Antibody Production

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Validation of a Gastrointestinal Explant System for Measurement of Mucosal Antibody Production

Validation of a Gastrointestinal Explant System for Measurement of Mucosal Antibody Production

The majority of our samples (80%) had ⬎1 ␮g of total IgA per ml per day produced in culture supernatants. At this level of antibody production coupled with the known limits of de- tection of antibody by our enzyme-linked immunosorbent as- say (3 to 5 ng/ml), the sensitivity of detection of specific anti- body by using pooled culture supernatants from 6 to 7 days of culture should achieve a level of 0.01% of the total antibody content. The lowest level of IgA antibody produced daily in our cultures was 0.4 ␮g/ml (seen in antral cultures), representing only 10% of the total samples tested. Even at this level of production, similarly high levels of sensitivity of detection of specific antibody could be attained by a simple concentration of pooled tissue culture supernatants. Current work is aimed at application of this system to specific antibody measurements. The immunocyte profile of IgA and IgG in gastric antral and duodenal explant cultures revealed, not surprisingly, that IgA cells predominate and, likewise, that IgA is preferentially present in culture supernatants. Interestingly, although the numbers of IgA ASCs in gastric and duodenal biopsies were 4 to 10 times higher and 10 to 20 times higher, respectively, than the number of IgG ASCs, IgA levels in culture supernatants were, overall, only 1 to 2 times higher and 4 times higher than IgG levels detected in gastric and duodenal cultures, respec- tively. The ASC data are in agreement with other published reports (1, 13). In contrast, the levels of IgG detected in antral culture supernatants were higher than one would expect from the numbers of ASCs extracted. The consistency of this finding will have to be verified. If true, it would suggest that IgG antibody production may be upregulated in gastric mucosa.

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B cells mediate chronic allograft rejection independently of antibody production

B cells mediate chronic allograft rejection independently of antibody production

Similarly, untreated WT and AID/μS KO recipients of Bm12 heart allografts developed significant CAV, whereas μMT recipients did not (Figure 1C). Neither circulating donor-reactive antibodies nor IgG deposits within allograft vessels were observed in AID/μS KO recipients (Figure 1D and Supplemental Figure 1B), confirming that CAV in these mice indeed occurred in the absence of anti- bodies. These results demonstrate that B cells are sufficient and antibodies are not necessary for development of CAV and suggest that B cells could contribute to CAV by mechanisms other than antibody production.

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Role of plant expression systems in antibody production for passive immunization

Role of plant expression systems in antibody production for passive immunization

There are several excellent transient expression systems devel- oped that enable large-scale production of recombinant proteins, including antibodies, in about eight days after cloning of the target gene DNA. Some of these include virus-based expression systems, like magnICON® (Gleba et al., 2005), Gemini (Huang et al., 2010) and Geneware (Pogue et al., 2010); and engineered vectors for Agrobacterium-infiltration, like the pEAQ system, in which the T-DNA also bears the p19 viral silencing suppressor (Sainsbury et al., 2010, Sainsbury et al., 2009, Voinnet et al., 2003). The possibility of high-scale antibody production in the limited space of greenhouses and over a short period of time enables multiple rounds of production. This also eases the downstream process- ing and enables a high recovery of purified antibody. Companies and institutions like Medicago (Quebec city, Quebec, Canada), Kentucky BioProcesses (Owensboro, Kentucky, USA), Texas A&M (college station, Texas, USA), Fraunhofer (Newark, USA) and Icon Genetics (Bayer, Halle, Germany) have established infrastructures for large-scale automated systems to grow tobacco plants in the greenhouse, infiltrate/infect, and harvest the protein (Whaley et al., 2011). Once the antibody genes are isolated and cloned, within two weeks the antibody can be administered to the patient (Paul and Ma, 2011, Whaley et al., 2011). A disadvantage of the transient system is that the leaves have to be processed immediately for optimal product recovery. Also, Agrobacterium infiltration entails the presence of a large amount of bacterial cells, introducing the risk of bacterial endotoxin contamination. However, innovative, cost-effective purification systems, like the tobamovirus–protein A fusion, are being developed (Werner et al., 2006). Taken all together, the tobacco leaf-based expression system, both stable as well as transient, seems very promising.

