Localization of monoclonal antibody TNT-1 in experimental kidney infarction of the mouse

An experimental kidney infarction model was developed in the mouse to study the uptake of a radiolabeled monoclonal antibody previously shown to bind to degenerating cells in malignant tumors. To determine if this approach is applicable to normal tissue and cell degeneration, kidney infarction was produced by clamping the mouse renal artery for 3 h using surgical procedures. Various groups of mice were injected with 131I-labeled TNT-1 F(ab')2 monoclonal antibody directed against nuclear histone antigens at varying intervals after surgery. Imaging, biodistribution, autoradiography, and histological studies were performed on each group of mice, including sham-operated controls, to quantitate the level of binding and localize the uptake of label in clamped and unclamped (contralateral) kidneys. As additional controls, clamped mice were administered radiolabeled irrelevant monoclonal antibody Lym-1 or mouse albumin. The results showed a marked selective uptake of radiolabeled TNT-1 F(ab')2 in the injured clamped kidney compared with the untreated kidney and other normal organs of the mouse. These studies define a model of normal organ necrosis that may be useful for study of the kinetics of antibody uptake in infarcted tissues.     

Immunotargeting of urothelial cell carcinoma with intravesically administered Tc-99m labeled HMFG1 monoclonal antibody

Ten patients with transitional cell carcinoma (TCC) of the bladder received 3–6 mCi of HMFG1 monoclonal antibody (MAb) intravesically. The antibody was labeled with Tc-99m using the 2-Iminothiolane method. All patients underwent transurethral resection of the bladder tumor within 12–20 h following intravesical administration of^99m-Tc-HMFG1. The presence of the radiolabeled MAb in the circulation was studied by measuring the radioactivity in the serum for a period up to 20 h. Three of 10 patients underwent immunoscintigraphy (SPECT) 2–3 h postadministration and cancerous areas could be easily localized. Biopsies were taken from the tumor sites as well as from normal bladder mucosa. Absolute uptake of the administered MAb expressed as percent administered dose/kg of tissue could be evaluated only in eight patients. Multiple specimen taken from various tumor sites in every patient gave a wide range of uptake values ranging from 0 to 9.29% adm. dose/kg, whereas normal tissue uptake values ranged from 0 to 1.63, respectively.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen

Summary The pharmacokinetics of ¹¹¹In-labeled 260F9, a murine monoclonal antibody directed against a breast-cancer-associated antigen, was determined in seven patients with advanced breast cancer. Six patients were administered 1 mg antibody containing 1 mCi ¹¹¹In. The seventh patient was administered 20 mg unlabeled antibody followed by 1 mg ¹¹¹In-labeled antibody all via a peripheral vein. Immunoprecipitation, HPLC and SDS-PAGE gels demonstrated the stability of radiolabel on the antibody. The serum clearance of the radiolabel closely fits (r ²>0.95) a two-compartment model for the first six patients. The apparent volume of distribution of the radiolabel approximated to the plasma volume (3 1) and its mean residence time was 23.7 h. The radiolabel had an average t _1/2 of 22.9±12.21 h at the 1-mg dose. At the 20-mg dose one-compartment elimination kinetics were observed with the radiolabel and antibody showing similar mean residence times (36–41 h) and a t _1/2 of 26–28 h. Whole-body imaging showed that the blood-pool:liver ratio of radioactivity increased fourfold (at 48 h postinfusion) at the higher dose and the percentage of the injected dose of radioactivity in the liver decreased from 25% to 8% (24 h postinfusion).In one patient 7–14 times more radioactivity was localized in a breast tumor than in fat (normal breast). Over the first 25 h an average (cumulative) 7.5% of the total dose was excreted in urine. A study of 260F9 in CDF-1 mice demonstrated that the radiolabel remained associated with the antibody in serum. The antibody, however, cleared 60-fold slower in mice than in patients and showed an increased mean residence time of 191 h. The disparity in the pharmacokinetics of the antibody seen in the mouse and in the clinic, points to the different behavior shown by murine monoclonal antibodies in humans. This points to the need for preliminary studies of antibodies in patients for preclinical evaluations of their effectiveness as drug-targeting agents.     

