Characterization Of Immunoglobulin Research Articles

Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis.

Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.

Identification and Characterization of Immunoglobulin T Heavy Chain in Large Yellow Croaker (Larimichthys crocea)

Three immunoglobulin (Ig) isotypes have been identified in teleosts, IgM, IgD, and IgT or IgZ. IgT, a new teleost Ig isotype, plays a vital role in mucosal immunity. However, information on molecular and functional characteristics of fish IgT is still limited. In this study, an IgT heavy chain (LcIgT) gene was cloned and characterized in large yellow croaker (Larimichthys crocea). Complete cDNA of LcIgT was 1930 bp in length, encoding a protein of 554 amino acids. The deduced LcIgT contains a VH region and only three CH regions (CH1, CH2, CH4), but no transmembrane region was predicted. Phylogenetic analysis showed that IgT heavy chain sequences from all fish species are grouped together. Homology comparison showed that LcIgT shares the highest amino acid identity of 58.73% with IgT heavy chain in Scophthalmus maximus. The VH domain of LcIgT has the highest identity of 72.50% with that of Scophthalmus maximus IgT. Relatively, each constant domain of LcIgT exhibits the highest amino acid identity with that of IgT in Oreochromis niloticus (67.61% identity for CH1, 61.11% identity for CH2, and 63.74% identity for CH4). LcIgT was constitutively expressed in various tissues tested, with the highest levels in mucosa-associated tissues such as gills and skin. After Cryptocaryon irritans infection, the mRNA levels of LcIgT were significantly up-regulated in the spleen (3.27-fold) at 4 d, in the head kidney (3.98-fold) and skin (2.11-fold) at 7 d, and in gills (4.45-fold) at 14 d. The protein levels in these detected tissues were all significantly up-regulated; the peak of its up-regulation was 6.33-fold at 28d in gills, 3.44-fold at 7d in skin, and 3.72-fold at 14d in spleen. These results showed that IgT response could be simultaneously induced in both systemic and mucosal tissues after parasitic infection and that IgT may be involved in systemic immunity and mucosal immunity against parasitic infection.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

A proteomics-based method for identifying antigens within immune complexes

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis.

The advent of mass cytometry (CyTOF®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5h, and titration requires an additional 3-5h.

Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory.

ABSTRACTTherapeutic monoclonal antibodies (MAbs) are an important class of drugs used to treat diseases ranging from autoimmune disorders to B cell lymphomas to other rare conditions thought to be untreatable in the past. Many advances have been made in the characterization of immunoglobulins as a result of pharmaceutical companies investing in technologies that allow them to better understand MAbs during the development phase. Mass spectrometry is one of the new advancements utilized extensively by pharma to analyze MAbs and is now beginning to be applied in the clinical laboratory setting. The rise in the use of therapeutic MAbs has opened up new challenges for the development of assays for monitoring this class of drugs. MAbs are larger and more complex than typical small-molecule therapeutic drugs routinely analyzed by mass spectrometry. In addition, they must be quantified in samples that contain endogenous immunoglobulins with nearly identical structures. In contrast to an enzyme-linked immunosorbent assay (ELISA) for quantifying MAbs, mass spectrometry-based assays do not rely on MAb-specific reagents such as recombinant antigens and/or anti-idiotypic antibodies, and time for development is usually shorter. Furthermore, using molecular mass as a measurement tool provides increased specificity since it is a first-order principle unique to each MAb. This enables rapid quantification of MAbs and multiplexing. This review describes how mass spectrometry can become an important tool for clinical chemists and especially immunologists, who are starting to develop assays for MAbs in the clinical laboratory and are considering mass spectrometry as a versatile platform for the task.

Characterization of Immunoglobulin A/G Responses During 3 Doses of the Human Papillomavirus-16/18 ASO4-Adjuvanted Vaccine.

