Polyclonal Ig Research Articles

Characterization of M proteins as a series of peaks by capillary zone electrophoresis with a linear polyacrylamide-coated capillary.

Human serum proteins including myeloma (M) proteins were analyzed by capillary zone electrophoresis (CZE) using a linear polyacrylamide-coated capillary at pH 7.4. Each of the M proteins, IgG-κ, IgA-κ, and IgA-λ, which were located on the β zone on the cellulose acetate electrophoresis (CAE), was resolved into a group of peaks with a unique profile in a wide area following the transferrin (TF) peak. While the IgG-κ showed a series of narrow peaks, IgA-κ and IgA-λ showed a wide peak with many successive spikes. On the other hand, the increased polyclonal IgA-κ and IgG-κ, which were located on the β zone on the CAE, showed only a broad peak around the TF peak. Another IgG-κ M protein, which was located on the γ zone on the CAE, did not appear on the CZE at pH 7.4, but did appear with several peaks by increasing the pH to 9.0. No peaks were observed in the area following the TF peak when healthy serum proteins including immunoglobulins (Igs) were analyzed at both pHs. The coexistence of albumin did not affect the detection of monoclonal and polyclonal Igs. These results demonstrate that the CZE system is capable of distinguishing IgG M proteins from IgA M proteins, and monoclonal Igs from polyclonal ones. Furthermore, it was found that the CZE is useful for discriminating between isoforms of IgG M proteins.

Monocytoid B Cells Are Distinct From Splenic Marginal Zone Cells and Commonly Derive From Unmutated Naive B Cells and Less Frequently From Postgerminal Center B Cells by Polyclonal Transformation

Monocytoid B cells represent a morphologically conspicuous B-cell population that constantly occurs in Toxoplasma gondii-induced Piringer's lymphadenopathy. Although widely believed to be closely related to splenic marginal zone B cells, neither this relationship, nor the B-cell differentiation stage of monocytoid B cells, nor their cellular precursors have been established. We have therefore examined monocytoid B cells for their expression of B-cell differentiation markers and the Ig isotypes at the RNA and protein level as well as for rearranged Ig heavy chain (H) genes and somatic mutations within the variable (V) region. The results obtained were compared with the corresponding features of other B-cell populations. The monocytoid B cells displayed immunophenotypical differences to all other B-cell populations. IgM and IgD expression was absent from most monocytoid B cells at the RNA and protein levels. Unrelated (polyclonal) Ig rearrangements were found in 85 of the 95 cells studied. Seventy-four percent of the rearranged VH genes were devoid of somatic mutations, whereas the remaining 26% carried a low number of somatic mutations. The majority of these showed no significant signs of antigen selection. This finding in conjunction with the predominantly unrelated Ig gene rearrangements indicates that most monocytoid B cells arise not by clonal proliferation but by transformation of polyclonal B cells. The B cells undergoing a monocytoid B-cell transformation are in the majority (74%) naive B cells, and only a minority are (26%) non-antigen-selected postgerminal center B cells. Thus, our data show that monocytoid B cells represent a distinct B-cell subpopulation.

Effect of polyclonal immunoglobulins on neutrophil phagocytic capacity and reactive oxygen production in patients with gram-negative septicemia.

The effect of immunoglobulin (Ig) preparations on neutrophil phagocytic ability and oxidative burst in response to Escherichia coli stimulation was analyzed in 14 patients with gram-negative septicemia by an ex vivo whole blood assay using flow cytometry. In patients, neutrophils exhibited a decreased capacity to phagocytize E. coli and generate reactive oxygen products compared to healthy controls (median -68%, P < 0.01). The addition of both 7S-Ig and 19S-Ig enriched preparations in vitro resulted in a dose-dependent increase in neutrophil reactive oxygen production at concentrations of 10 g/l (median +153% and +211%, P < 0.01, respectively) and 20 g/l (median +205% and +282%, P < 0.01, respectively). A decreased neutrophil phagocytic ability was seen in patients with septicemia (median -58%) compared to healthy controls (P < 0.01). Again, the addition of 7S and 19S-Igs enhanced the phagocytic ability in a dose-dependent manner (10 g/l: median +56 and +126%; 20 g/l: median +126% and +165%, P < 0.01 for all). It can be concluded that both polyclonal Igs can increase depressed neutrophil reactive oxygen production and neutrophil phagocytosis in patients with gram-negative septicemia.

