Immunochemical analysis of IgG subclasses and IgM in South American camelids

Simone, Emilio De; Saccodossi, Natalia; Ferrari, Alejandro; Leoni, L.; Leoni, Juliana

doi:10.1016/j.smallrumres.2005.03.009

Cited by 16 publications

(18 citation statements)

References 22 publications

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“…IgG 1 titers had much higher values than those of IgG 3 in all animals, and its progress pattern resembles the behavior of total Igs. This difference in the relative contribution of each subisotype is reinforced by the fact that normal serum IgG 3 concentration, although being the most abundant HCAb isotype, has been previously reported to be 5 to 10 times lower than that of IgG 1 (De Simone et al, 2008). Activity modulation assays with purified Igs IgG 1 from both L1 APM and L2 APM produced a positive modulation of APM activity ( Figure 7); on the contrary, IgG 3 had no effect on the enzyme.…”

Section: Antigen Purificationmentioning

confidence: 78%

“…Purification of llama Igs IgG 1 and IgG 3 were purified from the selected serum samples as previously described (De Simone et al, 2005;Ferrari et al, 2007), using a HiTrap-protein G column (GE Healthcare, Piscataway, NJ, USA) and low pH elution buffers (pH 3.5 for IgG 3 and pH 2.7 for IgG 1 ). All Ab fractions were neutralized with Tris 1 M, dialyzed against PBS and protein purity was assessed by 10% SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…These Abs were first reported by Hamers-Casterman et al (1993), and they have been extensively described for all the members of the Camelidae family (dromedaries, camels, llamas, guanacos, vicunas and alpacas; Muyldermans et al, 2001;Nguyen et al, 2002;Su et al, 2002). In these studies, it was reported that conventional Abs (IgG 1 and IgM) constitute about 60% to 80% of total mass of serum Igs, and HCAbs (IgG 2 and IgG 3 ) the remaining 20% to 40% (De Simone et al, 2008).…”

Section: Introductionmentioning

confidence: 95%

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Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Ferrari

Weill

Paz

et al. 2012

Animal

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Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a b-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG 1 ), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy -neither as a biotechnological strategy nor in the biological context of an immune response against infection -as raising inhibitory rVHHs.

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Section: Antigen Purificationmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

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Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Ferrari

Weill

Paz

et al. 2012

Animal

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“…As indicated in Fig. 1, corresponding to heavy chains of IgG (H) with a molecular weight (MW) of 50 kDa and light chains of IgG (L) with MW of 25 kDa [20], two bands of 50 and 25 kDa, appeared in the sample from the peak 1. An exceeded 95% (w/w) purity of IgG determined by a gel-pro analyzer, indicating that substantial proportion of rabbit IgG existed in the nonretained (flowthrough and washing) effluents.…”

Section: Ft-iec Developmentmentioning

confidence: 86%

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

et al. 2011

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A one-step purification process using flowthrough mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min -1 . Ft-IEC using TrisHCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris-HCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.

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“…IgG1 occurs in the classical structure form of antibodies, while IgG2 and IgG3 represent heavy-chain antibodies [4], and they form about 75% of the IgG in camel serum [5]. Heavy-chain antibodies in camels are significantly effective in antigen recognition; in spite of losing their light chains [6][7][8], also, they have lower molecular weight than conventional antibodies. In addition, these antibodies are less immunogenic than other mammalian antibodies [9], that means when they are injected into experimental models, it will be less likely to induce adverse reactions during the course of treatment [10].…”

mentioning

confidence: 99%

Simple Protocol for immunoglobulin G Purification from Camel “Camelus dromedarius” Serum

et al. 2017

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Open Life Sci. 2017; 12: 143-155 was at 450 rpm. Overall, the results suggest that caprylic acid purification of camel serum IgG is more effective and safe than ammonium sulfate method in simplicity, purity, and lower non-IgG proteins in the final preparation with lower protein aggregates.Keywords: Camelus dromedarius; camel IgG purification; caprylic acid; temperature; stirring IntroductionCamels produce several immunoglobulin classes in their serum, colostrum and milk, including IgM, IgA and IgG (IgG1, IgG2 and IgG3) [1][2][3]. Camels are special animals in antibody production as they possess a unique type of antibody in their serum which lack light chains as well as the heavy chain constant domain "CH1", so-called heavychain antibodies [4]. IgG1 occurs in the classical structure form of antibodies, while IgG2 and IgG3 represent heavy-chain antibodies [4], and they form about 75% of the IgG in camel serum [5]. Heavy-chain antibodies in camels are significantly effective in antigen recognition; in spite of losing their light chains [6][7][8], also, they have lower molecular weight than conventional antibodies. In addition, these antibodies are less immunogenic than other mammalian antibodies [9], that means when they are injected into experimental models, it will be less likely to induce adverse reactions during the course of treatment [10]. The above pharmaceutical features recommend the camel antibodies as a substitute for other animals' therapeutic antibodies [11,12], or alternatively, it can be converted to a complete humanized antibody [13].Antibody purification from different biological fluids such as serum, ascites and milk is an important step in the production of antibodies used for diagnostic, therapeutic, and research applications [14]. There are different methods for purification and the first reported method for purifying camel IgG from serum, by , used protein A-Sepharose and protein G-Sepharose. More Abstract: The present study aimed to describe and standardize a simple and efficient protocol for purification of camel IgG from serum, which can be applied for Camilidae antibody production in research laboratories, the preindustrial stage. Camel serum IgG was separated with caprylic acid and ammonium sulfate, then the effect of four variables studied: caprylic acid concentration, pH, stirring time, and stirring intensity. Camel IgG prepared by standardized caprylic acid fractionation method for camel serum was compared with commercial anti-sera products. Camel IgG purification from undiluted sera using caprylic acid at concentration of 8% v/v gave the best results. Purification at different pH values using caprylic acid at 8% v/v revealed that pH 5.5 was optimal. Investigating purification at different stirring time intervals using 8% v/v caprylic acid at pH 5.5 demonstrated that stirring for 90 min gave the optimum results. Finally, studying purification at different stirring intensities using 8% v/v caprylic acid at pH 5.5 for 90 min, the best stirring intensity

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Immunochemical analysis of IgG subclasses and IgM in South American camelids

Cited by 16 publications

References 22 publications

Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

Simple Protocol for immunoglobulin G Purification from Camel “Camelus dromedarius” Serum

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