Precipitation Of IgG Research Articles

Isolation of IgG from chicken serum using a combination of coconut caprylic triglyceride and ammonium sulfate precipitation

This study aimed to isolate IgG from chicken serum using a combination of coconut caprylic triglyceride and ammonium sulfate precipitation techniques. This study used serum from chickens that had previously been vaccinated repeatedly using a commercial rabies vaccine. Initially, the serum was treated with 2.5% coconut caprylic triglyceride, followed by serum IgG precipitation using 40% ammonium sulfate. The 10% SDS-PAGE analysis showed that a protein of about 190 kDa with a purity above 95% was successfully isolated. Whether the isolate protein is IgG and specific to rabies antigen still needs to be investigated further.

Isolation of IgG from chicken serum using a combination of coconut caprylic triglyceride and ammonium sulfate precipitation

This study aimed to isolate IgG from chicken serum using a combination of coconut caprylic triglyceride and ammonium sulfate precipitation techniques. This study used serum from chickens that had previously been vaccinated repeatedly using a commercial rabies vaccine. Initially, the serum was treated with 2.5% coconut caprylic triglyceride, followed by serum IgG precipitation using 40% ammonium sulfate. The 10% SDS-PAGE analysis showed that a protein of about 190 kDa with a purity above 95% was successfully isolated. Whether the isolate protein is IgG and specific to rabies antigen still needs to be investigated further.

Ammonium Sulfate Fractionation of Antibodies.

Before the widespread availability and use of Protein A (rabbit) or Protein G (rodent) for the purification of IgG, the use of an ammonium sulfate "cut" was the standard method to isolate IgG and other serum proteins. The addition of ammonium sulfate reduces the effective solubility of proteins through direct competition for binding sites on the surface of the protein. The resulting precipitated proteins can be isolated by centrifugation. An ammonium sulfate concentration between 40% and 50% results in the precipitation of IgG from most species, and thus 50% is usually used. Because other proteins can be "trapped" within the aggregated protein, the use of ammonium sulfate does not result in a purified antibody fraction and as such should be considered a first step in a multistep antibody purification protocol.

Circadian clock cryptochrome proteins regulate autoimmunity

The circadian system regulates numerous physiological processes including immune responses. Here, we show that mice deficient of the circadian clock genes Cry1 and Cry2 [Cry double knockout (DKO)] develop an autoimmune phenotype including high serum IgG concentrations, serum antinuclear antibodies, and precipitation of IgG, IgM, and complement 3 in glomeruli and massive infiltration of leukocytes into the lungs and kidneys. Flow cytometry of lymphoid organs revealed decreased pre-B cell numbers and a higher percentage of mature recirculating B cells in the bone marrow, as well as increased numbers of B2 B cells in the peritoneal cavity of Cry DKO mice. The B cell receptor (BCR) proximal signaling pathway plays a critical role in autoimmunity regulation. Activation of Cry DKO splenic B cells elicited markedly enhanced tyrosine phosphorylation of cellular proteins compared with cells from control mice, suggesting that overactivation of the BCR-signaling pathway may contribute to the autoimmunity phenotype in the Cry DKO mice. In addition, the expression of C1q, the deficiency of which contributes to the pathogenesis of systemic lupus erythematosus, was significantly down-regulated in Cry DKO B cells. Our results suggest that B cell development, the BCR-signaling pathway, and C1q expression are regulated by circadian clock CRY proteins and that their dysregulation through loss of CRY contributes to autoimmunity.

Separation of immunoglobulins from colostrum using methods based on salting-out techniques

This study aimed to obtain a purified IgG fraction from bovine colostrum using salting-out precipitation methods as base techniques. As the first step after skimming of colostrum, isoelectric precipitation or rennet coagulation were used for casein removal. IgG concentrations in raw material and final products were determined using the ELISA method. SDS-PAGE electrophoresis was used for purity determination of the final fraction of IgG. According to the obtained results, the combination of precipitation, gel filtration, and cross-flow filtration techniques enables to separate the IgG fraction with purity up to 90% and yield up to 91%. Acid whey has a higher yield in comparison with sweet whey and both ammonium sulphate and sodium sulphate have similar effectivity for precipitation of IgG. The final concentrate of IgG can be used as a standalone product or as an additive to other food supplements.

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies

ObjectivesAutoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic...