Biodistribution and plasma survival in mice of anti-melanoma monoclonal antibody cross-linked to OKT3

Summary Anti-melanoma monoclonal antibody XMMME-001 was cross-linked to anti-CD3 monoclonal antibody OKT3 with succinimidyl 3(2-pyridyldithio)propionate (SPDP), and 2-iminothiolane. The dimer heteroconjugate was purified by HPLC gel filtration, labeled with¹³¹I, and 10 µg was injected into each of 24 BALB/c mice. The dimeric heteroconjugate's blood survival in sequentially bled mice (n = 3) and its biodistribution in organs of sacrificed mice (n = 21) were studied. In plasma, the heteroconjugate showed an phase with a half-life of 4 h, and a phase with a half-life of about 18 h. Electrophoretic analysis of labeled heteroconjugate in plasma showed that the half-life of disulfide bonds linking the monoclonal antibodies was approximately 7–8 h. Radioactive heteroconjugate accumulated primarily in the liver; significant uptake was also seen in white blood cells and spleen. Very little radioactivity accumulated in kidney, lung, or colon. As a comparison, SPDP-derivatized anti-melanoma antibody was studied by the same methods. It showed an average -phase half-life of 12.5 h; its maximum accumulation in liver or white blood cells was less than 30% of that of the heteroconjugate. Very low levels accumulated in other tissues. The results imply that the shorter half-life in plasma of the heteroconjugate is primarily determined by clearance due to its larger size and conformation, not the lability of cross-linking disulfide bonds.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA     

Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts

Summary We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11–13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an¹²⁵I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4, using a high or a low protein dose (500 µg or 5 µg). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as thecoefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-µg protein doses had substantially lower CV values, 0.327±0.027, than did tumors from animals given 5-µg protein doses, 0.458±0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.     

Postural effects on peritoneal transport and systemic uptake of radiolabeled monoclonal antibodies

 To optimize the regional delivery advantage with i. p. administration of monoclonal antibody (mAb) for radioimmunotherapy, it may be possible to delay the rate and extent of mAb absorption from the peritoneal cavity by simply altering the position of a patient after radioantibody administration. It has been shown that the hydrostatic pressure against the diaphragm plays a major role in the rate of egress of radioantibodies from the peritoneal cavity and that fluid removal from the peritoneal cavity can be altered by posture. The current study examined postural effects in normal rats following the i. p. injection of ¹²⁵I-5G6.4 murine IgG2a anticarcinoma antibody (45 μCi). A 10-ml injection volume of the radioantibody solution was administered to rats restrained in either a supine or inclined (reverse Trendelenburg; feet down at a 45° angle) position. Pharmacokinetic analysis confirmed that the appearance of the radioantibody into the systemic circulation was delayed in the inclined group. The time to peak blood concentration was prolonged from 14.7 (supine) to 19.2 (inclined) hours (P = 0.005). All other pharmacokinetic parameters were equivalent across the treatment groups. The mean half-life of 166 h, mean blood clearance of 9 ml/min, and mean steady-state volume of distribution of 36 ml were consistent with previous experience with this radioantibody in the rat. The intrinsic absorption profile indicated that the mean percentage absorption from the peritoneal cavity to the blood stream was greater in the supine animals from 4 h after i. p. injection until absorption was complete. By 10 h after injection, absorption from the peritoneal cavity was essentially complete in the supine-dosed animals, while those restrained in an inclined position had cleared only 50% of the total absorbed dose. Hence, the regional delivery advantage afforded by intraperitoneal administration of the radioantibody may be further exploited by maintaining an inclined position throughout the absorption phase, a strategy that may be applicable to radioimmunotherapy of patients. Received: 26 July 1996 / Accepted: 27 January 1997     

Systemic perfusion: a method of enhancing relative tumor uptake of radiolabeled monoclonal antibodies

