Human IgG Class Research Articles

Preliminary Tests of Multiplex Immunoassay for Detection of TORCH Infections in Human Blood Serum Using Flow Cytometry

Preliminary tests of multiplex immunoassay for detection of TORCH infections in human blood serum using flow cytometry are presented. The immunoassay was intended to provide qualitative detection of the specific human IgG class antibodies to Toxoplasma gondii, rubella virus, and cytomegalovirus in human blood serum. The multiplex analysis is performed in vitro on polystyrene microbeads labeled with an organic dye. The diagnostic sensitivity of the kit is 97%.

Evaluation of mice, sheep and human IgG and IgE antibody responses against the mice Crude Hydatid Cyst Fluid (HCF) antigens

Hydatidosis is a dangerous parasitic disease which commonly occurs in humans and animals. It annually causes many economic and health problems throughout the world. The existing diagnostic kit uses sheep cystic fluid antigen as an available antigen to evaluate the human IgG antibody. The current study was designed to evaluate an alternative antigen which is the mice HCF antigen for such diagnosing kit. For this purpose the IgG responses in sera of human, mice and sheep against the mice crude hydatid fluid antigens was applied using ELISA and Western Blot. Thirty Balb/c mice were immunized using sheep hydatid cyst and complete freunds adjuants. The hydatid cyst fluid antigens of human and sheep were obtained from naturally infected human and sheep and for mice from experimentally infected mice with protoscolices of hydatid cysts (HC). Antigens were used in ELISA and SDS PAGE after their concentrations were measured applying Bradford procedure. Thirty positive samples sera from mice, sheep and human were employed as the case and 30 healthy sera from each as the control group. The statistic analysis tests were ANOVA and post Hoc Analysis Model. The highest mice antibody response against mice HCF antigens was IgGAM antibody (with mean OD value 0.4) while the lowest was IgE (with mean OD value 0.26). The best antibody response was seen for human total IgG against mice HCF with mean OD value of 0.71 while the lowest response against this antigen belonged to human IgG3 and IgE each with mean OD value of 0.12. ANOVA analysis indicated significant differences between the human IgG class and subclass responses to mice HCF antigens (P < 0.05). The sensitivity and specificity of the ELISA for antibody responses to this antigen was mostly more than 90% almost for all sera of different hosts. The mean OD value of sheep IgG against mice HCF antigen was 0.34. The studied sera (mice, human and sheep) considerably showed a positive response against mice HCF antigens by ELISA test. The human and mice sera showed a higher response over the sheep sera in that their mean OD values and their OD ratio were significantly higher than that for the sheep. Totally there are some immune responses from the serum of each animal recognizing the crude hydatid cyst fluid antigen of mice. According to the results of the current study the mice HCF is an appropriate candidate for diagnosing of hydatidosis of human and animal. Key words: Hydatid cyst, cross-reaction, IgG response, hydatid cyst fluid.

Cross-Reaction between the Crude Hydatid Cyst Fluid Antigens of Human and Animals Origin in Response to Human IgG Class and Subclasses

The current work aimed to evaluate the cross-reactivity of human immune sera against crude hydatid fluid antigens of sheep, human, mouse, cattle, as well as B fraction of cystic fluid antigen. 30 balb/c mice were infected with sheep hydatid cyct fluid antigen containing protoscolex after the viability of these protoscolices was assessed. ANOVA was used to test the difference of themean of optical density (OD) values among case and control groups. The highest human IgG class antibody was against antigen B (0.93) and the lowest against cattle HCF antigen (0.32). The differences between responses to these antigens were statistically significant (P < 0.001). The sensitivity and specificity of ELISA test used for evaluating the responses of human total IgG to different hydatid cyst fluid (HCF) antigens among the case and control groups were 100 and 95.8%, respectively. Cross-reaction of human IgG class and subclasses responses was found almost for all the antigens with the best reaction against human and cattle (HCF) antigens and antigen B using a ratio of mean OD value to each antigen divided by the cut-off point value for the same antigen. Human sera showed a considerable cross-reactivity against all antigens by using ELISA.

Severe combined immunodeficient (SCID) mice: a model for investigating human thyroid autoantibody synthesis

We have studied the ability of lymphocytes from the blood, thyroid and lymph nodes of patients with autoimmune thyroid disease (AITD) to produce autoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were detectable in plasma from SCID mice 7 days after transfer of 15-25 x 10(6) cells/mouse and the highest levels were recorded 2-3 weeks later. In contrast, Tg and/or TPO antibodies were undetectable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the most antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounts of Tg and/or TPO antibody detected were in accordance with the ability of thyroid and lymph node lymphocytes to secrete these autoantibodies spontaneously in culture (indicating the presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tg and/or TPO antibodies in culture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1, 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from patients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of human antibodies to Tg and TPO.

Evaluation of human IgG class and subclass antibodies to a 24 kDa antigenic component of Gnathostoma spinigerum for the serodiagnosis of gnathostomiasis

The immunoglobulin G class (total IgG) and subclass (IgG1, IgG2, IgG3, and IgG4) antibody responses to the spirurid worm Gnathostoma spinigerum were analyzed by immunoblotting technique for the antibodies' potential use in the serodiagnosis of human gnathostomiasis. Serum samples from patients with proven gnathostomiasis and from clinically suspected cases of gnathostomiasis with migratory swelling were tested. Sera from patients with other parasitic illnesses and from healthy volunteers were also analyzed. The total IgG antibody to an antigenic band of approximately 24 kDa from a somatic extract of G. spinigerum advanced third-stage larvae (GS24) gave the highest sensitivity (91.6%) and 87.8% specificity. Individual IgG subclass detection had a lower sensitivity than the detection of specific total IgG antibody, but IgG4 had a slightly higher specificity (93.9%). However, for cost effectiveness, we suggest that anti GS24 total IgG is sufficient for the routine serodiagnosis of human gnathostomiasis as well as for seroepidemiological studies in developing countries.

Data on the isolation of immunoglobulin from the serum of the green frog: Rana esculenta

Even though the green frog (Rana esculenta) is often used as an experimental model for further studies of physiological laws, little is still known about its serum proteins and their role in immunity. Its serum proteins have been studied quite extensively, as when they are taken up into the organism of another animal they represent antigens themselves. In this work, an attempt was made to isolate some frog serum proteins and to investigate the electrophoretic qualities of the isolated components (electrophoresis on agar gel and immunoelectrophoresis). IgG was isolated using the same procedure applied for human sera and one of the components found in the beta globulin zone was isolated from frog serum by the same procedure. Immunoelectrophoretic analysis, carried out with the full antiserum of a rabbit, obtained by immunization of the rabbit with the frog serum, showed that the isolated component was pure. The obtained results confirm the fact that there are no slow gamma globulins in the frog serum and point out that this procedure, used for isolating human IgG class, is also suitable for isolating one protein component from the serum of the frog, which has the electrophoretic speed of beta globulin and which may represent one category of frog immunoglobulins.

Immunochemical study of human immunoglobulin G Fc region.

Fifteen murine monoclonal antibodies (mAb) were produced that reacted with conformational epitopes present on the Fc portion of all human IgG subclasses (PAN-Fc). Inhibition and sandwich ELISA were used to elucidate overlapping and distinct epitopes. The epitopes were aligned to form a continuous "chain" of overlapping determinants from the CH2-CH3 interface to the direction of hinge. The five more hinge-proximal epitopes demonstrated high lability both in competitive and sandwich assays, being completely or partially destroyed when any of these 15 other mAb bound the IgG first. Heat-inactivation of IgG caused full disruption of these epitopes. In contrast, epitopes situated at the opposite distal of the "chain" were more stable and mAb binding could only be affected by occupying an overlapping epitope. Under heat-inactivation these epitopes were affected, but not completely destroyed. Human IgG class anti-DNA autoantibodies were bound to insolubilized dsDNA and their reaction with PAN-Fc mAb was studied. mAb titration plots on IgG and dsDNA-IgG were compared. Five epitopes proved to be altered by antigen (dsDNA) binding. Two of these were the labile hinge-proximal epitopes and the other three were situated near the CH2-CH3 interface. Cross-reactivity of mAb with xenogeneic IgG was also studied. An "epitope map" of the crystallographic model of human IgG Fc portion drawn was based on these experimental data and printed matter, concerning the location of subclass-specific amino acids and homology regions of human and animal IgG.

Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type

IgG class thyroid peroxidase (TPO) autoantibodies with kappa light (L) chains predominate in serum and the genes for a large repertoire of such autoantibodies have been characterized. The present study was performed to clone and characterize TPO autoantibodies with lambda L chains which comprise ∼20% of serum TPO autoantibodies. From a combinatorial IgG H/lambda L chain cDNA library in the phage display vector pComb3, 24 TPO-binding clones with lambda L chains were isolated, comprising three different heavy (H) and light (L) chain combinations. These combinations utilized two genes from the Vlambda II and IIIb families (closest germline genes DPL11 and hsigg11150) and three genes from the VH1, VH3 and VH4 families ( VH26, 4.34 and hv1L1). The deduced amino acid sequences of these H chains were quite different from those of kappa F(ab) isolated using the same H chain library. We expressed the proteins for these three lambda F(ab), as well as for a lambda F(ab) (Humlv318 L chain/DP10-like H chain) previously isolated from another patient. The affnities for TPO of the lambda F(ab) ( K d 8 × 10 −10 M to 10 −7 M) were lower than those of the kappa F(ab) ( K d ∼ 10 −10 M). For two lambda F(ab), both H and L chain genes were close to germline configuration, but there was no straightforward relationship between the extent of somatic mutation from germline configuration and affinity for TPO. All four lambda F(ab) bound less well to denatured TPO as to native TPO. The three F(ab) for which sufficient protein could be expressed for competition studies all recognized domain B within the immunodominat region on TPO previously identified using F(ab) with kappa L chains. Aside from these TPO-specific F(ab), only a few other human IgG class, organ-specific autoantibodies with lambda L chains have been characterized at the molecular level. Our study significantly augments the small database on this category of autoantibodies in general.

Expression of recombinant antibody against cancer-specific carbohydrate

Starting with a previously established hybridoma producing a monoclonal antibody (F1α75) against a synthetic glycolipid, in which the carbohydrate moiety was artificially designed to be putatively cancer-specific, we cloned complementary DNA (cDNA) for active variable regions of both heavy and light chains of the antibody. We established a Chinese hamster ovary (CHO) cell line expressing a recombinant F1α75 of human IgG class. The recombinant F1α75 retained the binding ability to its antigen and was easily purified. The result suggests that the conversion into the IgG class by genetic engineering is useful for antibodies of the IgM class which are difficult to be purified due to aggregation. Sequence analysis of variable regions of F1α75 revealed no strong homology with an antibody against galactose, the terminal residue of the synthetic glycolipid, suggesting the clinical usefulness of the recombinant F1α75.

Efficacy of orally administered immune serum globulin against type III group B streptococcal colonization and systemic disease in an infant rat model.

We established an experimental animal model of the gastrointestinal colonization and systemic disease following oral challenge of type III group B streptococcal strain in 3-day-old newborn rats. Two type III group B streptococcal strains isolated from the cerebrospinal fluid of septic newborn infants produced colonization in 57-87% of the challenged animals and 13-31% of these colonized animals developed systemic disease. Using this new animal model, we evaluated the effect of orally administered human immune serum globulin on the colonization and systemic disease. This antiserum contained 21 micrograms/ml of type III group B streptococcal antibody of human IgG class. Animals fed with immune serum globulin developed significantly lower rates of colonization and systemic disease than those of control (albumin or saline) (23 versus 71%, p less than 0.001 for colonization; 7 versus 31%, p less than 0.05 for systemic disease). However, rates of the development of systemic disease among the colonized animals were not significantly different between the two groups (33 versus 43%, p greater than 0.1). These findings suggest that orally administered immune serum globulin is beneficial in the prevention of colonization and systemic disease in this rat model and that this protective effect of oral immune serum globulin occurs primarily at the mucosal level.