Anticuerpos anti-estreptavidina: Confusión diagnóstica por interferencia bioquímica / Anti-estreptavidin antibodies. Diagnostic confusion by biochemical interference

Se presenta el caso de una paciente que, durante los estudios por búsqueda de fertilidad y posterior embarazo, mostraba un perfil tiroideo alterado con niveles elevados de T4 libre y TSH normal. Luego de descartar un adenoma tirotropo y ante la ausencia de sintomatología clínica de hipertiroidismo, se investigó la posibilidad de interferencias analíticas en los inmunoensayos utilizados para la medición de las hormonas. Se han descrito interferencias causadas por anticuerpos heterófilos, macro TSH, anticuerpos anti-tiroideos, biotina, y en menor medida anticuerpos anti-estreptavidina y anti-rutenio. Los análisis de la paciente se realizaron en autoanalizador cuya plataforma emplea el sistema estreptavidina-biotina que es muy susceptible a varios interferentes. Un algoritmo propuesto incluye una serie de pruebas simples de realizar e interpretar que permiten detectar o descartar la presencia de interferentes. De acuerdo al mismo, se efectuó la comparación con una plataforma analítica diferente (que no utiliza el sistema estreptavidina-biotina), diluciones seriadas, precipitación con polietilenglicol 6000 y tratamiento con micropartículas recubiertas con estreptavidina. Los resultados obtenidos confirmaron la presencia de anticuerpos anti-estreptavidina en el suero de la paciente. Ante discordancias entre las manifestaciones clínicas y los resultados de laboratorio, se debe investigar la posibilidad de interferencias metodológicas para evitar el riesgo iatrogénico potencial que implica una interpretación bioquímica errónea.

We present the case of a patient who, during studies for fertility and subsequent pregnancy, showed an altered thyroid profile with elevated levels of free T4 and normal TSH. After ruling out a thyrotropic adenoma and in the absence of clinical symptoms of hyperthyroidism, the possibility of analytical interference in the immunoassays used to measure hormones was investigated. Interferences caused by heterophile antibodies, macro TSH, anti-thyroid antibodies, biotin, and to a lesser extent anti-streptavidin and anti-ruthenium antibodies have been described. The analysis of the patient was carried out in a self-analyzer whose platform uses the streptavidin-biotin system that is very susceptible to several interferents. A proposed algorithm includes a series of simple tests to perform and interpret that allow detecting or ruling out the presence of interferents. Accordingly, a comparison was made with a different analytical platform (which does not use the streptavidin-biotin system), serial dilutions, precipitation with polyethylene glycol 6000 and treatment with microparticles coated with streptavidin. Results obtained confirmed the presence of anti-streptavidin antibodies in the patient's serum. In the case of disagreements between clinical manifestations and laboratory results, the possibility of methodological interferences should be investigated in order to avoid the potential iatrogenic risk involved in an erroneous biochemical interpretation.

Leprosy: prevalence and factors associated with seropositivity for anti-NDO-LID antibodies in children under 15 years of age

Abstract: Background: A high prevalence of leprosy among children under 15 years of age indicates the need to implement actions to prevent new cases of the disease. Serological tests have been developed with the aim of helping to control the disease by indicating, through seropositivity, the presence of infection. Objective: To analyze the prevalence and factors associated with seropositivity rate for anti-NDO-LID antibodies in children under 15 years of age, contacts of leprosy patients. Method: We performed a cross-sectional study with 210 children under 15 years old of age. Of them, 50 were household contacts and 160 were neighborhood contacts living in the municipality of Cuiabá, state of Mato Grosso, in 2016. The data were obtained from interviews and the NDO-LID rapid test during home visits from February to July 2016. For the analysis, we used Poisson regression and prevalence ratio. Results: Seropositivity in contacts was 6.2%. Variables associated with seropositive tests included sex (PR = 1.05; 95% CI: 1.01 - 1.08), race/skin color (PR = 0.95; 95% CI: 0.90 - 0.99), residence area (PR = 1.05; 95% CI: 1.01 - 1.09), and number of people per household (PR = 1.06; 95% CI: 1.02 - 1.08). Study Limitations: The small sample size, besides leading to wide confidence intervals, may have been a limitation for the identification of associated factors. Conclusions: The prevalence of seropositivity was high. Variables associated with NDO-LID seropositivity included female sex, not to be brown skinned, live in urban areas, and live with five or more people.