We evaluated the feasibility of systemic vascular perfusion with saline (mimicking plasmapheresis) as a method to enhance tumor-specific monoclonal antibody (MoAb) tumor/background ratios. Initially, groups of rats were injected intravenously (i.v.) with ¹³¹I-5G6.4 MoAb (murine IgG2aK reactive with ovarian carcinoma). These animal's radioactivity levels were determined by dose calibrator and they were imaged before and after perfusion which was conducted at 4 or 24 h post-antibody injection. Animals were sacrificed after perfusion, as were controls, and normal organ radioactivity levels determined. In addition, nude mice bearing HTB77 ovarian cancers subcutaneously were injected i.v. with ¹³¹I-5G6.4 MoAb and were imaged before and after systemic perfusion with saline 24 h post-5G6.4 injection. Perfusion in rats dropped whole-body 5G6.4 levels significantly at both perfusion times (P < 0.0005). The drop in whole-body radioactivity with perfusion was significantly greater for the animals perfused at 4 h post i.v. 5G6.4 antibody injection (48.3 ± 5.1%) than for those perfused at 24 h post i.v. antibody injection (32.9 ± 2.9%) (P < 0.025). In the nude mice with ovarian cancer xenografts, γ camera images of tumors were visually and quantitatively (by computer image analysis) enhanced by perfusion, with a 2.33-fold greater decline in whole body uptake than in the tumor (P < 0.05). These studies show that (1) much background antibody radioactivity can be removed using whole-body perfusion with saline, (2) that the decline in whole body activity is larger with 4 than 24 h perfusion and (3) tumor imaging can be enhanced by this approach. This and similar approaches that increase relative tumor antibody uptake such as plasmapheresis may be useful in imaging and therapy with radiolabeled antibodies.     

Studies on the metabolic fate of 111In-labeled antibodies

Indium-111-labeled antibodies, though providing superior photon flux to iodine-labeled antibodies, can exhibit high levels of accumulation in some non-target organs. In an effort to understand the nature of this non-target uptake we have evaluated the molecular weight of ¹¹¹In species retained in several tissues by radio-FPLC (sizing chromatography) following injection of [¹¹¹In]DTPA 5G6.4, a murine monoclonal antibody, into normal mice. Blood, liver and kidneys were removed, and liver and kidneys were homogenized at several time points after antibody injection. The proportion of ¹¹¹In-containing species was found to vary with the tissue and with time. Analysis of blood showed only radiolabeled antibody. In the liver, several ¹¹¹In species were identified with molecular weights compatible with intact antibody, [¹¹¹In]transferrin, and low molecular weight complexes, with an increase in the proportion of [¹¹¹In]transferrin and low molecular weight species occurring over time. While the same molecular weight species were also identified in the kidneys, the kidneys contained the largest percentage of low molecular weight species which increased over time. When ¹²⁵I-labeled 5G6.4 was injected and the tissues similarly analyzed, only radioactive material with the molecular weight of intact antibody was detected. Comparison of two methods of purification of [¹¹¹In]labeled antibody after labeling revealed a significant difference in the organ uptake of radiolabeled products for ¹¹¹In. Although dialysis was sufficient for the removal of labile ¹¹¹In, as determined by TLC, subsequent sizing chromatography on Bio-Gel P-60 dramatically dropped the hepatic and renal uptake of ¹¹¹In relative to blood and diminished the proportion of the low molecular weight species present on sizing FPLC of extracts from tissues. These data indicate that low and intermediate molecular weight ¹¹¹In compounds are accreted in the liver and kidneys following the i.p. injection of ¹¹¹In-labeled monoclonal antibodies and that their uptake can be diminished by more stringent radioantibody purification. This knowledge may be valuable in developing methods for reducing non-target ¹¹¹In uptake.     

Radioimmunotherapy of small-cell lung cancer xenografts using131I-labelled anti-NCAM monoclonal antibody 123C3

We have studied the therapeutic efficacy of¹³¹I-labelled monoclonal antibody 123C3 in human small-cell lung carcinoma xenografts established from the NCI-H69 cell line in nude mice. Several radiation does were administered intraperitoneally and different treatment schedules were tested. The maximal tolerated dose, 2×500 Ci, resulted in complete remission of tumours smaller than 200 mm³ and long-lasting remission (more than 135 days) of the larger tumours. In control experiments, treatment with unlabelled monoclonal antibody 123C3 did not affect the tumour growth rate, while the effect of radiolabelled non-relevant, isotype-matched, monoclonal antibody M6/1 was minor and transient. Regrowth of the tumours occurred in all cases and could not be attributed to loss of neural cell adhesion molecule (NCAM) expression. Tumour recurrence is probably caused by insufficient radiation dosage. Radiation-induced toxicity was monitored by assessment of weight and bone marrow examination. Weight loss was observed in all treatment groups, but the mice regained their initial weight within 14 days, except for the group receiving the highest radiation dose (3×600 Ci). In this group all mice died as a result of radiotoxicity. Of the mice injected with 600 Ci radiolabelled control antibody, 50% died within 2 weeks after administration. Apparently the higher uptake of the radiolabelled monoclonal antibody in the tumour reduced systemic radiation toxicity.     