Two-dimensional electrophoretic analysis of cryoproteins: a report of 335 samples.

Cryoproteins are defined as proteins precipitating at low temperature. Most frequently, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Three types of cryoglobulins have been described: type I contains a single monoclonal Ig, whereas type II is a mixture of a monoclonal Ig with polyclonal Igs, and type III is a mixture of polyclonal Igs of different isotypes, most frequently IgG and IgM. Type II and type III are also called mixed cryoglobulins. A new type of cryoglobulins, containing polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM has recently been described after two-dimensional polyacrylamide gel electrophoresis (2-DE). This type of cryoglobulin has been called type II-III cryoglobulin. In this study, we report on 2-DE analysis of 335 cryoproteins from patients with heterogeneous clinical conditions. In 69 out of 335 samples (20.7%), 2-DE revealed patterns that were inadequate to characterize the cryoproteins. Out of 335 (79.3%) cryoproteins, 266 were identified according to their two-dimensional patterns: 265 samples contained Igs and were diagnosed as cryoglobulins, and one sample consisted of fibrinogen, and was identified as cryofibrinogen. Among the 265 cryoglobulins, types I, II, and III were observed in 9 (3.4%), 69 (26%), and 116 (43.8%) cases, respectively, whereas type II-III was detected in 71 (26.8%) cases. Eleven of the latter consisted of oligoclonal Igs (IgM in 10 cases, IgA in 1 case) mixed with traces of polyclonal IgG. These cryoproteins were tentatively named type II-IIIvariant cryoglobulins. Taken together, our result clearly show that 2-DE is a suitable technique to analyze cryoproteins.

Bacterial DNA Containing CpG Motifs Stimulates Lymphocyte-Dependent Protection of Mice Against Lethal Infection with Intracellular Bacteria

Bacterial DNA containing unmethylated CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig. These include IFN-gamma, IL-12, TNF-alpha, and IL-6, which are important in the control of intracellular bacterial infection. Here, we show that bacterial DNA, as well as synthetic oligonucleotides containing CpG motifs, induce protection against large lethal doses of Francisella tularensis live vaccine strain (LVS) and Listeria monocytogenes. Methylation of DNA at CpG dinucleotides or inversion of the motif abolished this protection. Surprisingly, DNA-mediated protection was highly dependent on lymphocytes, particularly B cells, as well as the production of IFN-gamma. Optimal protection was elicited 2-3 days after inoculation with DNA and persisted for up to 2 wk. Further, animals surviving lethal challenge developed pathogen-specific secondary immunity. These findings indicate that host innate immune responses to bacterial DNA may contribute to the induction of protective immunity to bacteria and the subsequent development of memory.

Immune reconstitution following allogeneic peripheral blood stem cell transplants.

Growth factor-mobilized peripheral blood stem cells (PBSCs) engraft rapidly in myeloablated recipients compared to conventional BM, but this procedure also mobilizes mature lymphocytes and monocytes which can impact immune reconstitution and GVHD. Hence, we serially evaluated immune reconstitution and cytokine expression in PBSCT recipients in the first year. Engraftment of neutrophils and monocytes stabilized early but NK cells, B cells and CD4+ T cell numbers were significantly (P < 0.05) low with persistently reversed CD4:CD8 ratios. NK function remained low throughout the first year. The quantitative decrease in CD4+ T cells resulted in significantly decreased proliferation in response to mitogens and alloHLA antigens. Yet, a qualitative analysis of T cell function measured by Ca++ influx after T cell activation with antiCD3 as well as T-dependent polyclonal Ig secretion by mitogen-stimulated B cells was preserved even early post transplant. TNF alpha mRNA was detected in almost all recipients in the first year. IL-10 mRNA was detected in 77%, IL-2 in 22% and IFN gamma in 44% of recipients in the first 6 months. Only 30% expressed IL-10 in the second 6 months post transplant while expression of IL-2 and IFN gamma was detected in 38% and 46% respectively. Thirty-seven percent of PBSCT recipients developed grades II-IV acute GVHD but 72% went on to develop chronic extensive GVHD at a median of 120 days. Sixty-two percent developed CMV viremia and 5.4% developed overt CMV disease in the first year post PBSCT. Lymphocyte engraftment is quantitatively delayed but CD4 functions are preserved while NK numbers and function are compromised post PBSCT. IL-10 expression decreases after the first 6 months post transplant while TNF alpha is continually expressed. The balance between quantitative lymphocyte reconstitution and qualitative lymphocyte functions as well as changes in lymphokine patterns may influence infection and GVHD and thus the clinical outcome post PBSCT.

