Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioMérieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

Transplantation in Highly Sensitised Patients Treated with Intravenous Immunoglobulin and Rituximab

Renal transplant in highly sensitised patients is associated with increased morbidity. The aim of this retrospective study was to evaluate the clinical evolution of 30 highly sensitised deceased donor kidney transplants and the influence of different timing of B cell directed treatment and its importance in the outcome of these patients. All recipients had negative complement dependent lymphocytotoxicity cytotoxic T cell crossmatch and no identified anti human leucocyte antigen class I donor specific antibodies. T cell flow crossmatch was performed within 24h of transplantation with serum obtained pretransplant (historic, recent or baseline). Posttransplant flow crossmatch were performed prospectively starting on the 3rd posttransplantation day. The immunosuppressive regime included thymoglobulin, tacrolimus, mycofenolate mofetil and steroids. Positive flow crossmatch occurred in 20/29 patients by the 3rd posttransplantation day, and in 17/27 patients after the 3rd posttransplantation day. All patients were started on intravenous immunoglobulin before transplantation: in nine patients (group A) at 400mg/kg/day for five days; in the remaining 21 patients (group B), as a continued infusion of 2g/kg during 48h. In group A, Rituximab was added only in the presence of antibody mediated rejection; in group B, introduced on the 3rd posttransplantation day whenever a positive flow crossmatch (with serum obtained pre or posttransplant) was reported. Antibody mediated rejection was observed in 44.4% of patients in group A, and 19% of those in group B. Mean follow-up was 12.2±5.5 months. Overall allograft survival was 76.6%, 81% in group B, and 66.6% in group A. At last follow up, mean serum creatinine was 1.3±0.6 mg/dl. Renal transplantation with pretransplant positive flow crossmatch is highly associated with antibody mediated rejection, despite introduction of intravenous immunoglobulin pretransplantation. However high dose intravenous immunoglobulin for 48h plus Rituximab by the 3rd posttransplantation day reduce the incidence of antibody mediated rejection by more than 50% and allowed for allograft survival of 81% at one year, with an excellent renal function.

PROTECTIVE LEVELS OF VARICELLA-ZOSTER ANTIBODY DID NOT EFFECTIVELY PREVENT CHICKENPOX IN AN X-LINKED AGAMMAGLOBULINEMIA PATIENT

SUMMARY We describe the case of an eight-year-old boy with X-linked agammaglobulinemia who developed mild varicella despite regular intravenous immunoglobulin (IVIG) therapy. He maintained protective antibody levels against varicella and the previous batches of IVIG that he received had adequate varicella-specific IgG levels. The case illustrates that IVIG may not prevent VZV infection.

Quantitative Analysis of Immunoglobulin E Reactivity Profiles in Patients Allergic or Sensitized to Natural Rubber Latex (Hevea Brasiliensis)

Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy

Diagnosis of rubella infection by detecting specific immunoglobulin M antibodies in saliva samples: a clinic-based study in Niterói, RJ, Brazil

This study was designed to investigate whether saliva could be a feasible alternative to serum for the diagnosis of recent rubella infection in a clinic setting. Forty-five paired blood and saliva samples collected 1 to 29 days after onset of illness were tested for specific immunoglobulin (Ig) M by antibody-capture radioimmunoassay (MACRIA). Rubella IgM was detected in all serum samples and in 38 (84.4%) saliva specimens. Forty-six serum and saliva samples from other patients with rash diseases were tested by MACRIA for control purposes and two saliva specimens were reactive. The saliva test had specificity of 96%. These results indicate that salivary IgM detection may be a convenient non-invasive alternative to serum for investigation of recent rubella cases, especially for disease surveillance and control programmes.