Maximizing the precision of the mathematically ideal radioimmunoassay at a specified dose

Mathematical analysis of the equation for the radioimmunoassay, R²+R(1+Kp+Kp^*–Kq)–Kq=0, gives a general solution for the dose of maximum precision, ²R/p², of the assay. This solution, (R–c₁+Kp)(R–c₂), and the radioimmunoassay equation can be used to determine theoretical conditions for maximum precision of a radioimmunoassay at any dose p. A theoretically optimal antibody concentration, q, can be calculated for precision at a critical concentration of interest, p. This is dependent on the affinity constant, K, of the particular antibody being used and, potentially, the amount of labeled tracer, p^*. This solution idealizes the radioimmunoassay to a one affinity constant system. Though this may be achievable, particularly with monoclonal antibodies or receptors, the applicability needs to be explored experimentally.     

Autoradiolysis of iodinated monoclonal antibody preparations

We investigated the effects of ionizing radiation on the immunointegrity of antibody fragments (Fab) because large amounts of high specific activity ¹³¹I may damage the proteins. We found that 1000 Gy of external ¹³⁷Cs γ radiation was sufficient to destroy 80–90% of the immunointegrity of the initial preparation. This effect was also produced by internally added [¹³¹I]NaI in a quantity sufficient to provide the same radiation absorbed dose. Since radioiodinated monoclonal antibodies labeled to high specific activity are being evaluated for radioimmunotherapy, the above observation is significant since high levels of internal radiation occur with therapeutic doses of ¹³¹I-labeled antibody. Human serum albumin in low concentration (2%) added to the iodinated antibody solutions was successful in preventing loss of immunoreactivity and can be used to protect and stabilize therapeutic quantities of radiolabeled monoclonal antibody preparations.     

Biodistribution Analyses of a Near-Infrared,Fluorescently Labeled,Bispecific Monoclonal Antibody Using Optical Imaging

In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.Abbreviations: EGFR, epidermal growth factor receptor; EIBS, EGFR–IGF1R bispecific monoclonal antibody; FMT, fluorescence molecular tomography; IGF1R, insulin-like growth factor 1 receptor; NIRF, near-infrared fluorophore; PET, positron emission tomographyOver the past 25 y, bispecific antibodies have been added to the arsenal of humanized antibodies that have been developed to treat cancer, infectious diseases, and inflammatory diseases. Bispecific biotherapeutics simultaneously recognize 2 different epitopes on the same or different cells or bacteria. This multispecificity offers several advantages over their monospecific counterparts in that cell or tissue specificity can be increased and thus toxicity reduced, 2 (or more) receptor pathways can be targeted simultaneously, and immune-mediated responses at clinically affected sites may be activated (cancer) or deactivated (inflammatory disease).^13,18 As the size and structure of monoclonal antibodies are altered from their natural state to create biotherapeutics, such as bispecific antibodies,⁸ the pharmacokinetic properties of the products may differ from that of their parental (natural) antibodies. Aside from the potential to increase immunogenicity or aggregate, very little is known about how these structural differences affect pharmacokinetics and biodistribution. Receptor-mediated uptake and cellular processing both are likely to play a role,²⁰ but identifying the tissues or cell types involved in recognizing these altered proteins is a necessary first step toward developing engineering strategies that can circumvent these impediments.At the organ or tissue level, the use of radiolabeling and imaging in clinical and preclinical studies has greatly facilitated our understanding of the pharmacokinetics and tissue distribution of biotherapeutics.^7,21 This advance is in part due to the ability to track and monitor the distribution of labeled compounds in the same animal over time. The increased sensitivity of positron emission tomography (PET) and the ability of radioisotope signals to travel through tissues without being significantly attenuated or diffracted has made PET the leading modality for preclinical and clinical biodistribution analyses. Disadvantages of PET are that its application is more expensive and requires skill in chemical conjugation, imaging technology, and radiation safety. Given that these resources are often limited or unavailable at several institutions (ours included), near-infrared optical imaging with improved tissue penetrance and quantitative imaging algorithms are demonstrating value when compared with ‘high-end’ imaging modalities.^19,33 The accessibility of a variety commercially available fluorescently labeled targeting proteins and peptides, activatable probes, and fluorescent protein labeling kits has made this technology more readily available. In addition, because multiple fluorophores of nonoverlapping emission spectrums can be administered simultaneously, colocalization studies and direct comparisons of 2 or more labeled compounds can be performed in the same animal. Here we show how fluorescence-based optical imaging was used to identify potential tissue depots or sinks and characterize the biodistribution profile of a bispecific antibody under preclinical development.     

