The diagnosis of atypical pheochromocytoma: a challenge for the biologist as well

Biological diagnosis of pheochromocytoma is relatively easy in those cases releasing great amounts of catecholamines with strong clinical features; instead, diagnosis could be more problematic in atypical or asymptomatic familial pheochromocytoma with small tumors secreting low catecholamine amounts. Several plasma and urine adrenergic markers must be used to confirm the clinical suspicion. We have discussed the biological data of three totally asymptomatic pheochromocytomas (cases no 2, 3, 4) and one case with a very discrete clinical manifestation (no 1). Three patients had very small tumors (4, 7 and 25 g) secreting preeminently adrenaline, one patient had a 45 g adrenal incidentaloma without clinical expression. Our study shows that, in these special cases, except for an inconstant increase of adrenaline, plasma and urine catecholamines and urine VMA can be normal. The most useful markers are plasma and urine methoxyamines. However, plasma methoxyamines are the most sensitive because their increase over reference values is by far greater than in urines. Several factors may explain these findings: a low tumoral secretion, the nature of the released amine, the short half-life of catecholamines in plasma and, in some cases, the involvement of intratumoral catecholamine metabolism. Analysis of the ratio NMN/MN in plasma provides an additional diagnosis tool to reveal adrenaline secretion abnormalities.     

Therapeutic drug monitoring: a challenge for biologists

Therapeutic drug monitoring may be a precious help for the clinician especially if rigorously performed, and it is used for many drugs. The leading role of biologists in the development of new tests is strengthened by the coming-up of new drugs, the increasing demands for clinical trials to study the concentration-clinical effects of drugs and the development of pharmacogenetics. These represent stimulating challenges to optimise therapeutic drug monitoring and favour collaboration between biologists and clinicians so as to improve the care quality of the patient.     

Preparation of Fab fragments from a mouse monoclonal IgM

A procedure is described for preparing and purifying Fab fragments from a mouse monoclonal IgM. The IgM was digested with trypsin in the presence of the reducing agent, cysteine. The resulting Fab fragments were alkylated with iodoacetamide and purified using a Sephacryl S-200 column. The Fab fragments had a molecular weight of 48 000 as determined by SDS polyacrylamide gel electrophoresis and molecular sieve chromatography. This procedure has been successfully used with five different mouse monoclonal IgM. The Fab fragments bound antigen with the same specificity as the whole IgM.     

人源Fab抗体库的构建和抗HBsAg抗体的筛选和鉴定

目的 构建人源Fab抗体文库,筛选抗HBsAg 抗体片段并进行初步鉴定.方法 收集20份临床检验废弃的成人乙肝感染者淋巴细胞,抽提总RNA ,逆转录成cDNA,构建抗乙肝病毒人源免疫型Fab抗体文库.以HBsAg包板进行4轮循环的吸附-洗脱-扩增,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,对阳性克隆进行可溶性表达,并用ELISA对其特异性进行鉴定.结果 构建的人源Fab型抗体文库的库容为2.0×108,并具有良好的多样性.经过4轮筛选,成功获得4株能与HBsAg结合的人源抗体克隆,分别命名为hFabHB1、hFabHB2、hFabHB3 和hFabHB4.对其中的hFabHB1进行可溶性表达,ELISA鉴定阳性.结论 成功构建了抗乙肝病毒人源免疫型Fa b抗体文库,从中筛选获得的4株人源Fab抗体片段有望在乙型肝炎的预防和治疗上发挥作用 .     

Nanotechnology for the biologist

Nanotechnology refers to research and technology development at the atomic, molecular, and macromolecular scale, leading to the controlled manipulation and study of structures and devices with length scales in the 1- to 100-nanometers range. Objects at this scale, such as "nanoparticles," take on novel properties and functions that differ markedly from those seen in the bulk scale. The small size, surface tailorability, improved solubility, and multifunctionality of nanoparticles open many new research avenues for biologists. The novel properties of nanomaterials offer the ability to interact with complex biological functions in new ways-operating at the very scale of biomolecules. This rapidly growing field allows cross-disciplinary researchers the opportunity to design and develop multifunctional nanoparticles that can target, diagnose, and treat diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses "nanotech" strategies and constructs that have already demonstrated in vitro and in vivo efficacy.     

