Search

An international project team (including members from US, Canada and UK) was formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results of the first comparison with eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology has been reported previously [1]. This report represents the addition of other instruments using a different run buffer. The relative migration times were different, as expected, prohibiting a direct comparison between companies. The within-organisation variability was low for both relative migration time (

An international project team (including members from US, Canada and UK) has been formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results from this exercise demonstrate the robustness of CE-SDS across eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology. Data generated from the analysis of a series of molecular weight markers showed very good precision with regards to relative migration time (RMT) both within and between organisations. The apparent molecular weight of bovine serum albumin (BSA) was measured with good precision to within approximately 2% RSD across the participants. A representative IgG sample showed similar results with regards to relative migration time of its 3 main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain. Fractional peak area for each peak also showed good agreement, with less than 9% RSD for all peaks. This exercise will facilitate both increased regulatory and industrial opinion of CE for biopharmaceutical analysis.

In order to evaluate the serum protein concentration of sheep experimentally poisoned by Ipomoea asarifolia, four groups of five animals received diet with inclusion of 25% (G1), 50% (G2), 75% (G3) and 100% (G4) of the plant replacing to Pennisetum purpureum. Blood samples for serum protein concentration evaluation were collected at seven moments (M): before of the I. asarifolia supply (M1), and five (M2), ten (M3), 15 (M4), 20 (M5), 25 (M6) and 30 (M7) days after I. asarifolia supply. The serum concentration of total protein (TP) was measured through spectrophotometry and the protein fractions by means of eletrophoresis in poliacrylamide (SDS-PAGE). Twenty-nine to 31 proteins were detected by densitometric tracing, with molecular weights varying from 19,160 to 250,000 Daltons (Da). Reduction of the transferrin concentration and increase of acid ?1-glycoprotein level were verified in sheep that ingested 75% and 100% of the plant, immediately before dying. Also, reduction of concentration of IgG heavy chain was evident in M3 and M4, in G3 animals, as well as the level IgG light chain in M2, M3 and M4 of this group, suggesting interference in the immune function in poisoned sheep, especially in those with higher level of the plant on the diet. It is inferred that these proteins can be utilized as auxiliary indicators to diagnose intoxication by I. asarifolia. KEYWORDS: Ipomoea asarifolia; poisonous plants; sheep.

We report a highly unusual case of monoclonal immunoglobulin deposition disease-associated nephrotic syndrome in which a patient developed both lambda light chain deposition disease and 6 years afterward IgG-heavy chain amyloidosis. The patient initially underwent autologous peripheral blood stem cell transplantation as treatment of the underlying plasma cell dyscrasia causing the light chain deposition disease-related nephrotic syndrome. After 6 years of clinical remission, recurrence of the nephrotic syndrome led to a renal biopsy demonstrating IgG-heavy chain amyloidosis. Interestingly, much of the characteristic nodular glomerular sclerosis seen in light chain deposition disease regressed between the time of the first biopsy and the second. Given the length of time between the development of the two diseases and the apparent success of stem cell transplantation in treating the first, we think that the patient produced two distinctly different abnormal plasma cell clones. To our knowledge, this is the first report of two different monoclonal immunoglobulin deposition diseases occurring in the same patient.

A 56K protein co-purified with bovine milk fat globule membrane (MF-GM) proteins bound to Wisteria floribunda agglutinin (WFA) like most MFGM glycoproteins. Treatment with N -glycanase or β- N -acetylhexosaminidase abolished the lectin binding to the protein. Amino acid sequence and immunoblot analyses revealed that the 56K protein is an IgG heavy chain. Lectin column chromatography of the oligosaccharides released by hydrazinolysis from the purified IgG heavy chains revealed that 0.08% of the total N -linked sugar chains bind to a WFA-agarose column, suggesting that they contain the β-N-acetylgalactosaminylated structure.

We typed coded sera from 135 healthy controls, seventy-six patients with autoimmune goitrous and seventy-three with atrophic thyroiditis for IgG heavy chain markers (Gm). All subjects were Caucasian from Newfoundland. An increase in the Gm phenotype ag was found in the 149 patients with thyroiditis compared to controls (chi 1(2) = 5.82, P less than 0.01); significance was, however, not maintained after correction for the number of variables tested. The difference in ag phenotype was more pronounced among the seventy-three patients with atrophic thyroiditis (chi 1(2) = 8.80 corrected P less than 0.05). Because the haplotype ag was not significantly increase in this group, we conclude that homozygotes for Gm ag are at an increased risk of developing atrophic thyroiditis.

A new method for the determination of IgG heavy chain by electroimmuno-diffusion is described: the sensitivity reaches 1 mg/1 using 5 μ1 only ( i.e .: 5 nanograms). Results of simultaneous determinations in serum and CSF are given and discussed: the study of a neurological population (102 patients = among these 61 cases of multiple sclerosis) suggests a general lack of correlation between serum and CSF IgG levels.

The properties of unstable RNA in Bacillus subtilis have been studied under a variety of conditions. It was found that: (1) The unstable fraction constitutes approximately 8% of the total RNA in Bacillus subtilis. The corresponding proportions in two strains of Bacillus megaterium are 6 and 9% of the total RNA. (2) After short pulses of radioactive uracil, most of the labeled RNA is distinguished from 16 and 23 s ribosomal, and from soluble RNA, by its sedimentation properties. (3) Completed 16 and 23 s ribosomal RNA molecules are stable in the presence of actinomycin. If there are ribosomal RNA molecules which are unfinished at the time actinomycin is added, they are degraded. (4) After a 20 to 30-second pulse of radioactive uracil, about 50% of the incorporated label is found to sediment with 70 s ribosomes and polysomes. When actinomycin is added to the medium, the degradation of the unstable RNA is accompanied by the disappearance of polyribosomal structures. (5) If the polysomes are separated by shearing forces, the unstable RNA originally associated with polysomes sediments with 70 s ribosomes. Using this technique, it has been estimated that in polyribosomes there are, on the average, 150,000 daltons or 500 nucleotides of messenger RNA per 70 s ribosomes. (6) After a 30-second pulse of radioactive uracil and subsequent incubation in actinomycin. the radioactivity stable to degradation is found entirely in 30 and 50 s ribosomal subunits and in soluble RNA. The specific activity of the 50 s particle is less than that of the 30 s particle and of soluble RNA. If the length of the pulse of radioactive precursors is increased to three minutes, then all the stable fractions have approximately the same specific activity after incubation in actinomycin. Possible explanations of this result are discussed. It is suggested that the transcription of RNA on the DNA template may be a slow process requiring 1 min to synthesize 1·5 to 2·0 x 106 daltons of RNA.

Streptococcus pyogenes employs an IgG specific endopeptidase, IdeS, to counteract the effector functions of specific IgG. The physiological significant step in disarming specific IgG is the cleavage of one IgG heavy chain. So far, characterizations of IdeS enzymatic activity have employed techniques that failed to differentiate between the first and the second cleavage step. The present data demonstrate that IdeS is active as a monomer and that IdeS activity follows classical Michaelis–Menten kinetics arguing against the previously proposed formation of a functional IdeS dimer. Our results show that IdeS inactivates IgG 100-fold faster than previously reported.

IN guinea pigs1 , 2 and mice, 3 4 5 the ability to develop humoral and cell-mediated immune responses to heterologous insulins is in part controlled by immune-response genes linked to the major his...

IgG heavy-chain (Gm) allotypes were determined in 11 Caucasian patients with the syndrome of demyelinating polyneuropathy associated with monoclonal IgM autoantibodies with affinity for myelin-associated glycoprotein. The frequency of Gm phenotypes in the patient group was significantly different from that seen in a normal Caucasian population.

Eighty-eight patients with insulin-dependent diabetes mellitus (IDDM) and seventy-two unrelated normal controls in Korea were studied for Gm allotypes and HLA-antigens. Ten Gm phenotypes were found among the Korean population. The phenotype frequencies of Gm axg (1, 2, 21), Gm ag (1, 21), Gm agb0b3b5st (1, 21, 11, 13, 10, 15, 16) and Gm agfb0b1b3b4b5 (1, 21, 3, 11, 5, 13, 14, 10) were higher than the other Gm allotypes, but there was no significant difference between patients and controls. HLA-BW54 was found in 15% of patients who had any of those Gm allotypes (Gm axg, Gm ag, Gm agb0b3b5st, Gm ab0b3b5st). However there was no significant difference in frequency as compared with controls.

Pigs with hereditary lymphosarcoma were found to show an increase in serum gamma globulin levels from 0.24 to 2.04 g/dl during a period from the 10 th to the 24 th week of life. Compared to normal litter mates this increase was significant ( P P All cases in the later stages of the disease had an IgG heavy chain (γFc) and light chain (γL) component in their serum and urine. This component could not be found in the serum or urine of normal litter mates, but could be extracted from mesenteric lymph nodes of normal pigs and lymphosarcoma cases. The molecular size of the main component in serum and urine was approximately 50 to 60 × 10 3 . Other tumour cell products similar to foetal albumin, alpha 2 and beta globulin were also present in the serum of lymphosarcoma cases. With the exception of trace amounts of albumin foetal proteins were not present in the serum of normal litter mates.

The frequencies of Gm allotypes a, x, f, b, g and n have been investigated in classical seropositive rheumatoid arthritis (RA), erosive definite seronegative RA, non-erosive definite RA and in healthy individuals with serum IgM-RF. No differences in the frequencies of these Gm allotypes were found between patients and healthy controls. It is concluded that any possible putative genes outside the HLA region involved in RA are most likely unrelated to the genes coding for the constant regions of IgG heavy (gamma) chains.

Binding constants for the human IgG-anti human IgG heavy chain were determined from elution profiles obtained by loading human IgG-Sepharose 4B columns with the antibody and eluting it at different NaI concentrations. The agreement between the value obtained at zero NaI concentration and the value determined in solution by equilibrium molecular sieving was good. An application of this column method should be useful for comparison of avidity values of antibody populations of the same specificity.

Publisher Summary This chapter describes using the regulation of idiotype production as a context for illustration of basic circuit properties (a) a core regulatory circuit (CRC) consisting of two pairs of Ts and Th that regulate each other so that the circuit maintains stably in either the help or suppression mode; (b) a macrophage-containing auxiliary regulatory circuit (ARC) sensitive to serum idiotype levels that can switch the CRC from help to suppression; (c) a carrier-specific Th circuit that regulates the supply of idiotype-specific help; and (d) an allotype-sensitive ARC that suppresses the production of idiotypes associated with an allotypebearing IgG heavy chain. It discusses the tentative nature of the detail with which the basic circuit structures can be constructed. Existentially, the presentation of detail is inescapable since abstract concepts such as circuit design require concrete models from which essential principles can be extracted. Furthermore, detail must be included to enable design of experiments that provide grounds either for rejection of the model or for its modification to conform to reality.

Publisher Summary This chapter discusses the separation of pooled human IgG into fractions by isoelectric focusing, and their electrophoretic and immunological properties. The starting material was human Cohn Fr. II. Preliminary experiments showed that it was possible to dissolve the Cohn Fr. II in 2 % ampholines, and that if the electrophoresis was carried out with the anode at the bottom of the column precipitation was negligible and that reproducible fractionation profiles could be obtained. The majority of the peaks of the fractionation were also examined for the known IgG heavy chain subclass types. It is found that IgG1 was present over a wide range of pH, IgG2 was restricted to the pH below 8–4, IgG3 was restricted to a pH range of 8–2–9–0. The original Cohn Fr. II did not contain much electrophoretically fast material, and as the incidence of IgG4 myelomas which have fast electrophoretic mobilities is only of the order of 6% this could account for the small amount of IgG4 found in separation

Despite being the least abundant immunoglobulin G in human plasma, IgG4 are used therapeutically when weak effector functions are needed. The increase in engineered IgG4-based antibodies on the market led us to study the patent landscape of IgG4 Fc engineering, i.e., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.