Prevalence and molecular heterogeneity of alfa+thalassemia in two tribal populations from Andhra Pradesh,India

Summary We describe here the screening of a small group of apparently healthy individuals belonging to the tribal communities of Koya Dora and Konda Reddi. A remarkably high incidence of deletion and nondeletion ⁺ thalassemia mutants has been found with allele frequencies and distributions characteristic to each tribe. We have confirmed the strict relationship between Hb S levels and the number of globin genes in double heterozygotes for the S gene and thalassemia. In this population sample we did not find either heterozygous carriers of ⁰ thalassemia (deletion of both alpha genes in cis) or individuals showing hemolytic anemia due to inactivation of three -globin genes (Hb H disease). Selection by malaria is most probably responsible for the prevalence of the various ⁺ thalassemia haplotypes among the two tribal populations of Andhra Pradesh.     

Rapid miniaturized chromatography for 111In labeled monoclonal antibodies: Comparison to size exclusion high performance liquid chromatography

Our laboratory investigated the use of a rapid miniaturized chromatography system, ITLC-SG with 0.9% NaCl, to assess the radiochemical purity of ¹¹¹In labeled monoclonal antibodies (MoAbs). Radiochemical analysis was performed on numerous ¹¹¹In labeled antibody preparations with labeling efficiencies ranging from 40 to greater than 95% and the results compared to those obtained with size exclusion high performance liquid chromatography (HPLC). The chromatographic procedure involved challenging radiolabeled antibodies with 0.05 M DTPA to chelate unbound and/or non-specific bound ¹¹¹In, spotting on miniaturized instant thin layer-silica gel chromatography strips, developing in 0.9% NaCl, and counting appropriate segments for radioactivity. Results of the study demonstrated that the miniaturized chromatography procedure was rapid, taking less than 4 min to complete, and accurate in assessing the amount of unbound or non-specific bound ¹¹¹In in ¹¹¹In labeled monoclonal antibodies, when compared to size exclusion HPLC.     

Specific macromolecular interactions between tau and the microtubule system

The microtubule-associated protein Tau, a major component of brain microtubules, shares common repeated C-terminal sequences with the high molecular-weight protein MAP-2. It has been shown that tau peptides V¹⁸⁷-G²⁰⁴ and V²¹⁸-G²³⁵ representing two main repeats, induced brain tubulin assembly in a concentration-dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides and site-directed monoclonal antibodies. Tau peptides appeared to compete with MAP-2 incorporation into assembled microtubules. The interactions of the tau fragments with -tubulin peptides bearing the tau binding domain on tubulin were analyzed by fluorescence spectroscopy. The specificity of the binding was further demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti-idiotypic antibody produced after immunization with the -11(422–434) tubulin peptide, as assessed by enzyme-linked immunoassay. Western blots confirmed the interaction of tau with the monoclonal antibody. In addition, immunoassays revealed a competition between the MAP-reacting monoclonal antibody and the tubulin peptide -11(422–434) for their interaction with the tau molecule.     

Metabolism of trimethylamines in kelp bass (Paralabrax clathratus) and marine and freshwater pink salmon (Oncorhynchus gorbuscha)