Human recombinant Fab fragments with sub-nanomolar affinities for acetylated histones

We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.     

Studies on the conjugation of horseradish peroxidase to Fab fragments

The two step method for conjugation of glutaraldehyde-activated horseradish peroxidase to isolated Fab fragments was evaluated. By increasing the molar excess of horseradish peroxidase nearly all available Fab fragments could be conjugated. However, with increasing excess of peroxidase more than one peroxidase molecule became conjugated to each Fab fragment with concomitant loss of antibody activity. The optimal yield of one-to-one conjugate was achieved at about 8-fold excess of peroxidase.     

Factors influencing bonding of bromelain agglutinators and their Fab fragments

The complex of bromelain agglutinators and their homologous Fab fragments is dissociated by gel chromatography under certain conditions. When albumin is present as a source of thiol groups, Fab fragments previously treated with N-ethylmaleimide (NEM) will dissociate from the agglutinators (fluid phase). If anti-Rh Fab fragments are bound to Rh-positive erythrocytes, the agglutinates are not dissociated by thiols (cellular phase). Prior to erythrocyte sensitization, the agglutinator site on Fab fragments can be blocked by thiol-disulfide exchange. Once the Fab fragments are coated on erythrocytes, the agglutinator site is more readily available than it was prior to sensitization, as evidenced by inhibition with 0.01 M NEM. The differences between the bonding characteristics of the fluid phase and the cellular phase and the influence of mercaptoalbumin on the agglutinator-Fab complex suggest that the agglutinators are not antibodies.     

Isolation of Fab and Fc fragments from mouse immunoglobulin Gl

Two methods of obtaining Fab and Fc fragments from mouse immunoglobulin Gl are described. In the first case the papain protein digest is fractionated on a column with DEAE-or DE-32-cellulose, equilibrated with 0.005 M potassium phosphate buffer, pH 8.0. The Fab fragment is eluted from the column in the starting buffer; the fragment is eluted when the ionic strength of the buffer is increased to 0.4 M. In the second case the protein is fractionated on an ion-exchange resin equilibrated with 0.004 M Tris-H₃PO₄ buffer, pH 8.5. The whole of the protein applied under these circumstances is bound by the column. The Fab fragment is eluted with 0.04 M Tris buffer containing 0.004 M of a mixture of K-phosphate salts at pH 8.5; the Fc fragment is eluted by increasing the ionic strength by means of phosphate to 0.4 M. Since neither method can yield absolutely pure Fab or Fc fragments, in order to obtain monospecific antisera against these fragments it is necessary to cross-exhaust the antisera with appropriate immunosorbents.Laboratory of Antibody Chemistry and Biosynthesis, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Sciences of the USSR P. A. Vershilov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp. 116–119, July, 1979.     

Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library

Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl beta-D-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.     

Human monoclonal Fab fragments specific for viral antigens from combinatorial IgA libraries

Background: IgA constitutes the first line of immune defense, interacting with a variety of environmental antigens. Following infection with respiratory syncytial virus (RSV) individuals frequently exhibit elevated serum IgA titers specific for the virus. Previously combinatorial IgG libraries have successfully been used to clone such human antibody responses. Objectives: Here we evaluate the possibility of constructing combinatorial IgA libraries on the surface of filamentous phage to retrieve human viral-specific IgA Fab fragments. Study design: Bone marrow from an HIV-1 seropositive donor was used as RNA source to construct combinatorial IgA κ and λ libraries of approximately 10⁷ clones. Results: By affinity selection using an immobilized recombinant RSV FG protein, two unique IgA Fab fragments producing clones (AD5 and AD23) reactive with the selecting antigen were isolated. One of the Fab fragments was found to be specific for RSV F glycoprotein and bind with high apparent affinity (2 × 10⁸ M⁻¹). The other binds with lower affinity and exhibits cross-reactivity with other antigens. Conclusion: The strategy described, involving construction of combinatorial IgA libraries on the surface of filamentous phage, should be generally applicable to the investigation of both mucosal and systemic human IgA immune responses, and may become an important tool for evaluation of mucosal vaccine regimes.     