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Antibody production and application for immunoassay development of environmental hormones: a review

Antibody production and application for immunoassay development of environmental hormones: a review

Environmental hormones, also called environmental endocrine-disrupting chemicals (EDCs), are produced by various human activities and environment pollution, and they are composed of industrial chemicals and environmental pol- lutants. They play a harmful role in breaking the balance of normal physiological metabolism, causing serious diseases and disrupting reproductive development in wildlife and human beings by inhibiting or imitating the action of normal gonadal hormones. Strict controls of maximum residue levels to some environmental hormones in foodstuff have been set up by many countries. Traditional detection methods, due to their high accuracy and maturity, are used extensively, such as HPLC, HPLC–MS, and GC–MS. However, these methods are time consuming and require extraor- dinary skills of operators, and sometimes they cannot meet the requirements of field testing. Immunoassay has the advantages of high specificity, sensitivity, simplicity, convenience, and the ability to achieve high throughput, playing an irreplaceable role in the field of rapid detection. This review focuses on the antibody production and the develop- ment of immunoassays to detect environmental hormones in food and environment. Therefore, estrogen, progester- one, and testosterone are selected as examples to introduce a series of procedures for the detection of environmental hormones including antigen synthesis, antibody production, and antibody-based detection methods.

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Norovirus P Particle, a Novel Platform for Vaccine Development and Antibody Production

Norovirus P Particle, a Novel Platform for Vaccine Development and Antibody Production

small epitope to a loop of the P particle enhanced immunoge- nicity of the inserted His tag in mice. Further study on rotavi- rus VP8 (159 amino acids) showed that the P particles can tolerate an insertion of large antigens. VP8 is a trypsin-cleaved product of the surface spike protein VP4, which plays an im- portant role in viral infectivity and neutralization of rotavirus, and several neutralizing epitopes have been mapped on its surface (11, 12, 14, 15, 27), indicating that the VP8 may be an excellent subunit vaccine candidate against rotaviruses. In ad- dition to enhanced immunogenicity in mice, antibodies in- duced by the P particle-VP8 chimeras revealed strong neutral- ization and protection against rotavirus replication/infection in cell culture and in a murine rotavirus challenge model. The resulting antibody also blocked norovirus VLP binding to HBGA receptors, providing an opportunity to develop a dual vaccine against both noroviruses and rotaviruses. Our data proved the principle that the P particle is a multipurpose plat- form for vaccine development and antibody production.

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Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects

Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects

fay-Barbe et al. (13), the administration of 1 booster dose of teta- nus toxoid in 10 to 11-year-old children who had received 3 doses of primary vaccination during early infancy induced the minimal antibody level of 0.1 IU/ml in only 55% of the patients studied, suggesting that the immaturity of the immune response to vacci- nation was applicable within the first months of life along with a failure to generate a long-term immune memory. Dorsey and Or- ange (14) demonstrated anti-tetanus antibodies present in 30% of children with transient hypogammagammaglobulinemia of in- fancy (THI) and considered this impaired immune response to immunization along with increased B cell number as the principal pathomechanisms of THI. In our study, 50% of patients with hy- pogammaglobulinemia did not achieve 0.1 IU/ml, that is, the minimal protective level of anti-tetanus IgG antibodies, and as many as 73% of patients did not achieve 1.0 IU/ml, the antibody level associated with long-term seroprotection. Likewise, a defec- tive response to vaccination with diphtheria toxoid related to a specific antibody level below 0.1 IU/ml was demonstrated in 27% of cases and a level below 1.0 IU/ml in 68% of the children studied. While in THI and in other frequent antibody production defects in children, such as IgG subclass deficiency, IgA deficiency, and specific anti-polysaccharide, an antibody deficiency response to protein and protein-conjugated vaccines is typically preserved, not least in a subset of pediatric patients with hypogammaglobu- linemia, in whom the response to vaccines is impaired, and a lack of evidence for specific diagnosis of PID may indicate a delayed maturation of the immune response with antibodies that can also be consistent with THI (10). In the case of a severe deficiency in congenital antibody production, such as common variable immu- nodeficiency (CVID), an insufficient response to vaccines is a typ- ical finding, displaying however wide intersubject variability of antibody responsiveness. A positive response to polypeptide vac- cines was assessed in 23% of CVID patients, with 18% of them demonstrating a similar response to polysaccharide vaccines (15); therefore, it may be assumed that hyporesponsiveness to vaccines and the lack of postvaccination specific antibodies is not an indis- pensable condition for a diagnosis of CVID.

