Monoclonal antibodies to the bovine immunoglobulin G2a allotypes A1 and A2.

Production of murine monoclonal antibodies (Mabs) to bovine IgG2a-A1 and A2 allotypes resulted in three Mabs being selected as anti-IgG2a-A1 and A2 reagents. Two Mabs recognize the A1 allotype and 1 recognizes the A2 allotype. Initial epitope mapping with the Mabs indicates that one of the A1 epitopes resides in the hinge region and the other epitope resides more toward the C-terminus of the immunoglobulin. The A2 epitope recognized by the A2 Mab does not appear to reside in the hinge region of the immunoglobulin but apparently resides more C-terminal.     

Altered immune response in mice deficient for the G protein-coupled receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.     

Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry

Bacterial Protein A (PrtA) and Protein G (PrtG) are widely used for affinity purification of antibodies. An understanding of how PrtA and PrtG bind to different isotypes of immunoglobulin type G (IgG) and to their corresponding Fc fragments is essential for the development of PrtA and PrtG mimetic ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration calorimetry. The results indicate that both protein ligands bind IgG and Fc fragments strongly with Ka values in the range of 10(7) -10(8) M(-1) and for both ligands, the interaction with both IgG isotypes is enthalpically driven though entropically unfavorable. Moreover, variation in the standard entropic and standard enthalpic contribution to binding between the two isotypes as well as between IgG and Fc fragment implies that the specific interaction with PrtA varies according to IgG isotype. In contrast to PrtA, PrtG bound to F(ab')(2) fragment with a Ka value of 5.1 × 10(5) M(-1) ; thus underscoring the usefulness of PrtA as a preferred ligand for generic antibody purification processes.     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

Validation of Brix refractometer to estimate colostrum immunoglobulin G content and composition in the sow

Colostrum is an essential source of immunoglobulin G (IgG) for neonate piglets. However, colostrum IgG content and nutritional composition can vary considerably among sows due to age, parity, feeding regime and immunological background. Currently, there is no practical way to obtain information about colostrum IgG concentration at herd level. We evaluated sows’ colostrum IgG content on-farm using a Brix refractometer and its performance was compared with that of an IgG ELISA. In addition, nutritional compositions of the colostrum samples were analyzed using Fourier transform IR spectroscopy. Colostrum samples (5 to 6 ml) (n=153) were obtained within 0 to 3 h of farrowing. However, to obtain a 24 h IgG profile for 11 sows, colostrum samples were collected at 0, 2, 4, 6, 8, 10, 16 and 24 h after farrowing. A 0.3 ml of freshly drawn colostrum sample was used for the on-farm measurement of Brix percentages using a digital refractometer shortly after collection. The remaining fractions of the samples were frozen and submitted to laboratory analysis for total IgG, using a commercially available pig IgG ELISA kit. For nutritional composition analysis, a 35 ml colostrum sample (n=34) was obtained immediately after birth of first piglet from the first three pairs of frontal teats. Colostrum concentrations of IgG averaged 52.03±30.70 mg/ml (mean±SEM) at 0 to 3 h after farrowing. Concentration of IgG decreased on average by 50% during the 1st day of lactation (P<0.01). Sow parity did not influence colostrum concentrations of IgG. Differences in colostrum composition were recorded between two herds and among the parity groups (P<0.05). The Brix refractometer measurement of colostrum and the corresponding log transformed IgG measurements from the ELISA were moderately correlated (r=0.63, P<0.001, n=153). Based on the classification we suggest here, low levels of IgG (14.5±1.8 mg/ml) were recorded for colostrum samples with Brix readings below 20%. Borderline colostrum IgG content (43.8±2.3 mg/ml) had Brix readings of 20% to 24%, adequate colostrum IgG content (50.7±2.1 mg/ml) had Brix % readings of 25% to 29% and very good IgG colostrum content (78.6±8.4 mg/ml) had Brix readings >30%. Colostrum IgG concentration is highly variable among sows, Brix measurement of a sows’ fresh colostrum is an inexpensive, rapid and satisfactorily accurate method of estimating IgG concentration, providing indication of differentiation between good and poor IgG content of colostrum.     

Binding site on human immunoglobulin G for the affinity ligand HWRGWV

Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383–Asn389 (SNGQPEN) located in the C_H3 domain. Subsequent modeling has suggested a possible three‐dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. Copyright © 2009 John Wiley & Sons, Ltd.     