CD4-immunoglobulin G2 protects Hu-PBL-SCID mice against challenge by primary human immunodeficiency virus type 1 isolates.

CD4-immunoglobulin G2 (IgG2) is a fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. Previous studies found that CD4-IgG2 potently neutralizes a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates in vitro and ex vivo. The current report demonstrates that CD4-IgG2 protects against infection by primary isolates of HIV-1 in vivo, using the hu-PBL-SCID mouse model. Passive administration of 10 mg of CD4-IgG2 per kg of body weight protected all animals against subsequent challenge with 10 mouse infectious doses of the laboratory-adapted T-cell-tropic isolate HIV-1(LAI), while 50 mg of CD4-IgG2 per kg protected four of five mice against the primary isolates HIV-1(JR-CSF) and HIV-1(AD6). In contrast, a polyclonal HIV-1 Ig fraction exhibited partial protection against HIV-1(LAI) at 150 mg/kg but no significant protection against the primary HIV-1 isolates. The results demonstrate that CD4-IgG2 effectively neutralizes primary HIV-1 isolates in vivo and can prevent the initiation of infection by these viruses.

CD8+/CD57 cells and apoptosis suppress T-cell functions in multiple myeloma.

The aim of this study was to evaluate the role of CD8+/CD57+ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8+/CD57+ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR+ and Fas+. Removal of CD8+/CD57+ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8+/CD57+ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8+/CD57+ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8+/CD57+ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8+/CD57+ population. Our results indicate that both the immunosuppressive effect of CD8+/CD57+ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.

RNA-unwinding and RNA-folding activities of RNA helicase II/Gu--two activities in separate domains of the same protein.

The human RNA helicase II/Gu protein (RH-II/Gu) is a member of the D-E-A-D box protein family. It is a unique enzyme, which possesses an ATP-dependent RNA-unwinding activity and has an RNA-folding activity that introduces an intramolecular secondary structure in single-stranded RNA. This report shows that these two enzymatic activities are distinct. ATP[S], GTP and low concentrations of ATP enhance the RNA-folding activity of RH-II/Gu but not the RNA-helicase activity. High concentrations of ATP are required for the helicase activity but are inhibitory to the RNA-folding activity. Mg2+ is required for the helicase activity but not for the RNA-folding reaction. Affinity-purified anti-(RH-II/Gu) polyclonal Ig inhibit the RNA-unwinding activity but not the folding activity. Mutations of the DEVD sequence, which corresponds to the DEAD box, and the SAT motif enhanced RNA-folding activity of RH-II/Gu but completely inhibited the RNA-helicase activity. A mutant that lacks the COOH-terminal 76 amino acid residues, including the four FRGQR repeats, had unwinding activity but did not catalyze the folding of a single-stranded RNA. The two enzymatic activities of RH-II/Gu reside in distinct domains. Amino acids 1-650 are active in the RNA-unwinding reaction but lack RNA-folding activity. Amino acids 646-801 fold single-stranded RNA but lack helicase activity. This report shows distinct RNA-unwinding and RNA-folding activities residing in separate domains within the same protein.

Distinguishable patterns of connectivity in serum immunoglobulins from SLE patients and healthy individuals.