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Characterization of differential antibody production against hepatitis C virus in different HCV infection status

Characterization of differential antibody production against hepatitis C virus in different HCV infection status

The association of indeterminate RIBA results with older age is consistent with what Makuria et al. [19] has also reported in their study. They reported that RIBA- indeterminate blood donors were older than spontan- eously recovered subjects or chronic HCV carriers. The older age of this group suggests that their HCV exposure might have been in the remote past allowing time for some anti-HCV antibody responses to have waned. Sup- porting this concept is the finding of Seeff et al. [20] who found that complete loss of antibody was shown in a retrospective-prospective study where 7 % of subjects who had anti-HCV in their original stored sample, no longer had antibody when recalled 23 years later. Inter- estingly, Sillanpää and coworkers [6] analysed sera from five HCV RNA and antibody positive patients during a period of 18 to 25 month. The antibody levels against the major immunogenic proteins were found to remain relatively constant. However, in three patients there were some changes in anti-HCV antibody levels, namely a weak decrease in the core and NS specific antibody levels during the follow-up period. Similar analysis by Muerhoff et al. [21] revealed that while in most cases anti-HCV antibodies remain at a constant level, there were some individuals whose antibody levels showed some fluctuation.

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Commensal Fungi are Involved in Antigen-Specific Antibody Production in the Elderly

Commensal Fungi are Involved in Antigen-Specific Antibody Production in the Elderly

prevalence of elevated antigen-specific IgE aging has also been reported [32], with some studies suggesting heterogeneity for each laxis [22]. An experimental model of allergic conjunctivitis demonstrated that continuous topical antigen IgG1/IgG2 production regulation of mast cells, IgE production, mast cell degranulation and histamine release, lymphoid [23]. Allergen- specific IgG may promote expansion of the secondary Th2 response through ligation of Rs on innate immune cells [24], and be involved in the development of airway suggesting that specific IgG could play an important role However, it remains controversial whether IgG contributes to the pathogenesis of or tolerance to allergy. In school children, allergy was associated with IgG antibodies to molds that can be found in gs. However, no association was found between IgG antibodies to molds and exposure to moisture and molds [26].

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Antibody Detection and Kinetics of Antibody Production during Early Stages of Immunization with Hepatitis B Virus Vaccine

Antibody Detection and Kinetics of Antibody Production during Early Stages of Immunization with Hepatitis B Virus Vaccine

After the primary vaccine injection, no reactivity was ob- served in the plasma of the susceptible students (Fig. 2). In contrast, after the second vaccine injection, plasma samples from eight volunteers contained anti-HBs and the plasma of one volunteer was equivocal when it was tested by the antigen sandwich assay (Murex; Abbott). The peak of the antibody reactivity was reached between 7 and 11 days postinjection. Contrary to what was observed with the lysates, in five cases, anti-HBs became detectable at 9 days and the reactivity con- tinued to rise, while in two cases, the antibody level initially detectable at 7 days stabilized at 9 and 11 days postinjection, respectively. The last positive case had clearly detectable anti- HBs on days 7 and 9, but it was no longer detectable on day 11. The ability of the antigen sandwich assay to detect anti-HBs in the lysates and the ability of the antigen sandwich assay to TABLE 1. Anti-HBs markers in B-cell lysates and plasma samples after the primary and first booster injections of HBV recombinant vaccine

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Gut dysbiosis in mice genetically selected for low antibody production