Selective determination of human immunoglobulin G by flow‐injection chemiluminescence

A novel flow‐injection chemiluminescence method was developed for the selective determination of human immunoglobulin G (IgG) in the presence of thiomersal by changing the flow rates of peristaltic pump. The study was based on the independence and additivity of the CL signals of human IgG and thiomersal in the galangin–potassium permanganate–polyphosphoric acid system. In meantime, two equations relating to the concentrations of mixing solutions of human IgG and thiomersal vs the CL intensity were established and solved, on the basis of which the content of thiomersal included in samples was simultaneously determined too. The enhanced CL intensity was in proportion to concerntrations in the range 8.0 × 10^?7 to 8.0 × 10^?5 g/mL for human IgG and 1.0 × 10^?7 to 2.0 × 10^?6 g/mL for thiomersal with the detection limits of 5.0 × 10^?7 g/mL for human IgG and 6.0 × 10^?8 g/mL for thiomersal, respectively. The relative standard deviation for 1.0 × 10^?5 g/mL human IgG was 0.8% and for 2.0 × 10^?7 g/mL thiomersal it was 2.0% (n = 10). The proposed method was applied to determine three synthetic samples with recoveries of 91.5–109.5%. In addition, the possible chemiluminescence mechanisms are discussed as well. Copyright © 2010 John Wiley & Sons, Ltd.     

The relationship between airway immunoglobulin activity and eosinophils in COPD

In chronic obstructive pulmonary disease (COPD), the effects of inhaled corticosteroids are predicted by blood eosinophil counts. We previously briefly reported increased immunoglobulin (Ig)A and IgM levels in bronchoalveolar lavage (BAL) of COPD patients with higher (eosinophil^high) compared to lower (eosinophil^low) blood eosinophils (>250/μL versus < 150/μL), suggesting differences in adaptive immune function. An inverse relationship exists between eosinophil counts and airway pathogenic bacteria levels. The mechanistic reasons for these associations between eosinophils, corticosteroids and pathogenic bacteria are unclear. IgA, IgM and IgG levels were assessed in BAL, bronchial biopsies and epithelium collected from eosinophil^high (n = 20) and eosinophil^low (n = 21) patients. Bronchial B-cell numbers were measured by immunohistochemistry. B-cell activity was assessed in bronchial samples and following exposure to BAL from eosinophil^high and eosinophil^low patients. BAL levels of non-typeable Haemophilus influenza (NTHi)-specific immunoglobulins were quantified. Results showed airway expression of IgA, IgG1 and IgM were lower in eosinophil^low compared to eosinophil^high patients, with lower levels of NTHi-specific IgA and IgM. Bronchial B-cell numbers were similar in both groups, but B-cell activity was lower in eosinophil^low patients. In conclusion, COPD eosinophil^low patients show differences in adaptive immune function compared to COPD eosinophil^high patients. These differences may cause different microbiomes in these COPD phenotypes.     

Echinostoma caproni: kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice

The kinetics of specific immunoglobulin M, A and IgG subclasses against Echinostoma caproni (Trematoda: Echinostomatidae) were analyzed in serum and intestinal fluid of two host species (Wistar rats and ICR mice) in which the course of the infection markedly differs. In rats, the worms were rapidly expelled, whereas E. caproni evokes in mice long-lasting infection. The pattern of antibody responses in both serum and intestinal samples was different in each host species. Serum responses in mice were characterized by significant increases of IgM, IgA, total IgG, IgG1 and IgG3, but not IgG2a. In contrast, serum responses in rats showed elevated levels of IgM, probably in relation to thymus-independent antigens, and slight increases of total IgG, IgG1 and IgG2a. At the intestinal level, increases of IgM and IgA levels were observed in mice. In regard to IgG subclasses, increases in both IgG1 and IgG2a were detected. Later decreases to normal values in IgG2a were also detected. In rats, only increases in total IgG and IgG2a were found. According to our results the development of long-lasting E. caproni infections in mice appears to be associated with a dominance of Th2 responses at the systemic level and balanced Th1/Th2 responses at the local level, characterized by initial increases in IgG1 and IgG2a levels. In contrast, the worm expulsion appears to be related to increases in local IgG2a levels.     

Molecular modeling and multi‐spectroscopic approaches to study the interaction between antibacterial drug and human immunoglobulin G

Mechanistic and conformational studies on the interaction of sulfamethoxazole (SMX) with human immunoglobulin G (HIgG) were performed by molecular modeling and multi‐spectroscopic methods. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HIgG. The binding parameters and thermodynamic parameters at different temperatures had been calculated according to the Stern?Volmer, Scatchard, Sips and Van ’t Hoff equations, respectively. Experimental results showed that the fluorescence intensity of HIgG was quenched by the gradual addition of SMX. The binding constants of SMX with HIgG decreased with the increase of temperature, which meant that the quenching mechanism was a static quenching. Meanwhile, the results also confirmed that there was one independent class of binding site on HIgG for SMX during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?14.69 kJ·mol^?1 and 22.99 J·mol^?1·K^?1, respectively, which suggested that the electrostatic and hydrophobic interactions were the predominant intermolecular forces in stabilizing the SMX?HIgG complex. Furthermore, experimental results obtained from three‐dimensional fluorescence spectroscopy, UV?vis absorption spectroscopy and circular dichroism (CD) spectroscopy confirmed that the conformational structure of HIgG was altered in the presence of SMX. Copyright © 2015 John Wiley & Sons, Ltd.     