Given that normal individuals maintain significant levels of serum autoantibodies that share many characteristics with those found in association with autoimmune diseases (AID), it has been proposed that disease could result from defects in supraclonal regulation, namely deviations of normal patterns of immunoglobulin (Ig) connectivity. Using conventional methods, together with a recently developed technique to quantitatively score a variety of V-region-dependent serum IgG interactions, the authors have now compared serum Ig connectivity in a group of patients with systemic lupus erythematosus (SLE) to healthy controls. The results demonstrate the existence of V-region interactions of serum IgG and IgM in SLE patients and healthy donors, with comparable connectivity titres, diversity and average affinities (microM range), but a wider individual variation and a tendency for higher F(ab')2 directed reactivities in the group of SLE patients. Multivariate statistics analysis of the data derived from reactivity patterns on F(ab')2 subsets, however, distinguished the two groups of donors, and demonstrated a larger dispersion and wider time-dependent variations in the patient population, as compared to healthy controls. The authors conclude that SLE is associated with circulating antibody repertoires that deviate from the patterns and levels of V-region connectivity characteristic of healthy individuals. These findings may shed light on the mechanisms of disease maintenance, and on the basis for the therapeutic effects of normal polyclonal Igs at high doses.

Polyphenols in Chocolate, Which Have Antioxidant Activity, Modulate Immune Functions in Humansin Vitro

We studied the effects of antioxidants from chocolate, cacao liquor polyphenol (CLP), on human immune functionsin vitro.CLP is an enriched polyphenol fraction purified from cacao liquor that is a major component of chocolate. It has been shown that polyphenols have antioxidant activity, and reactive oxygen species (ROS) are involved in immune responses. CLP inhibited both hydrogen peroxide and superoxide anion, typical ROS, production by phorbol myristate acetate-activated granulocytes. CLP also inhibited menadione-induced production of both hydrogen peroxide and superoxide anion in normal human peripheral blood lymphocytes (PBL). CLP treatment of normal PBLin vitroinhibited mitogen-induced proliferation of T cells and polyclonal Ig production by B cells in a dose-dependent manner. CLP treatment inhibited both IL-2 mRNA expression of and IL-2 secretion by T cells. These results suggest that antioxidant CLP has immunoregulatory effects.

Synergy between anti-CD40 MAb and Epstein-Barr virus in activation and transformation of human B lymphocytes

For human B lymphocytes, Epstein-Barr virus (EBV) is a polyclonal activator, inducing both proliferation and Ig secretion. It is also a transforming virus capable of generating immortalized B cell lines. These early and late functions of EBV are not apparently connected. The receptor for EBV, CD21, also serves as a receptor for some complement components and is called CR2. This molecule associates with CD19 and TAPA-1 on the surface of B cells. This complex is involved in signaling B cells and participates in many responses. We have observed that simultaneous ligation of CD40 and the CD21 complex, by exposure to anti-CD40 MAbs and EBV, enhances both the short-term proliferation as well as the long-term transformation rate of human B lymphocytes. B cell proliferation shows synergy between anti-CD40 MAb and EBV. CD19 also appears to be involved in the synergistic activation of B cells through CD40 and CD21, since ligation of CD19 with anti-CD19 MAbs, either prior to or concomitant with exposure to anti-CD40 and EBV, markedly inhibits both proliferation and subsequent B cell transformation. These observations do not elucidate the mechanisms of B cell transformation employed by EBV but the do suggest a relationship between early proliferation and later transformation induced by the virus. Anti-CD40 enhances both these effects and anti-CD19 is capable of inhibiting both.

Humoral immunoglobulins of the white sturgeon, Acipenser transmontanus: Partial characterization of and recognition with monoclonal antibodies

