低pH孵放病毒灭活处理对马破伤风免疫球蛋白F(ab')_2质量的影响

目的考察低pH孵放病毒灭活处理对马破伤风免疫球蛋白F(ab')_2质量的影响。方法取马破伤风免疫球蛋白F(ab')_24份,分别调整pH至3.8、4.1、4.4及6.5,于(25±1)℃条件下放置21 d后取样测定抗体效价、聚合物(二聚体、多聚体)及F(ab')_2含量,评估低pH孵放病毒灭活法对上述质量指标的影响;以1 mL/瓶的规格分装经低pH孵放病毒灭活处理的马破伤风免疫球蛋白F(ab')_2,于(25±1)℃条件下存放6个月,定期取样并进行抗体效价、聚合物(二聚体、多聚体)及F(ab')_2含量检测,评估其稳定性。结果马破伤风免疫球蛋白F(ab')_2经低pH孵放病毒灭活后,其抗体效价、聚合物(二聚体、多聚体)及F(ab')_2含量未发生明显变化;样品于(25±1)℃条件下存放6个月后,抗体效价和F(ab')_2含量均符合《中华人民共和国药典》2015版(三部)的要求。结论低pH孵放病毒灭活法适用于马破伤风免疫球蛋白F(ab')_2病毒的灭活。     

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。     

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.     

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.     

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.

     

Sequential adsorption of F(ab')(2) and BSA on negatively and positively charged polystyrene latexes

The aim of the present work is to study the sequential adsorption of F(ab')(2) and bovine serum albumin (BSA) molecules adsorbed onto positively and negatively charged polystyrene latexes. Cationic and anionic latexes were prepared by emulsifier-free emulsion polymerization. Adsorptions of F(ab')(2) on both latexes at a low ionic strength and different pHs were performed. The cationic latex showed a higher adsorption of F (ab')(2) molecules over a range of pH, which could be due to the formation of multilayers. Sequential adsorption of anti-CRP F(ab')(2) and monomeric BSA were performed at two different pre-adsorbed F(ab')(2) amounts on both types of latex. Displacement of F(ab')(2) occurred only when the preadsorbed amounts were larger than a certain critical value, which depends on the adsorption pH. A greater displacement of larger preadsorbed amounts might be the result of a weaker contact between the protein molecules and the polystyrene surface. The displacement of F(ab')(2) previously adsorbed onto both latexes occurred due to pH changes, an increase of ionic strength and the presence of BSA molecules. The effect caused by these three factors was studied independently. The main factors in the desorption of F(ab')(2) on the anionic latex are the changes in pH and ionic strength, whereas on the cationic latex the desorption is mainly caused by the increase of the ionic strength and the presence of BSA. The colloidal stability of the immunotatex was improved by BSA adsorption, especially on cationic latex. (c) 1995 John Wiley & Sons, Inc.     

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.     

Adsorption of 125I-labeled immunoglobulin G, its F(ab')2 and Fc fragments onto plasma-polymerized films

Plasma-polymerized films were formed on flat glass plates using allylamine, acrylic acid, acrolein, and allylcyanide as monomers. Adsorption of (125)I-labeled-proteins such as immunoglobulin G (IgG), its F(ab')(2) and Fc fragments, and human serum albumin (HSA) was measured on these plasma-polymerized (PP) films covering the glass plates and on commercially available polymer plates. The adsorption isotherm followed the Langmuir equation, from which the binding constant and amount of saturation binding were estimated. We found that, in general, a cationic surface had higher affinity for protein adsorption than an anionic surface. Among the surfaces examined, the PP-allylamine surface showed the highest binding capacity (264.2 nmol/m(2)) for F(ab')(2) fragment: it was remarkably high. Of the surfaces examined, the PP-acrylic acid surface showed the lowest binding capacity (12.8 nmol/m(2)) for F(ab')(2) fragment. The PP-acrylic acid surface also indicated the lowest protein binding capacity for IgG (16.5 nmol/m(2)), Fc-IgG (32.4 nmol/m(2)) and HSA (16.7 nmol/m(2)), respectively. These imply that the PP-acrylic acid film is useful to fabricate as a low protein adsorption material which expected to decrease cell adhesion. Results of our investigation indicate that the plasma-polymerization technique is promising for fabrication of a smart NanoBio-interface which can control the protein adsorption on a solid-phase substrate using a suitable monomer such as allylamine for the large adsorption and acrylic acid for the small adsorption.     

Distribution, kinetics and immunoscintigraphy of 131-I labelled intact antifibrinogen-fibrin antibody (AbFbg) and its F(ab')2 fragment

131-I-labelled anti fibrin-fibrinogen antibody (AbFbg) was compared with its F(ab')2 fragment in distribution studies and by immunoscintigraphy with a view to tumour visualization in tumour bearing rats. The distribution studies indicated that the intact antibody is more concentrated in tumour tissue than the F(ab')2 fragment. By 168h after injection, when tumour-to-tissue ratios were highest in the majority of tissues, the tumour concentration of intact antibody was 3 to 4 times that of the F(ab')2 fragment. The intact antibody is more suitable than the F(ab')2 fragment for tumour imaging especially in the abdominal region where the highest tumour-to tissue ratios were obtained with intact antibody in liver, spleen, intestines and kidneys.     