A CpG oligodeoxynucleotide enhances the immune response to rabies vaccination in mice

Background

Rabies is a fatal disease that is preventable when post exposure prophylaxis (PEP) is administered in a timely fashion. CpG oligodeoxynucleotides (ODNs) can trigger cells that express Toll-like receptor 9, and their immunopotentiation activity in an inactivated aluminum-adjuvanted rabies vaccine for dogs has been identified using mouse and dog models.

Methods

A human diploid cell rabies vaccine (HDCV) of humans and a CpG ODNs with cross-immunostimulatory activity in humans and mice were used to evaluate the immunogenicity and protective efficacy of CpG ODN in a mouse model that simulates human PEP.

Results

HDCV combined with CpG ODN (HDCV–CpG) stimulated mice to produce rabies virus-specific neutralizing antibody (RVNA) earlier and increased the seroconversion rate. Compared with HDCV alone, either HDCV–1.25 μg CpG or HDCV–5 μg CpG increased the levels of RVNA. In particular, 5 μg CpG ODN per mouse significantly boosted the levels of RVNA compared with HDCV alone. IFN-γ producing splenocytes generated in the HDCV-5 μg CpG group were significantly increased compared to the group treated with HDCV alone. When the immunization regimen was reduced to three injections or the dose was reduced to half of the recommended HDCV combined with CpG ODN, the RVNA titers were still higher than those induced by HDCV alone. After viral challenge, 50% of mice immunized with a half-dose HDCV–CpG survived, while the survival rate of mice immunized with HDCV alone was 30%.

Conclusions

The immunopotentiation activity of CpG ODNs for a commercially available human rabies vaccine was first evaluated in a mouse model on the basis of the Essen regimen. Our results suggest that the CpG ODN used in this study is a potential adjuvant to rabies vaccines for human use.

     

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G

We report a flow‐injection biosensor system with a capacitive transducer for assay and quality control of human immunoglobulin G (hIgG). The sensing platform is based on self‐assembled monolayers (SAMs) of carboxylic acid terminated alkyl‐thiols with covalently attached concanavalin A. The electrochemical characteristics of the sensor surface were assessed by cyclic voltammetry using a permeable redox couple (potassium ferricyanide). The developed biosensor proved capable of performing a sensitive label‐free assay of hIgG with a detection limit of 1.0 µg mL^?1. The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 µg mL^?1, in a logarithmic plot. Typical measurements were performed in 15 min and up to 18 successive assays were achieved without significant loss of sensitivity using a single electrode. In addition, the biosensor can detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content (5.0 µg mL^?1). Hence, it represents a potential post‐size‐exclusion chromatography–UV (post‐SEC–UV) binding assay for in‐process quality control of hIgG, which cannot be detected by SEC–UV singly at concentrations below 0.3% of the total hIgG content. Biotechnol. Bioeng. 2009; 104: 312–320 © 2009 Wiley Periodicals, Inc.     

Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi‐spectroscopic approach in vitro

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ ? HIgG complex. Fluorescence spectroscopy, ultraviolet–visible (UV–vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?19.12 kJ · mol^?1 and 20.22 J · mol^?1 · K^?1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ ? HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three‐dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G

Potential applications for functional RNAs are rapidly expanding, not only to address functions based on primary nucleotide sequences, but also by RNA aptamer, which can suppress the activity of any target molecule. Aptamers are short DNA or RNA folded molecules that can be selected in vitro on the basis of their high affinity for a target molecule. Here, we demonstrate the ability of RNA aptamers to recognize--and bind to--human IgG with high specificity and affinity. An optimized 23-nucleotide aptamer, Apt8-2, was prepared, and was shown to bind to the Fc domain of human IgG, but not to other IgG's, with high affinity. Apt8-2 was observed to compete with protein A, but not with the Fcgamma receptor, for IgG binding. NMR chemical-shift analyses localized the aptamer-binding sites on the Fc subdomain, which partially overlaps the protein A binding site but not the Fcgamma receptor binding site. The tertiary structures of the predicted recognition sites on the Fc domain differ significantly between human IgG and other species of IgGs; this, in part, accounts for the high specificity of the selected aptamer. Apt8-2 can therefore be used as a protein A alternative for affinity purification of human IgG and therapeutic antibodies. Using Apt8-2 would have several potential advantages, raising the possibility of developing new applications based on aptamer design.     