White sturgeon ( Acipenser transmontanus) immunoglobulin (Ig) was purified from serum by two methods, ion-exchange chromatography and gel filtration and precipitation of the euglobulin fraction. The purity of these immunoglobulin preparations was confirmed by gel electrophoresis. Sequence analysis of the N-terminal amino acids confirmed that the purified protein was immunoglobulin. The major portion of the immunoglobulin preparation consisted of two proteins with estimated molecular weights (m.w.) of 870 and 170 kDa. The m.w. of the H- and L-chains of the purified Ig were 73 and 27–30 kDa, respectively, as determined by SDS-PAGE. Ion-exchange purified Ig was used to immunize mice for the production of monoclonal antibodies. This resulted in the production of six stable hybrids that recognized sturgeon Ig, two specific for heavy chain and four specific for light chain. The two anti-H-chain mabs were highly specific for white sturgeon Ig while all four anti-L-chain mabs cross reacted with Ig from green sturgeon ( A. medirostris), Atlantic sturgeon ( A. oxyrhynchus oxyrhynchus), shovelnose sturgeon ( Scaphirhynchus platorynchus), and paddlefish ( Polyodon spathula), (all Chondrosteans), but not with channel catfish ( Ictalurus punctatus), rainbow trout ( Oncorhynchus mykiss) or striped bass ( Morone saxatilis). The mabs were used to enumerate the percentage of sIg + lymphocytes in the peripheral blood of white sturgeon by flow cytometry. The percentage of cells positively stained with the mabs ranged from 12 to 28%. In a comparison of mabs with polyclonal rabbit anti-sturgeon Ig serum by ELISA the mabs produced a larger signal and less background than the polyclonal serum.

Detection of Bence-Jones protein in serum by immunoblotting.

To detect Bence-Jones protein (BJP) in serum we precipitated intact immunoglobulins (Ig) using polyethylene glycol (PEG) and subjected the BJP in solution to electrophoresis in agarose gel, followed by transfer to a polyvinylidene difluoride membrane, and immunoenzymatic staining (successively using rabbit anti-human light/heavy chain of Ig, biotinylated swine anti-rabbit Ig, and alkaline phosphatase-conjugated streptavidin). Treatment with PEG effectively reduced background staining of polyclonal Ig in the immunoblots, although intact monoclonal Ig was not always completely removed. To compare the present method with immunoelectrophoresis (IEP), we selected samples from patients demonstrating BJP by IEP in both serum and urine (n = 40), serum only (n = 18), and urine only (n = 32); 21 of these patients had BJP alone and 69 had BJP in addition to intact monoclonal Ig. Efficiency of detection of BJP in serum was increased by the present method: serum BJP was detected in 70 patients by the present method versus 58 by IEP. The present method demonstrated single BJP bands in the samples from 16 patients (kappa, n = 7; lambda, n = 9) and multiple BJP bands (range: 2-9) in the samples from 54 patients (kappa, n = 31; lambda, n = 23). This method could be useful for detecting BJP in serum from patients suspected of having light chain gammopathy (without the need for urine testing) and may complement urine testing in patients excreting polyclonal free light chains of Ig.

Role of Different Hematologic Variables in Defining the Risk of Malignant Transformation in Monoclonal Gammopathy

The presenting clinico-hematologic features of 386 patients with nonmyelomatous monoclonal gammopathy (MG) were correlated with the frequency of malignant transformation to evaluate the most important variables conditioning its evolution into multiple myeloma (MM) or Waldenström macroglobulinemia (WM). Most of the patients (335) had monoclonal gammopathy of undetermined significance (MGUS: 39 IgA, 242 IgG, 54 IgM): the remaining 51 patients (12 IgA, 39 IgG) fulfilled all of the MGUS diagnostic criteria (according to Durie) except that bone marrow plasma cell (BMPC) content was 10% to 30%, and so they were defined as having monoclonal gammopathy of borderline significance (MGBS). There were no significant differences between the MGUS and MGBS groups in terms of age, sex, or median follow-up. After a median follow-up of 70 and 53 months, respectively, 23 of 335 MGUS and 19 of 51 MGBS patients had undergone a malignant evolution. Univariate analysis of the IgA and IgG patients showed that the cumulative probability of the disease evolving into MM correlated with diagnostic definition (MGBS v MGUS), BMPC content (> or = 10% v < 5% and < or = 5% v > 5%) and reduced serum polyclonal Ig. In the IgG cases, there was also a significant correlation with detectable Bence Jones proteinuria, serum monoclonal component (MC) levels and age at diagnosis (> 70 v < = or 55 years). In the IgG cases as a whole, the same variables remained in the Cox model where the BMPC percentage was considered after natural logarithmic transformation and the monoclonal component as g/dL value. The relative risks of developing MM are the following: 2.4 for each 1 g/dL increase of IgG, serum MC, 3.5 for detectable light chain proteinuria, 4.4 for the increase of 1 unit in log. BMPC percentage, 6.1 for age > 70, 3.6 and 13.1 for a reduction in one or two polyclonal Ig. In conclusion, our study allows the identification of a particular subset of MGUS patients (MC < = or 1.5 g/dL, BMPC < 5%, no reduction in polyclonal Ig and no detectable light chain proteinuria) at very low-risk of evolution, who can be considered as having benign monoclonal gammopathies. We also describe a previously undefined group of MG patients (with monoclonal gammopathy of borderline significance) who are at high-risk of malignant evolution. These findings could have a considerable impact on the cost/benefit ratio of monitoring programs in these patients.