Circadian Clock Protein CRY Controls B-Cell Intrinsic Tolerance

The circadian system regulates numerous physiological processes including adaptive immune system. Here we show that mice deficient for the circadian genes Cry1 and Cry2, (Cry double knockout [DKO]) display an autoimmune phenotype including higher serum IgG concentration than wild type (WT) mice, presence of serum anti-nuclear antibodies, precipitation of IgG, IgM and complement 3 (C3) in glomeruli, and massive infiltrations of leukocytes into the lung and kidney. A large panel of autoantigens demonstrated that the sera of the Cry DKO mice but not the WT mice, had autoantibodies covering most of the specificities reported to be present in patients with SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome and other autoimmune disorders. Taken together, lost of the CRY circadian protein leds to severe autoimmunity. Furthermore, flow cytometry analysis of lymphoid organs showed lower pre-B cell numbers and higher mature recirculating B cells in the bone marrow as well as increased number of B2 B cells in the peritoneal cavity of Cry DKO mice.The BCR-proximal signaling pathway plays a critical role in peripheral B cell tolerance and activation. Activation of splenic B cells from the Cry DKO mice elicited markedly enhanced and prolonged tyrosine phosphorylation of cellular proteins compared to WT mice, suggesting that a very active BCR signaling pathway may contribute to impaired B cell tolerance in the Cry DKO mice. In summary, our results suggest that B cell development, as well as the intrinsic checkpoints of immune tolerance, are under direct circadian control. DisclosuresNo relevant conflicts of interest to declare.

Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor.

We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup untill attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody.

False positive troponin result caused by a true macrotroponin

A 35-year-old female presented with complaints of tiredness, shortness of breath and recent onset of chest pain. The cardiac troponin I (cTnI) concentration was elevated at 6.4 μg/L (Abbott Architect reference value: <0.03 μg/L) with a normal ECG. Physical examination, radiological imaging and routine laboratory investigations did not provide an explanation for the elevated cTnI concentration. Use of different troponin assays, dilutions with assay diluent, addition of mouse serum, polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC) were used to investigate possible interference. Troponin concentrations were below the level of detection when measured using all other troponin immunoassays. Serial dilutions of sample and addition of mouse serum did not alter the results. However, PEG precipitation and GFC showed the presence of a high molecular weight immunoreactive protein. Using GFC and protein-A IgG precipitation, the interference could be identified as a macrocomplex containing IgG and (fragments of) cTnI. We report a false positive cTnI result caused by a true macrotroponin, containing IgG and (fragments of) cTnI. This macrotroponin was only immunoreactive in the Abbott Architect cTnI immunoassay. Clinicians should be aware of analytical interference when troponin results are constantly elevated in the absence of (non)coronary causes of an increased troponin.

High‐throughput screening techniques for rapid PEG‐based precipitation of IgG4 mAb from clarified cell culture supernatant

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high-throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG(4) monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96-microwell filter plates combined with high-throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG(4) precipitation. To aid the DoE, a set of preliminary range-finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG(4) as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG(4) concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high-throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations.

Porcine ulcerative dermatitis syndrome in sows: a form of vesicular cutaneous lupus erythematosus?

Severe ulcerative lesions were observed in the skin of two sows in a herd of 540 hybrid sows. Annular to polycyclic, severe crusting dermal ulcerations were found on the abdomen...

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

Orchitis: A Complication of Severe Acute Respiratory Syndrome (SARS)1

Severe acute respiratory syndrome (SARS) coronavirus has been known to damage multiple organs; however, little is known about its impact on the reproductive system. In the present study, we analyzed the pathological changes of testes from six patients who died of SARS. Results suggested that SARS caused orchitis. All SARS testes displayed widespread germ cell destruction, few or no spermatozoon in the seminiferous tubule, thickened basement membrane, and leukocyte infiltration. The numbers of CD3+ T lymphocytes and CD68+ macrophages increased significantly in the interstitial tissue compared with the control group (P < 0.05). SARS viral genomic sequences were not detected in the testes by in situ hybridization. Immunohistochemistry demonstrated abundant IgG precipitation in the seminiferous epithelium of SARS testes, indicating possible immune response as the cause for the damage. Our findings indicated that orchitis is a complication of SARS. It further suggests that the reproductive functions should be followed and evaluated in recovered male SARS patients.

IgM rheumatoid factor removal and performance of the FTA-ABS (IgM) test in congenital syphilis.