Study of protein-protein interactions by fluorescence of tryptophan analogs: application to immunoglobulin G binding domain of streptococcal protein G

The protein-protein interaction system often contains many fluorophores that may significantly interfere with the quantitative determination of the binding abilities. To solve this perplexing problem, we biosynthetically incorporated the two tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into the immunoglobulin G (IgG) binding domain of streptococcal protein G (PGBD). The exclusive excitation and novel fluorescence changes in both the intensity and anisotropy are beneficial to reporting the details of the interactions between PGBD and the IgG fragments and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 microM for the binding of human Fc and 8.0 microM for mouse Fc. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the binding abilities and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex.     

The effects of cobalt and iodine supplementation of the pregnant ewe diet on immunoglobulin G, vitamin E, T3 and T4 levels in the progeny

Sixty twin-bearing ewes were allocated to one of four dietary treatments investigating the effects of supplementary iodine or cobalt during late pregnancy on lamb serum immunoglobulin G (IgG), triiodothyronine (T3), thyroxine (T4) and vitamin E concentrations, and lamb IgG absorption efficiency. Ewes were offered grass silage ad libitum supplemented with 800 g per ewe per day of a 190 g/kg crude protein (CP) concentrate from day 126 of gestation until parturition plus one of the following supplements (n = 15 per treatment); no supplement (C); 26.6 mg iodine per day for final 3 weeks pre partum (I-3); 26.6 mg iodine/day for final week pre partum (I-1); 20 mg cobalt/day for final 3 weeks pre partum (Co-3). Lambs were blood sampled at 24 and 72 h post partum for serum IgG and vitamin E concentrations. Ten lambs from C and I-3 were blood sampled at 1 h post partum for serum IgG, vitamin E, T3 and T4 concentrations. There were no differences in serum IgG, vitamin E or T4 values (P > 0.05) at 1 h post partum between lambs born to the C and I-3 ewes. T3 levels were lower in I-3 compared with C progeny (P < 0.05). Supplemental iodine reduced colostral IgG absorption efficiency (P < 0.001) and lamb serum IgG concentrations at 24 and 72 h post partum (P < 0.001). Serum vitamin E concentration in I-3 and I-1 lambs was lower than in Co-3 lambs at 24 h post partum, while at 72 h post partum I-3, I-1 and Co-3 lambs had significantly lower concentrations than C lambs (P < 0.001). Supplementing the ewe's diet with 26.6 mg/day of iodine for the final week of pregnancy reduced lamb serum IgG concentration at 24 and 72 h post partum. The lower total and free T3 values in the progeny of I-3-treated ewes suggest interference in the synthesis and metabolism of thyroid hormones when ewes receive excessive dietary iodine for 3 weeks immediately pre partum. Based on these findings, the indications are that the toxicity level for iodine in the diet of the pregnant ewe should be lowered to 20 mg per ewe per day, equivalent to 40% of its current level. The finding that high-level cobalt supplementation during the final 3 weeks of pregnancy will have a negative effect on serum vitamin E concentration at 72 h post partum is a new and significant finding and previously has not been reported in the literature.     

The fate of immunoglobulin G fed to larvae of Ostrinia nubilalis

Since plants can be transformed genetically to produce functional antibodies, an immunological approach may be developed for controlling their arthropod pests. Specific antibodies would protect plants from arthropods if they could gain access to the pest antigen in sufficient amounts such that the normal function of the antigen is disrupted. In order to study the fate of ingested antibodies in the body of the European corn borer (ECB), Ostrinia nubilalis (Hübner), (Lepidoptera: Pyralidae), we fed the larvae on serum-containing diet. When larvae were fed on the serum-containing diet for various lengths of time between 12 and 96 h, no significant differences were noted in the immunoglobulin G (IgG) concentration in their body. Immediately after the larvae stopped feeding, the concentrations of the IgG in their midgut was about one half that of the diet itself, but it decreased significantly after 6 h and again after 18 h (about 3 and 10 fold, respectively). Immediately after the larvae stopped feeding, the concentration of the IgG in their hemolymph was about 1/500 that in the diet. The concentration of IgG in the hemolymph of ECB larvae was influenced directly by the titer of antibodies in their diet. During the first 6 h after the larvae stop feeding the concentration of IgG in their hemolymph did not decrease significantly; however, it did so after 18 h (about 6 fold). The possibility that specific antibodies will gain access to antigens in the ECB body is discussed.