The secretory immunoglobulin A response to Mycobacterium tuberculosis in a childhood population

We report on the measurement of saliva anti-Purified Protein Derivative sIgA and 38kDa antibodies from 127 children, of whom 31 were strong tuberculosis suspects and 96 were healthy contact children. The results concerning the percentage of children with antibody reactivity to PPD and 38kDa antigens showed that, of these 2 antigens, 38kDa induced higher reactivity in patients positive and negative for the Tuberculin Skin Test (28% and 16.6%, respectively) in comparison to controls positive and negative for the TST (11.7% and 7.1%, respectively). There was a statistically significant difference between patients positive and controls negative for the TST. In relation to the Purified Protein Derivative antigen, while 14.2% of patients positive for the TST showed antibody reactivity to the PPD antigen, no patients negative for the TST had reactivity to this antigen. The findings suggest that these two antigens seem be associated with a different development of the mucosal defence mechanisms mediated by sIgA against Mycobacterium tuberculosis.

Human serum antibody reactivity towards Paracoccidioides brasiliensis antigens treated with sodium metaperiodate

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

Lagochilascaris minor: antibody production in experimentally infected mice

Lagochilascaris minor is the causative agent of lagochilascariosis, a disease that affects the neck region and causes festering abscesses, with eggs, adult parasites and L3/L4 larvae within the purulent exudates. Today, mice are considered to be intermediate hosts for the parasite. C57BL/6 mice produce immunoglobulin IgM, IgA and IgG against the crude extract of the parasite; on the other hand, antibodies produced against the secreted/excreted antigens of Lagochilascaris minor present lower levels of IgM, IgA and IgG. This is the first description of antibody detection against different antigens of Lagochilascaris minor.

Interpretation of the presence of IgM and IgG antibodies in a rapid test for dengue: analysis of dengue antibody prevalence in Fortaleza City in the 20th year of the epidemic

INTRODUCTION: The diagnosis of dengue and the differentiation between primary and secondary infections are important for monitoring the spread of the epidemic and identifying the risk of severe forms of the disease. The detection of immunoglobulin (Ig)M and IgG antibodies is the main technique for the laboratory diagnosis of dengue. The present study assessed the application of a rapid test for dengue concerning detection of new cases, reinfection recognition, and estimation of the epidemic attack rate. METHODS: This was a retrospective, cross-sectional, descriptive study on dengue using the Fortaleza Health Municipal Department database. The results from 1,530 tested samples, from 2005-2006, were compared with data from epidemiological studies of dengue outbreaks in 1996, 2003, and 2010. RESULTS: The rapid test confirmed 52% recent infections in the tested patients with clinical suspicion of dengue: 40% detected using IgM and 12% of new cases using IgG in the non-reactive IgM results. The positive IgM plus negative IgG (IgM+ plus IgG-) results showed that 38% of those patients had a recent primary dengue infection, while the positive IgG plus either positive or negative IgM (IgG+ plus IgM+/-) results indicated that 62% had dengue for at least a second time (recent secondary infections). This proportion of reinfections permitted us to estimate the attack rate as >62% of the population sample. CONCLUSIONS: The rapid test for dengue has enhanced our ability to detect new infections and to characterize them into primary and secondary infections, permitting the estimation of the minimal attack rate for a population during an outbreak.