To determine the performance of the FTA-ABS (IgM) test in congenital syphilis after eliminating interference by IgM rheumatoid factor (RF) and preventing competitive inhibition by IgG. The FTA-ABS (IgM) test was carried out before and after RF removal (achieved by immunoprecipitation of the IgG) in infants with congenital syphilis and controls. Newborns delivered in the Peninsula Maternal and Neonatal Services in Cape Town and infants presenting at Red Cross War Memorial Children's Hospital. Infants with congenital syphilis aged 0-4 months were divided into those with clinical signs at presentation and those who were asymptomatic at delivery. In addition, patients without congenital syphilis but with similar clinical signs at presentation were investigated as were control infants. The diagnosis of congenital syphilis was based on the criteria suggested by Kaufman et al (1977). Amongst symptomatic infants with congenital syphilis the FTA-ABS (IgM) test was positive in 34 (92%) of 37 cases prior to abolishing the RF effect and in 29 (78.4%) of 37 cases afterwards (p = 0.19). In 12 cases of congenital syphilis who were asymptomatic at birth, 10 had positive FTA-ABS (IgM) tests before RF removal and only three had positive tests afterwards (p = 0.006). False positive tests were not found amongst 15 symptomatic infants whose clinical features mimicked those of the infants with congenital syphilis. Among 51 healthy infants the test had a false-positive rate of 2% in newborns and 13% in older infants. The false positive reactions were eradicated by IgG precipitation. Following IgG and RF removal there was an improvement in the specificity of the FTA-ABS (IgM) test but this was at the expense of a loss of sensitivity, particularly in asymptomatic newborns. For newborns, if the FTA-ABS (IgM) test was positive, the patient was likely to require treatment for congenital syphilis, regardless of whether the result was due to the presence of RF or specific IgM.

Immunoglobulins from the sera of immunologically activated persons with pairs of electrophoretic restricted bands show a greater tendency to aggregate than normal.

From in vitro data, it has been speculated that pairs of endogenous restricted bands migrating in close proximity in the gamma region upon high resolution serum electrophoresis (HRE) represent circulating immune complexes (CIC). Using a polyethylene glycol (PEG) method to separate CIC, we found a very high correlation between the presence of such band pairs and elevated levels of CIC (CHI2 = 25.7, p less than 0.001) in 51 sera. HRE appears to be a good screening technique to identify, with a high degree of certainty, samples with elevated levels of CIC for delineation by more specific methods. Yet, examination by gel-filtration chromatography and precipitation with PEG indicated that the molecules comprising the band patterns were not CIC, but polyclonal 7S IgG. These bands are usually found in patients with chronic activation of the immune system. Fractionation of the sera from 5 such patients with various cuts of PEG indicated that the average IgG concentration in the 2.5-5%, and 5-7.5% cuts from patients was 3.99 g/l and 2.2 g/l, while from healthy subjects the concentrations were 0.68 g/l and 2.88 g/l. This reversed precipitation pattern was seen both for absolute levels of IgG and for percent of total IgG. On the average the amount of precipitation of IgG in the 2.5-5% fraction of patients was about 5-fold above that seen in the healthy subjects. The endogenous bands were not associated with any specific cut of PEG, but appeared to be proportionally distributed in accord with the levels of IgG. The data is consistent with the idea that immunologically activated patients exhibit a greater tendency for immunoglobulins to associate than normal. This propensity to aggregate may cause CIC to form in situ in local compartments even though CIC do not appear to be present upon analysis by biochemical techniques.

Surface thermodynamic properties and chromatographic and salting-out behavior of IgA and other serum proteins

By means of contact angle determinations with two liquids, on hydrated as well as on dried protein layers, the long-range and the short-range contributions to the protein surface tensions, and from these the protein (ΔG131) and the protein-ligand (ΔG132) free energies of interaction in aqueous media, were determined. For human serum albumin (HSA), human IgG, and human IgA, the differences between ΔG 131 HYDRATED and ΔG 131 DRY were connected with the behavior of these proteins in low concentrations of (NH4)2SO4 versus saturated (NH4)2SO4 solutions. By interpolation, intermediate points are found that correlate well with the known salting-out properties of these three proteins. On the basis of the data, it is predicted that the precipitation of IgG by 1/3 saturated (NH4)2SO4 is preventable, or reversible, by the admixture of 15% dimethylsulfoxide; both predictions are confirmed experimentally. From the ΔG132 values found, it is shown that HSA and IgG should attach to phenyl ligands under physiological conditions, but that IgA is so hydrophilic that it only can adhere to phenyl ligands after partial dehydration brought about when admixed with 1 M (NH4)2SO4. Closer analysis of the values obtained for the long-range and short-range components of the surface tensions of HSA, IgG, and IgA allow deeper insight into their functional, chemical, and physicochemical properties.