Frecuencia de anticuerpos anti-transglutaminasa y antiendomisio en pacientes con diabetes tipo 1 / Association of type 1 diabetes mellitus with celiac disease

Background: Type 1 diabetes mellitus and celiac disease share common genetic and immunological aspects and celiac disease is more common among type 1 diabetic patients. Aim: To determine the frequency of anti endomysial and anti transglutaminase antibodies among patients with type 1 diabetes. Material and Methods: Anti endomysialantibodies determined by indirect immunofluorescence an anti transglutaminase antibodies determined by ELISA were measured in 410 serum samples of patients with type 1 diabetes. Results: Seventy one samples (17 percent) had positive anti transglutaminase antibodies. Among these, 17 had also positive anti endomysial antibodies. In 11 of these 17 patients, the presence of celiac disease was confirmed. Conclusions: Among patients with type 1 diabetes mellitus, the frequency of celiac disease is three times higher than in the general population.

Triagem sorológica para doença celíaca em adolescentes / Serologic screening of celiac disease in adolescents

OBJETIVO: Este estudo objetivou identificar a soroprevalência da doença celíaca em adolescentes de escolas públicas da cidade de Salvador, Bahia. MÉTODO: Trata-se de um estudo transversal com amostra probabilística de 1.213 adolescentes de 11 a 17 anos, de ambos os sexos. O índice de massa corporal foi utilizado para o diagnóstico do estado nutricional, adotando-se os percentis segundo idade e sexo, propostos pela World Health Organization. O anticorpo anti-transglutaminase humana da classe imunoglobulina A (anti-tTG-IgA) foi adotado como teste sorológico para triagem da doença celíaca e foi determinado pela técnica do ensaio imunoabsorvente ligado à enzima (ELISA). Foi realizada análise descritiva, utilizando-se a proporção e a média (desvio padrão). RESULTADOS: O sexo feminino predominou entre os adolescentes, e a maioria encontrava-se com adequado estado nutricional. O anticorpo anti-tTG-IgA foi positivo em 6/1.213 (0,49%) adolescentes. CONCLUSÃO: A soroprevalência de doença celíaca entre os adolescentes estudados foi 0,49%. Novas investigações são necessárias para confirmar a prevalência de doença celíaca nessa faixa etária. .

OBJECTIVE: This study aimed to identify the seroprevalence of celiac disease in adolescents from public schools in the city of Salvador, Bahia. METHODS: This was a cross-sectional study with probabilistic sample of 1,213 adolescents, aged 11 to 17 years old, of both genders. The body mass index was used to determine the participants' nutritional status based on the percentiles for age and gender recommended by the World Health Organization. Measurement of the anti-human transglutaminase immunoglobulin A (anti-tTG-IgA) antibody was established as the specific screening test for celiac disease, which involved an enzyme-linked immunosorbent assay (ELISA). Descriptive analysis was performed using proportions and means (standard deviation). RESULTS: The female gender prevailed in the sample, and most of the participants had normal weights. The anti-tTG-IgA antibody was positive in 6/1,213 (0.49%) adolescents. CONCLUSION: The seroprevalence of celiac disease was 0.49% in the investigated adolescents. Further studies are necessary to establish the prevalence of celiac disease in this age range. .

An Immunoglobulin G4-Related Sclerosing Disease of the Small Bowel: CT and Small Bowel Series Findings

Immunoglobulin G4 (IgG4)-related sclerosing disease is rare and is known to involve various organs. We present a case of histologically proven IgG4-related sclerosing disease of the small bowel with imaging findings on computed tomography (CT) and small bowel series. CT showed irregular wall thickening, loss of mural stratification and aneurysmal dilatation of the distal ileum. Small bowel series showed aneurysmal dilatations, interloop adhesion with traction and abrupt angulation.

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.

A pilot study on parvovirus B19 infection in paediatric haematological malignancies.

Background & objectives: Leukaemia and lymphoma are common paediatric haematological malignancies acquiring human parvovirus B19 (B19) infection. In some studies anaemia has been found in children with acute lymphoblastic leukaemia (ALL) during maintenance therapy and rarely in lymphoma. We studied frequency of B19 infection and its implications in new onset acute leukaemia (mostly ALL) and lymphoma in children. Methods: Seventy serum samples from 35 children (age <12 yr, 29 males) newly diagnosed with haematological malignancies (on induction therapy) were collected together with 34 controls (solid tumours). Children were examined clinically and for anti-B19 IgM antibodies by quantitative ELISA and B19 DNA by PCR (VP1-VP2) and nested-PCR (VP1 unique). Bone marrow aspirates were examined histopathologically, whenever possible. Results: Of the 35 children, 22 had acute leukaemia while 13 had lymphoma. B19 infection was seen in six (17.1%) of 35 children (5 ALL, 1 NHL), two at diagnosis and four during follow up compared to none in the control. Among five B19 IgM positive ALL (n=18) children, two had B19 genome and two had giant pronormoblasts (lantern cells; but one lacked B19 DNA). Of the 70 serum samples tested, eight (11.4%) had anti-B19 IgM as two children had persistent B19 infection and one showed atypical maculopapular rashes (lower limbs) while 12 (34.3%) had anti-B19 IgG antibodies. B19 infected children had unexplained anaemia (80%), required more blood transfusions (6.6 ± 4.8 Units vs 3.0 ± 2.6 Units) besides induction chemotherapy was delayed (60%) and required longer duration of therapy (29.2 ± 20 vs 6.3 ± 7.8 days) (P<0.02). Five children (2 ALL, 2 AML, 1 NHL) died but none were infected with B19. Interpretation & conclusions: B19 infection should be considered in children with ALL as it frequently caused unexplained anaemia and delay in induction chemotherapy.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

Retinitis pigmentosa and congenital toxoplasmosis: a rare coexistence.