The use of digoxin-specific Fab fragments for severe digitalis intoxication in children.

BACKGROUND. Because life-threatening digitalis intoxication is unusual in children, treatment with digoxin-specific-antibody Fab fragments (Fab) has rarely been reported. We describe the efficacy of Fab in the treatment of children with severe digitalis intoxication. METHODS. Twenty-nine children with intoxication due to digoxin (28) or digitoxin (1) received Fab at 21 participating hospitals between 1974 and 1986. Data were gathered about the patients' medical illnesses, doses and serum concentrations of digitalis, responses to Fab therapy, and outcomes. RESULTS. In the infants and young children with acute digoxin intoxication, the digoxin doses ranged from 0.30 to 0.96 mg per kilogram of body weight; two adolescents had severe intoxication after doses of only 0.20 and 0.26 mg per kilogram. The serum digoxin concentrations ranged from 3.0 to greater than 100 ng per milliliter (mean, 13.8). Atrioventricular block (present in 22 patients [76 percent]) was the most common sign of toxicity. All the patients in this series had severe disturbances of cardiac rhythm, hyperkalemia (mean serum potassium concentration, 5.4 mmol per liter), or both. In 27 patients (93 percent), digitalis toxicity resolved after the administration of Fab. Of the 19 patients for whom data were available on the timing of the response to Fab, 15 responded within 180 minutes. Three patients required retreatment with Fab. Seven died of complications unrelated to the administration of Fab. CONCLUSIONS. We recommend that Fab be used in the treatment of digitalis poisoning in infants and young children who have ingested greater than or equal to 0.3 mg of digoxin per kilogram, who have underlying heart disease, or who have a serum digoxin concentration of greater than or equal to 6.4 nmol per liter (greater than or equal to 5.0 ng per milliliter) in the elimination phase; and who also have a life-threatening arrhythmia, hemodynamic instability, hyperkalemia, or rapidly progressive toxicity. Adolescents, who are more sensitive to the toxic effects of digoxin than younger children, may require treatment with Fab after ingesting lower doses.     

A novel expression vector for production of epitope-tagged recombinant Fab fragments in bacteria

Labeling of recombinant Fab molecules is an important yet cumbersome and time-consuming procedure that is needed in many immunological experimental designs. This work describes the development of a novel expression vector fusing to the carboxyterminal of the Fab heavy chain fragments a tag peptide (FLAG) that is consistently recognized by a mouse monoclonal antibody. The presence of the FLAG peptide does not alter the binding characteristics of the unmodified Fab molecule, as demonstrated by relative affinity determinations and competition experiments. This new method is suitable for extensive utilization in immunological experimental work using recombinant Fabs.     

Digitalis intoxication and treatment with digoxin antibody fragments in renal failure

Summary Severe digitalis intoxication today is preferentially treated by intravenous infusion of Fab fragments of digoxin antibodies (Digitalis Antidot BM). The kinetics of Fab fragments in the circulation are well known when kidney function is normal or slightly impaired. There are no data available, however, in complete renal failure. We observed a patient with life-threatening digitalis intoxication (serum digoxin, 3.7 ng/ml) and anuria, who was treated successfully by 160 mg Fab fragments i.v. Serum digoxin and Fab fragment concentrations could be followed for 229 h. The extrarenal clearance of Fab fragments was lower (5.6 ml/min) than in patients with normal kidney function (10.9 ml/min). This finding suggests that lower doses than usual might be sufficient for treating patients with severe digitalis intoxication and renal failure.Abbreviations AUC area under the curve - FAB fragments digoxin antibody fragments - h hour     