Individuals receiving the human papillomavirus (HPV) vaccine develop high levels of circulating neutralizing antibodies. However, data about antibody responses in the cervix are limited. This study was designed to describe the course of IgA/IgG responses in cervical secretions and in serum after intramuscular administration of the HPV16/18 AS04-adjuvant vaccine. An enzyme-linked immunosorbent assay for detection of IgA and IgG anti-HPV-VLP was developed for this purpose. Immunoglobulin G seroconversion after the second dose was observed in 100% of the participants and remained 1 month after the third dose. Regarding IgG reactivity in cervical secretions, conversion was observed in 85% of women after the final dose. Immunoglobulin A seroconversion was observed in 76.7% of women after the third dose. Lower levels of IgA were detected in the cervical mucus (28.3%) and decreased to 23.3% after the last dose. Comparing local and systemic IgG responses, positivity in both serum and cervical samples was observed in 85%, whereas in 15% only, the serum was IgG antibody positive. A weak agreement between local and systemic IgA responses was observed. Only 18.3% of participants were local and systemic IgA positive, 58.4% were positive only in serum, 5% were positive only in the cervix, and 18.3% were both local and systemic IgA antibody negative. After the third vaccination, there is a strong agreement between cervical and systemic IgG antibody responses and a weak agreement between cervical and systemic IgA antibody responses. The induction of IgA antibodies seems to be secondary to that of IgG antibodies in response to HPV intramuscular vaccination.

Characterization of immunoglobulin E plasma cells that are elevated in the upper airway mucosa of nonatopic patients with chronic rhinosinusitis without nasal polyps.

The immunologic mechanisms driving inflammation in the upper airways of patients with chronic rhinosinusitis (CRS) are poorly understood. Previous studies have shown that B cells and immunoglobulin E (IgE) levels are elevated in the nasal tissue of patients with atopic chronic rhinosinusitis without nasal polyps (CRSsNP). However, less is known regarding B cell subsets and IgE-producing plasmablasts in nonatopic CRSsNP patients. Human blood and ethmoid sinus mucosa samples were analyzed from control (n = 6) and nonatopic CRSsNP (n = 11) patients. Tissue samples were evaluated using high-dimensional flow cytometry. A population of IgE antibody secreting cells is significantly increased in situ within inflamed nasal tissue of nonatopic CRSsNP subjects when compared to control nasal tissue and the circulating peripheral blood (p < 0.05). This IgE plasma cell population displays ∼90% cell surface Ig lambda light chain, is mitotically active (Ki-67(+)), and displays intracellular IgE expression. The predominant B cell population expressing IgE are plasmablasts (CD38(high), CD138(-)) not typically found in the blood or peripheral tissue of these patients. The nasal mucosa from nonatopic CRSsNP patients demonstrate a significant regional spike in resident in situ IgE plasmablast cells not seen in control nasal tissue or peripheral blood from the same patient. The restricted expression of Ig lambda light chain in this mitotically active IgE plasmablast population supports the hypothesis of aberrant B cell proliferation in the context of CRS. These findings suggest the presence of a unique regional immune microenvironment for B cell priming and/or selection within chronically inflamed airway tissues.

Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS

The aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a polyclonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, glycopeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is discussed for retention of sialic acid residues on peptide acidic glycoforms.

Molecular cloning and characterization of the α-chain gene of goose immunoglobulin heavy chain

A novel gene encoding the α-chain of goose immunoglobulin heavy-chain (Igα) was cloned by reverse transcription-PCR. The cDNA had 1,760 bp and encompassed a partial V-D-J region of the heavy chain, a constant region (Cα) and 3'-untranslated region of α-chain. The Cα gene contains four constant region domains (CH1-CH4). Phylogenetic analysis indicated that goose IgCα has a close genetic relationship with duck, ostrich and chicken IgCα. Three-dimensional modeling and glycosylation analysis revealed the goose Igα is consistent with the characterization of immunoglobulin. Western blotting suggested the goose IgCα has the same antigenicity to natural IgA. In general, the identification of goose immunoglobulin not only provides insights into the evolution of the Ig heavy-chain gene family, but may also benefit future studies of the avian immune system.

