Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa.

The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.     

Anti-rabies immunoglobulin preparation based on F(ab')2 fragments

Technology of manufacturing of new anti-rabies immunoglobulin preparation based on F(ab')2 fragments has been developed. This preparation is characterized by low reactogenicity, increased virus-neutralizing activity and stability.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.     

Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose

In this paper, a novel amperometric glucose biosensor was constructed by alternative self-assembly of positively charged poly(diallydimethylammonium chloride) (PDDA) and negatively charged glucose oxidase (GOx) onto a 3D Nafion network via electrostatic adsorption. The amount of Nafion in the electrode and the number of the (PDDA/GOx)_n multilayers were optimized to develop a sensitive and selective glucose biosensor. Under optimal conditions, the glucose biosensor with (PDDA/GOx)₅ multilayers exhibited remarkable electrocatalytic activity, capable of detecting glucose with enhanced sensitivity of 9.55 μA/mM cm² and a commendably low detection limit of 20 μM (S/N = 3). A linear response range of 0.05–7 mM (a linear correlation coefficient of 0.9984, n = 20) was achieved. In addition, the glucose biosensor demonstrated superior selectivity towards glucose over some interferents, such as ascorbic acid (AA) and uric acid (UA), at an optimized detection potential of 0.6 V versus Ag/AgCl reference.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

A study on the use of caprylic acid and ammonium sulfate in combination for the fractionation of equine antivenom F(ab')(2)

This study involved the fractionation of equine antivenom F(ab′)₂ by combined stepwise ammonium sulfate (AS) and caprylic acid (CA) precipitation without intermediate separation of precipitate. Using a microplate format, 55 conditions with combinations of AS (0–20% saturation) and CA (0–5% v/v), were tested. AS significantly reduced the turbidity raised by CA. High specific antibody activity was observed in the area containing 2–5% CA and 10–20% AS. From these results, 12 precipitation conditions were selected for detailed quantitative studies. Two combinations, one with 4% CA and 15% AS and another with 5% CA and 20% AS, gave the highest fold-purification (1.79 and 1.83) with antibody recoveries at 68% and 59%, respectively. These combinations offered a benefit over CA alone in reducing the turbidity and in increasing the purity but not the recovery of antibody. The conditions giving more favorable overall results were with 2% CA alone and another with a combination of 1.5% CA and 10% AS. These preparations of F(ab′)₂ were homogeneous and without protein aggregate under size-exclusion HPLC. Lastly, 1 h precipitation showed better results than those of overnight precipitation. These results could be useful for the production of therapeutic antivenoms.     

Anti-DNA activity of IgG F(ab')2 from normal human serum.

The components of normal human serum (NHS) which bound DNA in a standard assay for anti-DNA antibody were characterized. IgG was the major detectable protein isolated from NHS by affinity chromatography on DNA-cellulose. A second adsorption of the whole serum IgG with DNA-cellulose did not remove additional gamma-globulin indicating that only a very small fraction of the IgG was capable of binding DNA. This binding activity was largely restricted to denatured DNA. IgG (Fab')2 bound DNA as well as the intact molecules demonstrating the antibody-like nature of the IgG binding. These results suggest that IgG antibody to denatured DNA is a normal component of human serum.     

Rabies post-exposure prophylaxis in the Philippines: health status of patients having received purified equine F(ab')(2) fragment rabies immunoglobulin (Favirab)

Background

Recommended treatment for severe rabies exposure in unvaccinated individuals includes wound cleaning, administration of rabies immunoglobulins (RIG), and rabies vaccination. We conducted a survey of rabies treatment outcomes in the Philippines.

Methods

This was a case series involving 7,660 patients (4 months to 98 years of age) given purified equine RIG (pERIG) at the Research Institute for Tropical Medicine (Muntinlupa, Philippines) from July 2003 to August 2004 following Category II or III exposures. Data on local and systemic adverse reactions (AR) within 28 days and biting animal status were recorded; outcome data were obtained by telephone or home visit 6–29 months post-exposure.

Results

Follow-up data were collected for 6,464 patients. Of 151 patients with laboratory-confirmed rabies exposure, 143 were in good health 6–48 months later, seven could not be contacted, and one 4-year-old girl died. Of 16 deaths in total, 14 were unrelated to rabies exposure or treatment. Two deaths were considered PEP failures: the 4-year old girl, who had multiple deep lacerated wounds from a rabid dog of the nape, neck, and shoulders requiring suturing on the day of exposure, and an 8-year-old boy who only received rabies PEP on the day of exposure.

Conclusions

This extensive review of outcomes in persons with Category III exposure shows the recommended treatment schedule at RITM using pERIG is well tolerated, while survival of 143 laboratory-confirmed rabies exposures confirms the intervention efficacy. Two PEP intervention failures demonstrate that sustained education and training is essential in rabies management.     