Immunoglobulin E-rheumatoid factor in juvenile rheumatoid arthritis

OBJECTIVES: To determine the presence of immunoglobulin E-rheumatoid factor in patients with juvenile rheumatoid arthritis and to correlate it with clinical and laboratory parameters. METHODS: A multicenter prospective study was carried out from January 1993 to January 1999 with the enrollment of 3 centers of pediatric rheumatology. Ninety-one children with juvenile rheumatoid arthritis diagnosed according to the American College of Rheumatology criteria were studied: 38 (42%) with systemic, 28 (31%) with pauciarticular, and 25 (27%) with polyarticular onset. Ages ranged from 2.1 years to 22.6 years (mean 10.5 ± 4.7), with 59 (65%) girls. The control group consisted of 45 healthy children. The detection of immunoglobulin E-rheumatoid factor was carried out utilizing an enzyme-linked immunosorbent assay. Associations of immunoglobulin E-rheumatoid factor with immunoglobulin M-rheumatoid factor (latex agglutination test), total serum immunoglobulin E, erythrocyte sedimentation rate, antinuclear antibody, and functional and radiological classes III or IV were analyzed. RESULTS: Positive immunoglobulin E-rheumatoid factor was found in 15 (16.5%) of the 91 children with juvenile rheumatoid arthritis: 7 (18.5%) with systemic, 5 (18%) with pauciarticular, and 3 (12%) with polyarticular onset. A significant correlation was observed between immunoglobulin E-rheumatoid factor and total serum immunoglobulin E in the juvenile rheumatoid arthritis patients. No correlation was found between immunoglobulin E-rheumatoid factor and positive latex agglutination slide test, erythrocyte sedimentation rate, antinuclear antibody, or the functional and radiological classes III or IV in any disease onset group. In 4 out of 45 control children (8.9%), immunoglobulin E-rheumatoid factor was positive but with no correlation with total serum immunoglobulin E levels. CONCLUSIONS: Immunoglobulin E-rheumatoid factor could be detected in 16.5% of juvenile rheumatoid arthritis patients, particularly in those with high levels of total serum immunoglobulin E, and immunoglobulin E-rheumatoid factor appears not to be associated with disease activity or severity.

Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37ºC, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.

Role of secretory immunoglobulin A and secretory component in the protection of mucosal surfaces.

The contribution of secretory immunoglobulin A (SIgA) antibodies in the defense of mucosal epithelia plays an important role in preventing pathogen adhesion to host cells, therefore blocking dissemination and further infection. This mechanism, referred to as immune exclusion, represents the dominant mode of action of the antibody. However, SIgA antibodies combine multiple facets, which together confer properties extending from intracellular and serosal neutralization of antigens, activation of non-inflammatory pathways and homeostatic control of the endogenous microbiota. The sum of these features suggests that future opportunities for translational application from research-based knowledge to clinics include the mucosal delivery of bioactive antibodies capable of preserving immunoreactivity in the lung, gastrointestinal tract, the genito-urinary tract for the treatment of infections. This article covers topics dealing with the structure of SIgA, the dissection of its mode of action in epithelia lining different mucosal surfaces and its potential in immunotherapy against infectious pathogens.

Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Immunoglobulin classes in aquatic mammals. Characterization by serologic cross-reactivity, molecular size and binding of human free secretory component.

On the basis of serologic cross-reactivity, three immunoglobulin classes homologous to human IgG, IgM and IgA were identified in two species of acquatic mammal representing the orders Cetacea (dolphin) and Pinnipedea (sea lion). Molecular size was estimated by sucrose density gradient ultracentrifugation and Sephadex G-200 chromatography, indicating a 7S IgG, 19S IgM and heterogeneous serum IgA. Human secretory component was readily bound to the IgM of both species and to an apparently lesser extent to the larger molecular size populations of IgA. No binding was observed with IgG. Several antisera specific for human γ-chains gave a single precipitin line with the sea lion IgG but when made to react with dolphin serum produced two lines, suggesting the presence of two different subclasses of IgG in this species.

Generation of a recombinant mouse-human chimaeric monoclonal antibody directed against human carcinoembryonic antigen.

A procedure was devised for the identification and specific cloning of functionally rearranged variable region immunoglobulin (Ig) gene segments from genomic DNA of a murine hybridoma cell line which produces a high-affinity monoclonal antibody (MAb) directed against human carcinoembryonic antigen (CEA). The cloned, functionally-rearranged murine Ig H-chain and L-chain variable region gene segments were incorporated into plasmid vectors capable of directing the expression of a chimaeric mouse-human antibody molecule with human (gamma 4, kappa) constant region sequences. Expression plasmids were transfected into a mouse myeloma cell line by electroporation and transfectomas secreting functional chimaeric antibody selected. Chimaeric antibody generated by transfectomas was analysed and shown to compete effectively with its murine counterpart for binding to the CEA epitope, and to have an equivalent antigen-binding affinity. This anti-CEA recombinant antibody should find application in in vivo diagnosis by immunoscintigraphy of human colonic carcinoma, and possibly also in therapy of the disease, overcoming some of the difficulties associated with the repeated use of non-human immunoglobulins in human patients.