Mouse IgG2b monoclonal antibody fragmentation. Preparation and purification of Fab, Fc and Fab/c fragments

Mouse IgG2b fragmentation has been poorly described in the literature because of the sensitivity of this subclass to proteases and the inability to produce F(ab')2 fragments. The fragments obtained include both Fc and Fab fragments and an intermediate of degradation, the Fab/c fragment, consisting of the Fc region and one Fab arm, which was first described by Parham (1983). Optimised pepsin digestion led to the formation of Fab/c in yields of up to 30% depending on the IgG2b antibodies susceptibility. DEAE-cellulose chromatography of the digested antibodies allowed, in all cases, separation of Fab fragments from Fc bearing fragments. Depending on the differences in pI between the fragments, Fab/c fragment purification was achieved either by CM-cellulose chromatography or by recycling HPLC gel filtration chromatography. Both procedures gave 97.5% purity Fab/c fragments. Fc fragments were purified by HPLC gel filtration chromatography. In cancer therapy the monovalent Fab/c fragments could be useful for drug targeting or for immunotherapy providing it retains a good affinity.     

Allotypic and isotypic specificities of rabbit IgM: localization to the Fab mu or Fc mu fragments

IgM from trypanosome-infected rabbits was digested with trypsin under different conditions to obtain Fab mu or Fc5 mu fragments suitable for analysis with anti-allotype and anti-isotype antibodies. The Fab mu but not the Fc5 mu fragment was shown to have the n-locus allotypic specificities, n80, n81, n82, n83 and n87, characteristic of the IgM class of immunoglobulins. Thus, the n82 and n83 allotypic specificities, conformationally dependent on the a VH locus for expression, and the n80, n81 and n87 allotypic specificities, independent of the a VH locus for expression, are in either the CH1 or CH2 domain of IgM heavy chains. In addition, two high-affinity mouse monoclonal antibodies (MoAbs) specific for IgM and able to bind IgM in direct-binding radioimmunoassays were produced and characterized. One MoAb (3C1) was specific for an isotypic determinant (epitope) in the Fab mu fragment, presumably in the CH1 or CH2 domain, whereas another MoAb (8C2) was specific for an isotypic epitope in the Fc5 mu fragment, presumably in the CH3 or CH4 domain. The proximity of the n-locus allotypic specificities (CH1 or CH2 domain) to the VH domain is consistent with the finding that some IgM allotypic specificities are expressed only in conjunction with certain a VH locus allotypic specificities.     

Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen

Serum titers of antibody to Epstein-Barr virus (EBV) viral capsid antigen (VCA) have been positively correlated with malignancies of lymphoid proliferation, such as Burkitt's lymphoma and Hodgkin's lymphoma. We have constructed a phage display combinatorial antibody Fab library from a patient with marginal zone B cell lymphoma associated with Sj?gren's syndrome and carrying high serum anti-EBV-VCA IgG titer. Fab fragments were selected by panning against EBV-VCA protein coated onto ELISA plates, and selected Fab clones were characterized by ELISA, western blotting (WB), indirect immunofluorescence assay and immunohistochemistry. We have established two Fab clones, Fab-aVCA1 and Fab-aVCA21, which specifically recognize EBV-VCA by ELISA and WB. Inhibition ELISA competition showed that both clones could significantly reduce the binding of specific anti-EBV-VCA mAb to its relevant proteins. Furthermore, these two Fab clones could localize VCA protein in the EBV-positive P3HR1 and Daudi cell lines, as well as in tissue samples from patients with EBV-infected lymphoid malignancies. These results indicate that our two Fab clones are novel human mAbs specific for EBV-VCA protein and may have potential benefits for development of novel diagnostic and therapeutic approaches in EBV-related lymphoid malignancies.