Albumin recovery with centrifugal adsorption technology (CAT)

Centrifugal adsorption technology (CAT) is a new compact, countercurrent technology for efficient adsorption from large liquid streams by using adsorbent particles in the micrometer range. CAT seems particularly suited for the recovery of macromolecules at low concentrations, because the small particle dimensions lead to fast mass transfer rates. In this work, the potential of CAT for protein recovery is studied by model and experiment. A predictive model for the separation performance of CAT is presented, incorporating mass transfer resistance and axial dispersion transport in the liquid and the adsorbent phases. The model calculations were compared to experimental data for the adsorption of bovine serum albumin (BSA) on a standard commercial anion-exchange resin with particle diameter d(p) = 50 microm in a pilot-scale CAT apparatus. The model calculations accurately predicted the separation efficiency of CAT. The experimental set-up is shown to be mass transfer limited for the conducted experiments, which agrees with the model predictions. The model was also used to estimate the dimensions and performance of a CAT apparatus for the large-scale recovery of human serum albumin (HSA) from fermentation broth at the scale of 40 tons per year. The resulting equipment dimensions proved to be very small indeed, making CAT a potentially very attractive technology.     

Higher protective activity of Fab' fragment compared to F(ab')2 and IgG on experimental tetanus

Summary Horse sera containing anti-tetanus whole IgG molecules, bivalent F(ab')₂ fragments and monovalent Fab' fragments were injected in 24 groups of 10–20 mice to compare their protective activity. When tetanus was induced in the mice, either with toxin or with spore suspension ofClostridium tetani 24 or 32 h prior to the injection of the antitoxins, monovalent Fab' was significantly more efficient in conferring protection against tetanus than F(ab')₂ or IgG.

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Resumen Afín de comparar la actividad protectora frente al tétanos de moléculas de IgG enteras, fragmentos F(ab')₂ bivalentes y fragmentos Fab' monovalentes, se inyectaron, con suero de caballo que contenía las distintas moleculas, 24 grupos de 10 a 20 ratones. Se indujo el tétanos en los ratones 24 o 32 horas antes de la inoculación con antitoxinas, mediante la toxina o bien con una suspensión de esporas deClostridium tetani. El fragmento monovalente Fab' confirió una protección frente al tétanos significativamente mayor que F(ab')₂ y IgG.

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Résumé Des sera de cheval contenant soit des molécules d'IgG entier, des fragments bivalents F(ab')₂ ou des fragments monovalents Fab' ont été injectés dans 24 groupes de 10 à 20 souris afin de comparer leur activité protectrice. Lorsque le tétanos a été induit chez les souris, que ce fût avec la toxine ou avec une suspension de spores deClostridium tetani, 24 ou 32 heures avant l'injection des antitoxines, le fragment monovalent Fab' s'est révelé plus efficace de manière significative que le fragment F(ab')₂ ou l'IgG dans l'octroi de l'activité protectrice contre le tétanos.

     

Adhesion of Azospirillum brasilense to polystyrene: Influence of ionic strength through protein adsorption

The influence of ionic strength on the adhesion of Azospirillum brasilense to polystyrene has been examined by comparing water and phosphate buffer saline (PBS) as suspending media. Polystyrene supports analysed by X‐ray photoelectron spectroscopy (XPS) after adhesion in PBS for 2 h or 24 h and detachment of adhering cells showed a higher protein surface concentration, reflected by the N/C atomic concentration ratio, compared to supports analysed after adhesion in water. It was shown that PBS both favours protein release by the cells into the solution and enhances the tendency of proteins to adsorb at the support surface.

After 2 h contact time, the increase in the concentration of adsorbed proteins in PBS was related to an increase in adhesion density. However, the observation that the adhesion density after 24 h was lower in PBS than in water indicated that the amount of proteins adsorbed at the support surface controls cell adhesion in a complex way. In PBS, a thick layer of proteinaceous material retaining the bacterial cells is formed; this leads to underestimation of the density of adhering cells as well as to a heterogeneous adhesion pattern and to a relatively low adhesion density due to detachment of pellicles upon rinsing.

The ionic strength thus influences bacterial adhesion in a more subtle way than simply through double layer interactions between the cells and the support.     

抗人肝癌单抗片段抗体HAb18F(ab')_2的冻干工艺研究

选择适合于HAb18F(ab')2片段抗体的冻干保护剂及其冻干工艺,是确保该生物制品的质量关键之一,对不同种类和不同浓度的保护剂进行了研究,结果表明10%蔗糖对HAb18F(ab')2片段抗体冻干样品的剂型、外观、残水量(<3%)、复溶时间(<10s)、纯度(>95%)及稳定性没有影响。研究优化与保护剂相结合的适用于HAb18F(ab')2片段抗体的最佳冻干工艺,为抗体片段扩大生产提供依据。     

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.     

Kinetics of whole serum and prepurified IgG digestion by pepsin for F(ab')2 manufacture

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.     

The role of A-cells in affecting the immunostimulating effect of F(ab')2-fragments

The immunoregulatory effect of F(ab')2 fragments on normal rabbit IgG and that preincubated with A-cells from spleen have been compared. Both products were tested for their ability to enhance primary immune response of rabbit spleen cells to SRBC. It was demonstrated that low molecular mass product appeared after F(ab')2 fragments incubation with A-cells at 37 degrees C and possessed immunostimulating activity similar to that of initial F(ab')2 fragments. In addition, it was shown that F(ab')2 reduction to monovalent Fab' fragment with the following alkylation of SH-group abolished the ability of Fab' fragment to enhance the immune response. It may signify that half cystein Fab' fragment residue is essential for processing of the fragment in A-cells and (or) for immune response enhancement.