Summary ³H or¹⁴C labeled tracers were used to investigate the metabolism of trimethylamine (TMA), trimethylamine oxide (TMAO), choline, and betaine in free swimming kelp bass (Paralabrax clathratus). An indwelling cannula in the ventral aorta was used to administer tracer and withdraw blood samples. The concentrations of TMA and TMAO were determined in liver, muscle, and plasma. The TMA liver content is higher than that of muscle (0.85 vs 0.01 moles/g wet tissue) while the amount of TMAO in muscle greatly exceeds its liver concentration (60 vs 0.04 moles/g wet tissue). Prolonged fasting (21 and 75 days) or feeding the fish a squid diet containing high levels of TMAO did not alter the tissue concentrations of TMA or TMAO, suggesting that these compounds are endogenous in origin and that their tissue concentrations are subject to regulation. Comparison of the radiospecific activities of TMA and TMAO, and the administered TMA tracer suggest that TMA is channled directly to TMAO in the liver without equilibration in the hepatic TMA pool. The conversion kinetics of TMA to TMAO and the distribution of these amines in liver and muscle with time suggest that labeled TMA is rapidly taken up into a sequestered pool from which it is slowly released, oxidized to TMAO in the liver, and then transported via the circulation to the muscle mass. The location of this proposed sequestered TMA pool was not determined. Experiments with labeled choline and betaine suggest that these compounds are interconverted in the liver and that enzymes are present for conversion of choline betaine TMA TMAO. Labeled dimethylamine (DMA) was not metabolized and is, therefore, probably not a precursor of TMA and TMAO. [¹⁴C]Trimethylamine (TMA) was also used to investigate the possible role of trimethylamine oxide (TMAO) as an osmoregulatory compound in migrating prespawning cannulated Pacific pink salmon (Oncorhynchus gorbuscha) taken from marine or fresh water environments. Marine and fresh water salmon oxidized administered [¹⁴C]TMA to TMAO; labeled metabolites other than TMA and TMAO were not detected. Four hours after [¹⁴C]TMA injection about 10% of the administered dose was present in muscle as labeled TMAO and about 33% as TMA. Unlike our finding in kelp bass, [¹⁴C]TMAO was not recovered in liver, although low amounts of labeled TMA were found (0.4% of administered dose). Labeled TMA and TMAO, however, were detected in liver after [¹⁴C]betaine adminstration to a marine salmon, indicating that TMA-mono-oxygenase is present in salmon liver. The presence of labeled choline indicates that choline and betaine are interconverted as in kelp bass. The amount of [¹⁴C]TMA oxidized to [¹⁴C]TMAO and then accumulated in the muscle mass is the same in marine and fresh water salmon, as is the amount of chemical TMAO present (4.6 moles/g muscle).     

Structural characterization of blood group A glycosphingolipids recognized by the antibody 3G9-A

In this study, the antibody 3G9-A was assayed for activity against human erythrocyte glycosphingolipids. The antibody was found to recognize glycosphingolipid components from blood group A erythrocytes but not glycosphingolipids from blood group B or O erythrocytes. Subsequent investigation revealed that the glycosphingolipid components recognized by the antibody were also recognized by a blood group A specific monoclonal antibody. The structures of two of the isolated active glycosphingolipid components were structurally characterized using proteon nuclear magnetic resonance (¹H NMR) and gas chromatography-mass spectrometry (GC-MS) techniques and were found to consist of two blood group A glycosphingolipids; the type 2 chain A^b and type 3 chain A^a glycosphingolipids. Subsequent analysis of the remaining active components by GC-MS and immunostaining techniques revealed that all of the active components were blood group A glycosphingolipids. Furthermore, structural studies of the active components suggested that the epitope of the antibody consisted of the group A trisaccharide, GalNAc1,3(Fuc1,2)Gal.Abbreviations GC-MS gas chromatography-mass spectrometry - ¹H NMR proton nuclear magnetic resonance - Gal d-galactose - Glc d-glucose - Fuc l-fucose - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Cer ceramide - mAb monoclonal antibody - BSA bovine serum albumin - PBS phosphate buffered saline - FID free induction decay - PMAA partially methylated alditol acetates     

An improved method of direct labeling monoclonal antibodies with 99mTc

An improved method of direct labeling MAbs with ^99mTc is described. Two murine monoclonal antibodies, designated Lym-1 and B72.3, have been successfully labeled with ^99mTc in 0.1 M borate buffer at pH 9.3. The choice of buffer and pH was essential for obtaining a radiolabeling yield ⩾98%. In vitro studies demonstrated that the radiolabeled antibodies were stable and retained their immunoreactivity. Imaging and biodistribution studies using Raji and LS174T human tumor-bearing nude mice demonstrated a significant tumor uptake at 24-h post-injection of ^99mTc-labeled MAbs. This improved labeling method showed better stability than those of previously published methods and resulted in significant improvement in the uptake of antibody in tumor. External images at 24 h post-injection revealed clearly visible tumors demonstrating the benefit of this method for tumor immunoscintigraphy.