B-lineage cell deficits in bone marrow of lpr/lpr mice.

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygous for the lpr mutation (B6.lpr) disclosed low numbers of pre-B and B cells, as compared with age-matched control B6 mice. BM depletion in B6.lpr mice was selective for B-lineage cells, appeared in young adults, and developed markedly with age and disease progression, contrasting with the peripheral lymphocyte hypercellularity. Normalization of pre-B and B cellularity in BM of B6.lpr mice was observed after administration of polyclonal Ig, that also markedly improved the clinical condition. Isolated pre-B (B220+ IgM-) cells from B6 or B6.lpr mice, however, showed essentially the same rates of IL-7-dependent proliferation and differentiation to B (IgM+) cells in culture, indicating that the BM B-lineage deficit is not the result of an intrinsic defect in B cell generation.

Human recombinant CD4 and CD4-derived synthetic peptides agglutinate immunoglobulin-coated latex particles. Evidence that residues 25–28 and 35–38 of human CD4 form two separate immunoglobulin binding sites

It has been shown previously that amino acid residues 21–49 of the first extracellular domain of human CD4 form the core of an immunoglobulin (Ig) binding site. Synthetic peptides of human CD4 that encompass this region also bind Ig and, with higher affinity, antigen/antibody complexes. Synthetic peptides also enhance binding of both monomeric and aggregated Ig to monocytic U937 cells and Staphylococcus aureus Protein A. To better characterize the nature of the Ig binding site on CD4, we tested the ability of human recombinant CD4 (rCD4) to agglutinate polystyrene particles coated with Ig. Evidence is presented that soluble rCD4 and CD4 peptide (p)21–49 were capable of specific agglutination of polystyrene particles coated with polyclonal Ig of either human or sheep origin. Agglutination could be blocked by soluble human polyclonal IgG or F(ab′) 2 fragments. Both heparin and sulfated dextrans also inhibit agglutination, suggesting that charged residues on rCD4 played an important role in agglutination mediated by rCD4 or CD4 peptide. Similarly, aurintricarboxylic acid (ATA) also blocked agglutination of Ig-coated particles by rCD4. Agglutination mapping studies performed using truncated peptides revealed the existence of two discrete, closely related Ig binding sites (residues 25–28 and 35–38).

An antigen detection immunoassay for big liver and spleen disease agent

An enzyme-linked immunosorbent assay (ELISA) able to detect an antigen associated with infection with big liver and spleen (BLS) agent was developed. The assay is a capture ELISA (C-ELISA) procedure using ELISA plates coated with mouse monoclonal antibody (mAb 1H 4) for antigen capture and a polyclonal chicken Ig preparation, extracted from yolk of BLS antibody positive birds, for detection of captured BLS antigen. mAb 1H 4 was produced to a partially purified antigen preparation from liver suspensions of naturally BLS infected birds. The C-ELISA was evaluated for specificity and sensitivity in naturally and experimentally BLS infected and uninfected broiler and layer breeds of domestic fowl.

Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis c virus antigens detected by using immunopurified polyclonal spontaneous human antibodies

Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather then experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies.

Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis C virus antigens detected by using immunopurified polyclonal spontaneous human antibodies

Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather than experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies.