Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Distributions of the use frequencies of amino acids in the hypervariable regions of immunoglobulins

Two types of distributions for the frequencies of occurrence of amino acids in each position of hypervariable regions CDR-1 and CDR-2 were obtained for 2,000 immunoglobulins. The results show that some positions fit an inverse power-law distribution, while others fit an exponential-type distribution. As a result of comparison with structural data in the literature it is proposed that sites in which the frequency distribution fits the inverse power law are critical to maintaining canonical shapes of the recognition regions or are involved in modulating these canonical conformations, while those sites where the distribution fits the exponential law are those which should be exclusively involved in the recognition mechanism. Correspondence to: F. Lara-Ochoa     

Echinococcus multilocularis: distribution and persistence of specific host immunoglobulins on cysts membranes

Cyst vesicles were obtained from larval cyst masses of Echinococcus multilocularis grown in Medium 858 in vitro or isolated from the intraperitoneal or subcutaneous larval cyst mass of C57BL/6J mice infected 12 weeks previously. Antigenic determinants were present on the outermost section of the laminated layer and throughout the germinal layer of the cysts. Specific host antibodies of the IgG1, IgG2a, IgG2b, and IgM classes and complement component C3 but not host IgA or C-reactive protein were detected on the intact cysts by the indirect immunofluorescent technique. Specific antibodies were bound to the epitopes on the laminated layer but were not detected on the germinal layer. Host albumin, however, was found on the laminated layer, germinal layer, and within the intact cyst. IgG2a and IgG2b were high-affinity antibodies but IgG1 and IgM were low-affinity antibodies as they were eluted easily from the laminated layer with two washes of sodium phosphate buffer (0.01 M, pH 7.2) containing 0.15 M NaCl. The significance of bound antibody in complement activation and antibody-dependent cell-mediated cytotoxicity of the proliferative phase (cyst) of alveolar hydatid cyst is discussed.     

Ascaris suum: specific antibodies in isolated intestinal loop washings from immunized swine.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.     

The first report on immunoglobulins A,E, G and M levels in cystic fibrosis patients in Saudi Arabia

BackgroundPrevious reports have shown that pulmonary and systemic hypergamma-globulinemia in CF patients is a reflection of chronic pulmonary infection. Infection with Pseudomonas aeruginosa is known to have major prognostic significance in patients CF. This study aims to identify the incidence of immunoglobulins (especially: IgG, and IgE) in a cohort of CF patients.MethodsA total of 297 patients recruited all over the country’s region for this study were a as part of the CF registry data from 1st January 1984 to 1st June 2016. All patients had their immunoglobulin levels measured by enzyme link immunosorbent assay (ELISA) in 3 stages, at presentation and two follow-ups.ResultsOf the 297 patients recruited, 139 (46.8%) were males while 158 (53.2%) were females. IgA and IgM levels were found not to have risen above the previously reported levels in healthy individuals in all stages. On the contrary, IgE level increased from 209.51 ± 32.30 KU/L to 303.58 ± 37.11 KU/L from baseline to stage 3 while IgG level rose from 12.26 ± 0.43 mg/mL to 17.17 ± 1.68 mg/mL for baseline and stage 3 respectively all above previously reported levels in healthy individuals.ConclusionThis study establishes a potential for the use of IgE and IgG in disease diagnosis as well as the prognostic implications. However, further study is needed to identify the role of infection or medications in relation to the rise of both IgE and IgG with advancement of age and progression of disease severity which may inherently confound the observed results.     

Structural differences between the repertoires of mouse and human germline genes and their evolutionary implications

 Although human and mouse antibodies are similar when one considers their diversification strategies, they differ in the extent to which kappa and lambda light chains are present in their respective variable light chain repertoires. While the Igk-V germline genes are preponderant in mice (95% or more), they comprise only 60% in humans. This may account for differences in the structural repertoire encoded in the Igk-V germline genes of these species. However, this subject has not been properly investigated, partially because a systematic structural characterization of the mouse Igk-V germline genes has not been undertaken. In the present study we compiled all available information on mouse Igk-V germline genes to characterize their structural repertoire. As expected, comparison with the structural repertoire of human Igk-V germline genes indicates differences. The most interesting is that the mouse Igk-V germline gene repertoire is more diverse in structural terms than its human counterpart: the mouse encodes seven canonical structure classes (combination of canonical structures in L1 and L3). In contrast, the human encodes only four. Analysis of the evolutionary relationships of human and mouse Igk-V germline genes led us to propose that the difference reflects a strategy of mice to compensate for the small lambda chain contribution to the repertoire of their variable light chains. Received: 1 June 1997 / Revised: 6 October 1997     

Sorbitol-mediated stabilization of human IgG against thermal inactivation

Sorbitol at 30% (w/w) stabilized human IgG to thermal denaturation from 60 to 85°C, increasing the protein's half life from 25 to 266 min at 70°C. A kinetic model based on the Lumry-Eyring inactivation scheme was developed and used to estimate the apparent rate constant and activation energy. © Rapid Science. 1998     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Dispensing an enzyme-conjugated solution into an ELISA plate by adapting ink-jet printers

The rapid and precise delivery of small volumes of bio-fluids (from picoliters to nanoliters) is a key feature of modern bioanalytical assays. Commercial ink-jet printers are low-cost systems which enable the dispensing of tiny droplets at a rate which may exceed 10⁴ Hz per nozzle. Currently, the main ejection technologies are piezoelectric and bubble-jet. We adapted two commercial printers, respectively a piezoelectric and a bubble-jet one, for the deposition of immunoglobulins into an ELISA plate. The objective was to perform a comparative evaluation of the two classes of ink-jet technologies in terms of required hardware modifications and possible damage on the dispensed molecules. The hardware of the two printers was modified to dispense an enzyme conjugate solution, containing polyclonal rabbit anti-human IgG labelled with HRP in 7 wells of an ELISA plate. Moreover, the ELISA assay was used to assess the functional activity of the biomolecules after ejection. ELISA is a common and well-assessed technique to detect the presence of particular antigens or antibodies in a sample. We employed an ELISA diagnostic kit for the qualitative screening of anti-ENA antibodies to verify the ability of the dispensed immunoglobulins to bind the primary antibodies in the wells. Experimental tests showed that the dispensing of immunoglobulins using the piezoelectric printer does not cause any detectable difference on the outcome of the ELISA test if compared to manual dispensing using micropipettes. On the contrary, the thermal printhead was not able to reliably dispense the bio-fluid, which may mean that a surfactant is required to modify the wetting properties of the liquid.     

The immunoglobulin allotypes (Gm and Km) of twelve Indian Tribes of Central and South America

The Gm and Km immunoglobulin allotypes are presented, for the first time, for six South American Indian tribes (Baniwa, Kanamari, Kraho, Makiritare, Panoa, and Ticuna) and one Central American tribe (Guaymi). Additional allotype information is presented for five previously reported South American tribes (Cayapo, Piaroa, Trio, Xavante and Yanomama). The distributions of the Gm and Km allotypes among all the tribal populations tested to date are reviewed and evidence is presented for the presence of a north (high) -south (low) cline in Km frequency. The wave theory of the populating of the South American continent was tested by an examination of the distribution of six alleles (Gm^ax;g, Gm^a;b0,3,t, Di^a, R^z, TF^D Chi, and 6PGD^C), absent in some populations but with polymorphic proportions in others. The present, limited, data failed to confirm the theory.