trenimon to immunoglobulins reduced systemic toxicity of the drug, with comparative retention of its antitumor activity. The antitumor action of Trenimon was potentiated by AHG irrespective of whether the drug was directly linked to AHG or free AHG was administered along with Trenimon linked to normal rabbit globulin (NRG). In vitro, Trenimon bound to AHG was less inhibitory to hepatoma cells than free Trenimon, but more inhibitory than Trenimon-NRG conjugates. There was no significant endocytosis of conjugates by the hepatoma cells. This suggests that unlike free Trenimon, the target molecules of Trenimon-immunoglobulin conjugates are not intracellular DNA but are located on the surface of the hepatoma cells.