Purification and Characterisation of Immunoglobulins from the Australian Black Flying Fox (Pteropus alecto) Using Anti-Fab Affinity Chromatography Reveals the Low Abundance of IgA

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

Characterization of immunoglobulin λ light chain variable region (IGLV) gene and its relationship with clinical features in patients with POEMS syndrome

The M protein of POEMS syndrome is essentially λ light chain restricted. Several studies have demonstrated the restrict usage of immunoglobulin λ light chain variable region (IGLV) genes in patients with POEMS syndrome. However, these studies only included a limited number of cases, and it is not clear whether the clinical features are influenced by the IGLV gene in POEMS syndrome. Here we demonstrated that the clonal IGLV genes were strictly derived from IGLV 1-40 (11 patients, 36.7 %) and IGLV 1-44 (19, 63.3 %) gene in 30 patients with POEMS syndrome. We further evaluated the relationship between clinical features and IGLV genes. Our study showed that patients with IGLV 1-44 were older than those with IGLV 1-40, and patients with IGLV 1-40 had more severe neuropathy, hypertrichosis, and papilledema. It was suggested that the IGLV gene influenced clinical characteristics in POEMS syndrome.

Isolation and characterization of immunoglobulin M of Asian sea bass, Lates calcarifer and its level in serum

Asian sea bass immunoglobulin M (IgM) was purified from the sera of Lates calcarifer by affinity chromatography. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions revealed that the sea bass IgM was a tetrameric protein with a molecular weight of 896 kDa; it contained an equimolar heavy chain and light chain with molecular weight of 83 kDa and 27 kDa respectively. However, besides the covalently linked tetrameric IgM, noncovalently linked tetramer dissociated into dimeric and monomeric forms also demonstrated by non-reducing SDS-PAGE. Carbohydrate moieties were found to be linked with both heavy and light chains. A polyclonal rabbit anti-Asian sea bass IgM was prepared which showed a specific reaction of anti-fish IgM antibody with IgM of sea bass. Sea bass IgM concentration was determined in the serum by indirect ELISA. The average IgM concentration in the sera of the healthy sea bass was 5.4±1.8 mg ml−1; it amounted to 16.7% of the total serum protein.

Isolation and characterization of immunoglobulin M of Asian sea bass, Lates calcarifer and its level in serum

Abstract Asian sea bass immunoglobulin M (IgM) was purified from the sera of Lates calcarifer by affinity chromatography. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions revealed that the sea bass IgM was a tetrameric protein with a molecular weight of 896 kDa; it contained an equimolar heavy chain and light chain with molecular weight of 83 kDa and 27 kDa respectively. However, besides the covalently linked tetrameric IgM, noncovalently linked tetramer dissociated into dimeric and monomeric forms also demonstrated by non-reducing SDS-PAGE. Carbohydrate moieties were found to be linked with both heavy and light chains. A polyclonal rabbit anti-Asian sea bass IgM was prepared which showed a specific reaction of anti-fish IgM antibody with IgM of sea bass. Sea bass IgM concentration was determined in the serum by indirect ELISA. The average IgM concentration in the sera of the healthy sea bass was 5.4±1.8 mg ml−1; it amounted to 16.7% of the total serum protein.

Characterization of immunoglobulin G antibodies to Plasmodium falciparum sporozoite surface antigen MB2 in malaria exposed individuals.