Human cytomegalovirus infection: recent developments in diagnosis and epidemiology.

Cytomegalic inclusion disease (CID) is caused by a horizontally or vertically transmitted human herpes virus infection and may persist for life without obvious clinical symptoms. A serious course of horizontal primary and recurrent infections, however, is often observed in immunocompromised persons such as recipients of organ transplants and patients receiving fresh blood transfusions. Vertical infection may cause fetopathies. The human cytomegalovirus (HCMV) is thought to inherit an oncogenic potential as lately discussed for AIDS and M. Kaposi. Laboratory diagnosis of HCMV infection is performed by light microscopy (inclusion bodies), electron microscopy, virus isolation in cell culture, demonstration of viral DNA and antigen in clinical specimens, by histochemical methods (e.g. immunoperoxidase technique) and by DNA and peptide analysis for identification of different isolates and viral finger prints. Evaluation of cell-mediated immunity in HCMV infection is performed quantitatively (assessment of Thelper/Tsuppressor ratios) or qualitatively (specific lymphocyte stimulation by the antigen). In most cases laboratory diagnosis is achieved by serological methods, i.e. demonstration and quantitation of HCMV-specific antibodies. In this context, a number of liquid- and solid-phase immunoassays have been developed, of which immunofluorescence and ELISA are most commonly used, besides complement fixation and passive haemagglutination. These procedures on the one hand allow the use of different antigen preparations as early and late viral proteins, and on the other hand permit a specific determination of different Ig classes and subclasses. A variety of assays has been established especially for determination of virus-specific IgM antibodies, which are predominantly found in active infection. These, however, at least in part may show non-specific results caused by interference of rheumatoid factor or IgG competition. Such problems have now been dealt with and are avoided by IgG precipitation or IgM immunosorption ("mu-capture" technique). These recent methods allow an exact epidemiological identification of risk groups for CMV infection. Results from our laboratory revealed 13% HCMV-IgM positive patients among pregnant women, 16% IgM positive patients among renal transplant recipients, 4% IgM positive cases in patients after cardiosurgery and 1.7% IgM positives among prostitutes. The prevalence of HCMV infection as indicated by specific IgG antibodies was 56%, 90%, 83%, and 90%, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement-dependent platelet injury by staphylococcal protein A.

A new example of complement-mediated platelet injury has been described. Staphylococcal protein A (SPA) causes rabbit platelet injury as manifested by release of platelet 5-hydroxytryptamine (5HT). This reaction is complement-dependent and occurs over a very small range of SPA concentration, larger amounts being inhibitory. Complement fixation by SPA demonstrates the same narrow SPA concentration requirement whereas precipitation of IgG by SPA is roughly proportional to SPA concentration over a wide concentration range. The reaction can be separated into a sensitization step which requires SPA and plasma but not complement, and a release step which does require complement. Complement-mediated platelet damage induced by SPA is a new biologic property of this common component of the cell wall of pathogenic staphylococci which may contribute to the development of inflammatory and thromboembolic reactions complicating intravascular staphylococcal infection.

Immunochemical Studies of the Phytohemagglutinin, Concanavalin A

Abstract Concanavalin A (Con A) has been demonstrated to be antigenic in goats and methods have been described for separating the nonspecific reaction of Con A with polysaccharides from the specific antigen-antibody reaction. Specific precipitating antibody was found to be associated with IgG in the animals studied, and isolated IgG also fixed C′ in the antigen-antibody reaction. The partial inhibition of IgG precipitation of Con A by methyl-α-d-mannopyranoside provides further evidence for the heterogeneity of the immune IgG studied.

Mise en evidence d'une reaction croisee entre les marqueurs allotypiques Aa1 et Aa2 du locus a chez le lapin

It is well known that, in rabbits, the allotypes of the b locus carried by the light chains of the IgG molecules cross-react. Nothing similar has been described for the allotypes of the a locus carried by the heavy chains of the IgG molecules. This paper describes a cross-reaction between the rabbit allotypic markers Aa1 and Aa2 of the a locus. This phenomenon became evident in successive I 125-labelled IgG precipitation experiments with a particular serum.