Gut dysbiosis in mice genetically selected for low antibody production

Proper functioning of the immune system (IS) depends on its modulation by body associated microorganisms, in a process starting early after birth, lasting through all life time and which contributes for the shaping of microbial ecosystem itself, ultimately influencing body homeostasis. The importance of a fine-tuned microbiota-immunity bal- ance can be illustrated by multiple human disorders hav- ing as an underlying basis defects in immune response and accompanying gut dysbiosis, such as autoimmune and inflammatory bowel diseases [1]. The use of animal mod- els has been essential for the works unraveling IS func- tions and for understanding of immune-related diseases. Examples of such models include mice lineages bi-direc- tionally selected for high and low quantitative antibody responses to a given antigen, but whose altered response extends to antigens unrelated to those employed in the selective process [2]. In addition to antibody produc- tion, these mice, which are represented by five selections

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Enhancing full-length antibody production by signal peptide engineering

Enhancing full-length antibody production by signal peptide engineering

Using signal peptide engineering, we increased the yields of hu5D5 in the shake flask cultures approximately 2.5–3 fold. Many factors remain to be tested to further optimize full-length mAb accumulation in the periplasm. For example, co-expression of key components of the secretion machinery may further increase antibody chain secretion. It’s been shown that over-expression of SRP (Ffh) increased the yields of full-length IgG [43], presum- ably due to increased secretion efficiency. Also, based on our preliminary results we suspect that heavy chain and light chain may compete for the limited secretion capac- ity. Therefore, the ratio of light chain to heavy chain may need to be finely tuned. Moreover, secreted light chain and heavy chain can form dimeric species or aggregates in the periplasm (Figs. 3, 5). Co-expression of periplasmic chaperones provides a feasible strategy to prevent protein aggregation and increase yields of mAbs [8, 43, 44].

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Lyme Meningitis in Children with Acute Serous Meningitis with no Clinical Signs of Lyme Borreliosis at Presentation

Lyme Meningitis in Children with Acute Serous Meningitis with no Clinical Signs of Lyme Borreliosis at Presentation

Medical history, physical examination, basic hematologic, biochemical and microbiologic investigations, and lumbar puncture with cerebrospinal fluid (CFS) investigation were performed at the time of admission. Abnormal CSF findings were defined as reported previously in our study of patients with multiple EM and central nervous system (CNS) involvement [12]. Briefly, a white blood cell count (WBC) ≥ 5 x 10 6 /l in CSF was considered as CSF pleocytosis, elevated protein level ≥ 0.45 g/l, albumin ≥ 300 mg/l, IgG ≥ 40 mg/l, IgM ≥ 0.7 mg/l, IgA ≥ 5 mg/l and decreased glucose concentrations < 50% of blood glucose. CSF flow rate and the presence of intrathecal antibody production were defined according to the criteria reported by Reiber et al [13]. In each patient venous blood and CSF samples were simultaneously taken for determination of CSF flow rate. In blood and CSF concentrations of albumin and immunoglobulins: G (IgG), A (IgA) and M (IgM) were determined.

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A search for genetic factors influencing immune responses to a killed Mycobacterium avium subspecies paratuberculosis vaccine in Australian fine wool merino sheep : thesis in fulfilment of the degree of Doctor of Philosophy in Animal Science, Institute of

A search for genetic factors influencing immune responses to a killed Mycobacterium avium subspecies paratuberculosis vaccine in Australian fine wool merino sheep : thesis in fulfilment of the degree of Doctor of Philosophy in Animal Science, Institute of Veterinary, Animal and Biomedical Sciences, College of Sciences, Massey University

Immune response trends in animals considered immune or susceptible to experimental infection with Map were investigated in two studies. Of the 14 Map infected lambs in a study (Gwozdz et al. 2000), seven animals tested positive in polymerase chain reaction (PCR) for the presence of acid-fast organisms (AFO) in necropsied ileum and ileo-caecal lymph nodes at 53 weeks post-infection. While the pattern of antibody production was similar in the AFO+ and AFO- sheep, IFN-γ responses in AFO- sheep were higher than those in AFO+ sheep between weeks 9-36 post-infection and differed significantly (P<0.05) at 18 weeks post- infection. In a different study (Begg and Griffin 2005) on experimental infection of Map, sheep considered to be immune to infection exhibited consistently higher lymphocyte transformation (LT) responses during 9-15 months post-infection, compared to diseased sheep. Antibody titres on the other hand remained at base line levels in all sheep except that the diseased sheep showed a small spike at nine months post-infection. The findings of these two studies further underlined the role of CMI responses in combating Map.