BackgroundMB2 protein is a sporozoite surface antigen on the human malaria parasite Plasmodium falciparum. MB2 was identified by screening a P. falciparum sporozoite cDNA expression library using immune sera from a protected donor immunized via the bites of P. falciparum-infected irradiated mosquitoes. It is not known whether natural exposure to P. falciparum also induces the anti-MB2 response and if this response differs from that in protected individuals immunized via the bites of P. falciparum infected irradiated mosquitoes. The anti-MB2 antibody response may be part of a robust protective response against the sporozoite.MethodsFragments of polypeptide regions of MB2 were constructed as recombinant fusions sandwiched between glutathione S-transferase and a hexa histidine tag for bacterial expression. The hexa histidine tag affinity purified proteins were used to immunize rabbits and the polyclonal sera evaluated in an in vitro inhibition of sporozoite invasion assay. The proteins were also used in immunoblots with sera from a limited number of donors immunized via the bites of P. falciparum infected irradiated mosquitoes and plasma and serum obtained from naturally exposed individuals in Kenya.ResultsRabbit polyclonal antibodies targeting the non-repeat region of the basic domain of MB2 inhibited sporozoites entry into HepG2-A16 cells in vitro. Analysis of serum from five human volunteers that were immunized via the bites of P. falciparum infected irradiated mosquitoes that developed immunity and were completely protected against subsequent challenge with non-irradiated parasite also had detectable levels of antibody against MB2 basic domain. In contrast, in three volunteers not protected, anti-MB2 antibodies were below the level of detection. Sera from protected volunteers preferentially recognized a non-repeat region of the basic domain of MB2, whereas plasma from naturally-infected individuals also had antibodies that recognize regions of MB2 that contain a repeat motif in immunoblots. Sequence analysis of eleven field isolates and four laboratory strains showed that these antigenic regions of the basic domain of the MB2 gene are highly conserved in parasites obtained from different parts of the world. Moreover, anti-MB2 antibodies also were detected in the plasma of 83% of the individuals living in a malaria endemic area of Kenya (n = 41).ConclusionA preliminary analysis of the human humoral response against MB2 indicates that it may be an additional highly conserved target for immune intervention at the pre-erythrocytic stage of P. falciparum life cycle.

Immunological Characterization of Immunoglobulin G towards House Dust Allergens

Immunological Characterization of Immunoglobulin G towards House Dust Allergens

Characterization of immunoglobulin G fragments in liquid intravenous immunoglobulin products

Intravenous immunoglobulin (IVIG) products formulated as a liquid instead of a powder have become commercially available. Preferably, such liquid products should not alter after storage outside the refrigerator. Therefore, a thorough characterization of immunoglobulin G (IgG) fragmentation at various storage temperatures is required. Storage experiments with liquid IVIG products from five manufacturers were performed at 4, 25, and 37 degrees C and IgG fragments were analyzed. Storage of liquid IVIG products at 4 degrees C resulted in negligible alterations, whereas an increase of IgG fragments was observed after prolonged storage at elevated temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of five liquid IVIG products revealed three IgG fragments (12, 26, and 54 kDa) in all products. Fragments of similar molecular mass were produced upon incubations of IgG with blood-derived proteases. N-terminal amino acid sequencing revealed the cleavage site of these fragments, suggesting human neutrophil elastase to cause the 12-kDa fragment. The presence of elastase in liquid IVIG was confirmed by enzyme-linked immunosorbent assay. The origin of the 26- and 54-kDa fragments, both with an aspartic acid residue at the cleavage site, could not be determined unambiguously. IgG fragmentation in liquid IVIG is negligible when stored in the refrigerator. Only after prolonged storage at elevated temperature does proteolytic degradation of IgG become apparent.

Characterization of immunoglobulin G and its subclass response to Indian kala-azar infection before and after chemotherapy.

Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity against Leishmania donovani membrane antigen (LAg). Heterogeneous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.

Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen.

Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.

Isolation and characterization of immunoglobulin in yolk (IgY) specific against hen egg white lysozyme by immunoaffinity chromatography.

Six hens were intramuscularly (im) immunized once a week for 3 weeks using chicken egg white lysozyme (LS) as antigen. Antibody (immunoglobulin in yolk, IgY) ELISA values of 10(3)-fold diluted yolk were almost as high as 1.879 in the sixth week and maintained a value of 0.756 in the eighth week after the initial immunization treatment. The purification efficiency (specific activity of purified IgY against LS/specific activity of antibody in yolk against LS) of IgY specific against LS isolated by laboratory-prepared LS-bound (IgY-) Sepharose 4 Fast Flow immunoaffinity column was approximately 3380. By applying various amounts (0-22 mg) of the thusly obtained IgY specific against LS to the immunoaffinity column, the binding capacity (q(m)) and dissociation constant (K(d), M(-1)) of such immunoaffinity gel for IgY against LS were found to be 0.68 mg of IgY/mL of wet gel (0.54 mg of IgY/mg of LS) and 7.13 x 10(-6) M, respectively, as determined by Langmuir-type adsorption isotherms.