Ligation of human CD46 with purified complement C3b or F(ab')(2) of monoclonal antibodies enhances isoform-specific interferon gamma-dependent nitric oxide production in macrophages

CD46, a complement regulatory protein widely expressed on human cells, serves as an entry receptor for measles virus (MV). We have previously shown that the expression of human CD46 in mouse macrophages restricts MV replication in these cells and enhances the production of nitric oxide (NO) in the presence of gamma interferon (IFN-gamma). In this study, we show that crosslinking human CD46 expressed on the mouse macrophage-like cell line RAW264.7 with purified C3b multimer but not monomer enhances NO production. The enhanced production of NO in response to IFN-gamma was observed again with C3b multimer but not monomer. The augmentation of NO production is human CD46-dependent with a CYT1>CYT2 profile. Thus, the reported MV-mediated NO production, irrespective of whether it is IFN-gamma-dependent or -independent, should be largely attributable to CD46 signaling but not to MV replication. Similar CYT1-dependent augmentation of NO production was reproducible with two CD46 ligating reagents, CD46-specific monoclonal antibodies (mAb) or their F(ab')(2) and MV hemagglutinin (H) and fusion (F) glycoproteins. Co-cultivation of mouse macrophages bearing human CD46 with Chinese hamster ovary (CHO) cells expressing MV H and F enhanced IFN-gamma-induced NO production. Yet, the NO levels induced by F(ab')(2) against CD46 or MV H/F on CHO cells were much lower than those induced by CD46-crosslinking mAb with Fc or MV infection. Removing the cytoplasmic tails of CD46 abrogated the augmentation of NO production triggered by all three stimulators. Thus, the CD46 CYT1 and CYT2 isoforms functionally diverge to elicit innate immune responses, which can be modulated by purified C3b multimer or anti-CD46 mAbs.     

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.     

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.     

Gelatin binding to the 6F1(1)F2(2)F2 fragment of fibronectin is independent of module-module interactions

Fibronectin, a large modular protein, interacts with many other proteins in the extracellular matrix and on the cell surface. It has previously been shown that interactions between noncontiguous modules exist in the collagen binding region. It is shown here that the interaction between the sixth type I module ((6)F1) and the second type II module ((2)F2) can be disrupted by mutation of a residue in the intermodule interface of the (6)F1(1)F2(2)F2 fragment. The perturbation of the interface and the binding of collagen-derived peptides to individual modules were assessed by high-resolution nuclear magnetic resonance (NMR) spectroscopy. Cooperativity between the modules in binding ligand was assessed by analytical gelatin affinity chromatography of the mutant and wild-type proteins. Differential scanning calorimetry (DSC) was used to probe the influence of the interface on module stability. It is shown that while the (6)F1-(2)F2 interface confers significant thermal stability to the (2)F2 module, it has little effect on gelatin binding activity of the (6)F1(1)F2(2)F2 fragment.     

Coupling of antibody-binding fragments to solid-phase supports: site-directed binding of F(ab')2 fragments.

A method to covalently bind antibody fragments, via their carboxyl termini to solid supports, is presented. The strategy involves: (1) reversibly blocking all the accessible carboxyl groups on the antibody molecule with phenylhydrazine, (2) exposing the carboxyl termini of the fragment by enzymatic digestion with pepsin and (3) subsequently coupling the fragment to an appropriate support. Experiments with an anti-bovine serum albumin monoclonal antibody and C-14 phenylhydrazine revealed that the blocking step was nearly completely reversible with a dilute solution of FeCl3. Radioiodinated blocked F(ab')2 fragments were then coupled to an amino-functionalized Sepharose 4B column, and characterized as to their coupling capacity (mass of protein coupled/ml of bead), and antigen-binding activity. The coupling capacity of the blocked fragments was found to be 12%, half the coupling efficiency of unmodified radioiodinated F(ab')2. The antigen-binding capacity (mol antigen bound per mol antibody coupled) for the blocked F(ab')2, on the other hand, was found to be 1.9, which was approx. 3.5-times greater than for the unmodified F(ab')2. Comparisons with other conventional coupling techniques were also made. These preliminary studies suggest that this technique can provide one with the means to obtain more uniform and active populations of immobilized antibody fragments.     

Differential T cell hyporesponsiveness induced by in vivo administration of intact or F(ab')2 fragments of anti-CD3 monoclonal antibody. F(ab')2 fragments induce a selective T helper dysfunction.

Induction of peripheral T cell anergy associated with stimulation through the TCR complex in vivo has been described in mice using chemically modified APC, staphylococcal enterotoxin B, and intact anti-CD3 mAb. In the latter two models, T cell proliferation, IL-2R expression, and lymphokine production have been demonstrated before subsequent induction of hyporesponsiveness, whereas in the former model, these events have not been observed. To further investigate the relationship between mitogenicity and induction of peripheral hyporesponsiveness, mice were treated with either mitogenic intact anti-CD3 mAb or nonmitogenic F(ab')2 fragments of anti-CD3 mAb. T cells from F(ab')2-treated mice demonstrated a selective decrease in helper functions, with minimal effect on CTL function. Specifically, a marked reduction in ability of Th cells to secrete IL-2 when challenged in vitro with mitogen or alloantigen was observed, which persisted for at least 2 mo after mAb administration and which was independent of T cell depletion. Proliferative function was decreased in CD4+ T cells and could not be fully restored with addition of exogenous IL-2. A helper defect was also evident in vivo, in that F(ab')2-treated mice were deficient in their ability to reject MHC-disparate skin grafts, and in vivo administration of IL-2 reconstituted their ability to reject skin grafts normally. In contrast, T cells from mice treated with intact mAb demonstrated a significant decrease in both CTL and helper functions. A long term reduction in TCR expression on CD4+ cells from F(ab')2-treated mice, and on both CD4+ and CD8+ cells from intact mAb-treated mice was observed. These findings demonstrate that peripheral T cell hyporesponsiveness can be induced in vivo by binding an identical epitope on the TCR complex in the presence or absence of initial proliferation, lymphokine secretion, or IL-2R expression, and that binding to the same epitope can result in varying long term effects on T cell function.