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Delayed hyperacute rejection in a patient who developed <em>clostridium difficile</em> infection after ABO-incompatible kidney transplantation

Delayed hyperacute rejection in a patient who developed <em>clostridium difficile</em> infection after ABO-incompatible kidney transplantation

Abstract: Over the past decade ABO incompatible transplantation has emerged as an important potential source for increasing living kidney transplantation in selected transplant centers. Early reports suggest that patients who have elevated serum anti-blood group antibody titers (anti-A/B) before transplantation and a rebound antibody production after antibody removal may be at high immunological risk. With currently available immune modulation protocols and immunosuppressive therapy, excellent short- and long-term patient and graft survival rates have been achieved even in those with high anti-A/B antibody titers before plasmapheresis or immunoadsorption. Nonetheless, acute infection with an organism possessing surface markers analogous to blood group antigens such as carbohydrate structures on the surface of bacterial cell wall occurring before the firm establishment of accommodation can trigger the onset of acute antibody-mediated rejection. We herein report a case of delayed hyperacute rejection in an A1 to O, ABO incompatible transplant recipient following an episode of Clostridium difficile infection.

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Local immune response in experimental pyelonephritis

Local immune response in experimental pyelonephritis

Experiments using an in vitro method of assessing protein synthesis by 14 C amino acid incorporation were designed to determine whether pyelonephritic kidneys were capable of local antibody production. Unilateral pyelonephritis was produced in rabbits by intravenous injection of E. coli 0-75 while one ureter was transiently occluded. The capability of protein and immunoglobulin synthesis by pyelonephritic kidneys, contralateral kidneys, normal kidneys, and spleens from normal and pyelonephritic animals was measured.

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Journal of Applied Pharmaceutical Science

Journal of Applied Pharmaceutical Science

Lohman laying hens are easily raised and have high egg productivity. After immunization with a small dose of antigen, a chicken can continuously produce eggs containing antigen-specific antibodies in their yolks. A chicken usually lays 280 eggs/year and an egg yolk (12–15 ml) usually contains 150–200 mg IgY, of which 2–10% are specific antibodies (Nguyen et al., 2010). Thus, a chicken is referred as a small “factory” for antibody production. In this study,We adopted a PEG extraction method followed by several steps of ammonium sulfate precipitation. The method is simple, efficient, and safe compared with water dilution, chloroform extraction, and gel-filtration chromatography and therefore, it is suited to large-scale isolation of IgY from egg yolk. The PEG used in this process, which is of low toxicity, is widely used in pharmaceutical production. After purifying of IgY from egg yolk, the IgY concentration in egg yolk determined by spectrophotometer (Biorad, USA) and Bradford method. IgY was successfully elicited by immunizing the hens with formalin- inactivated HIV antigen emulsified in Freund’s adjuvant. The IgY concentration in egg yolk increased during the immunization period until week 6 where it began to increase dramatically at 2 weeks and it reached a plateau at 4 weeks after immunization. After week 6 the levels decreased gradually. These results indicated that chickens require about two weeks for antibody production and it also indicates that lohman hens, as the host for the production of anti HIV IgY, show the remarkable ability to rapidly and efficiently generate an abundant IgY and provide specific IgY in a noninvasive way. The injection of the antigen by the intramuscular route results in higher antibody levels by day 28 after immunization, and the resulting antibodies also exhibit higher specificity, being over 10 times more specific when compared with chickens immunized with the same antigen (Pauly et al., 2009; Hirai et al., 2010). Chickens, immunized by the intramuscular via, continue producing specific antibodies during more than 200 days (Sui et al., 2011). Chickens can also tolerate the use of common immunological adjuvants, such as Freund’s adjuvant. AGPT are able to detect the presence or absence of antibodies to any virus. AGPT reactions between dengue antigen and IgY after immunization showed that IgY positive anti HIV if the precipitation lines from the IgY into the well. One line of

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Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft vs host disease

Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft vs host disease

spontaneous immunoglobulin production in vitro, and associated splenomegaly. Antibody production remained inhibited for extended periods of time after termination of anti-gp39 administration. Antiallogeneic CTL responses induced in a GVHD were also prevented by the in vivo administration of anti-gp39 as was the associated splenomegaly. These data suggest that CD40-gp39 interactions are critical in GVHD and that CD40-gp39 may be a valuable ligand-receptor pair for targeting immunotherapeutic agents to control GVHD.

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The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

Many additional applications can be envisaged if an inex- pensive and simple production system is available, yield- ing large amounts of antibody fragments that can be purified easily. The highly specific antigen-binding ability could be used for inactivating bacteria or specific enzymes that can cause spoilage of food. Other suggested applica- tions are the use in biosensors, treatment of wastewater [78], industrial scale separation processes such as separa- tion of chiral molecules [79], purification of specific com- ponents (proteins) from biological materials or the use as abzymes [80,81]. They have also been considered as com- ponents of novel consumer goods with new improved functionalities, in oral care and personal hygiene (e.g. in toothpaste or mouthwashes [82]). For dental applications antibody fragments can be coupled to enzymes to increase the concentration of antimicrobials like hypothiocyanate and hypohalites, for example glucose oxidase (GOX; [83]), galactose oxidase (GaOX; [84]) or lactate oxidase (LOX; [85]). Other examples are targeted bleach in laun- dry washing (e.g. detergents containing antibodies cou- pled to molecules that specifically remove difficult stains) or the use in shampoos where antibodies act to prevent dandruff by inhibiting growth of specific microorganisms causing this [3].

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Production of recombinant antibody fragments in microorganisms.

Production of recombinant antibody fragments in microorganisms.

From an engineering viewpoint the entire process from host organism, vector construction and its expression characteristics, to the final product (in a suitable form for its intended application) must be considered for complete and optimal process design. The starting point for investigations in this study were strains o f Escherichia coli which could express Fv and scFv fragments o f the D1.3 monoclonal antibody and secrete them into the periplasmic space where accumulation o f functional antibody fragments occurred. Subsequent leakage o f these proteins to the extracellular culture medium was however observed during fermentation, the extent o f which was initially unpredictable. Investigations reported in Chapter 3 were concerned with a fed-batch process for production o f scFv fragments. In a 14 L fermenter, controlled exponential feeding to a low specific growth rate resulted in a process which routinely achieved E. coli biomass to cell densities o f 50 g/L dry cell weight in a reproducible manner. Induction o f this fermentation was performed by addition o f IPTG to activate expression o f scFv which was under the control o f the tac promoter. Feeding o f yeast extract solution at a uniform flow rate throughout induction was initiated simultaneously with IPTG addition. Increasing concentration o f the yeast extract feed affected both titre and location o f scFv fragments after 12 h o f induction, resulting in a 40-fold increase in scFv activity to titres o f 200 mg/L (40 mg scFv/g dry biomass) with almost 80% o f this located within the periplasm.

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Production and characterization of a cytotoxic monoclonal antibody reacting with rat islet cells

Production and characterization of a cytotoxic monoclonal antibody reacting with rat islet cells

We have produced a murine monoclonal antibody (F43 A1D2) that binds to the cell surface of both rat islet tumor cells (RINm clone 5F and RINm clone 14B) and normal rat islet cells. This antibody is cytotoxic in the presence of complement for RIN tumor cells as well as A, B, and D pancreatic polypeptide rat islet cells. Antibody A1D2 does not bind to rat thymus cells, pancreatic acinar cells, or fibroblasts. Antigen A1D2 (termed ICSAn-1 [islet cell surface antigen 1]) is a trypsin-sensitive glycoprotein with a molecular mass of

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