We describe a previously unreported co-existence of retinitis pigmentosa and congenital toxoplasmosis. An eight year old male presented to our center with complaints of decreased night vision. Fundus evaluations in both the eyes demonstrated features typical of retinitis pigmentosa. There were well-defined punched out healed chorio-retinal scars suggestive of congenital toxoplasmosis. On the basis of history, clinical findings and reduction of a and b wave amplitudes on scotopic and photopic electroretinograph, a diagnosis of retinitis pigmentosa with congenital toxoplasmosis was made. Retinitis pigmentosa may co-exist with congenital toxoplasmosis that may affect the patient's overall ocular morbidity and visual acuity.

Anti-myeloperoxidase antibodies in patients with microscopic polyangiitis.

BACKGROUND & OBJECTIVES: Anti-neutrophil cytoplasmic antibodies in active necrotizing and crescentic glomerulonephritis are associated with systemic vasculitides like Wegener's granulomatosis, Microscopic polyangitiis and Churg Strauss Syndrome. This study shows the incidence of ANCA with specificities to Myeloperoxidase and Proteinase3 in MPA cases and gives the correlation of ANCA with Birmingham Vasculitis Activity Score. MATERIAL & METHODS: Eighteen cases of MPA were diagnosed as per Chapel Hill Consensus Criteria. ANCA was detected by indirect immunofluorescence microscopy using fluorescence and Confocal Laser Scanning Microscopes. Anti-MPO and anti-PR3 were identified by commercial ELISAs and anti-MPO subclass and IgG isotypes were also detected. RESULTS: MPA patients showed a male preponderance with BVAS ranging from 17-30. Systemic involvement was seen in 88.9%, lower respiratory tract involvement in 77.8% and upper respiratory tract in only 33.3% cases. All these patients had perinuclear pattern on IIF, where titers ranged from 80-640 and ELISA showed anti-MPO; values ranging from 20-80 units/ml. IIF and ELISA showed a good correlation (r=0.77). Two patients having FPGN had dual specificities and had both anti-MPO and anti-PR3 which could be picked up only by ELISA. A good correlation (r=0.78) was observed between BVAS and ANCA levels as well. IgG ANCA was detected in 88.7% and 11.1% had IgG+IgM and IgG1+IgG4 ANCA was detected in 50% patients. CONCLUSION: p-ANCA with anti-MPO is highly specific for MPA; both IIF and ELISA should be carried out for true positivity and to identify rare cases of dual specificities. Confocal laser scanning microscopy is useful in identifying ANCA patterns especially when ANA is also positive. ANCA testing with BVAS assessment will surely help in early diagnosis and estimating the severity of this life threatening disease.

Seroprevalence of cytomegalovirus infection in children born to HIV-1 infected women.

Cytomegalovirus (CMV) is a frequent opportunistic infectious agent in children infected with human immunodeficiency virus type 1 (HIV-1). It has been implicated as a factor in the progression of HIV-1 disease. The aim of the present study was to evaluate the prevalence of CMV infection in Thai children born to HIV-1 infected women. The prevalence of CMV infection was 13, 89 and 84% in HIV-infected children and 9, 61 and 75% in HIV uninfected at age ranges of 0-12, 13-36 and 37-79 months, respectively. The prevalence of CMV infection was significantly different between HIV infected children (89%) and HIV uninfected (61%) at the age of 13-36 months (p < 0.05). The presence of CMV IgM in some children of age < 1 year suggested that CMV infection could occur early in life. Early co-infection may be important as they remain a risk factor for reactivation of latent CMV infection throughout the course of the HIV diseases. Clinical monitoring and appropriate work up may be of benefit in the early diagnosis and treatment of CMV disease.

Lymphoproliferative responses of splenocytes before and after challenge with schistosoma haematobium in C57BL/6 mice vaccinated with human anti-idiotypes

Anti-idiotypic vaccines [anti-Id or antibody 2; Ab2] in experimental schistosomiasis engender varying degrees of resistance to challenge infection. To further characterize the mechanisms involved in the induction of protective immunity associated with such a vaccine model, spleen cells of mice vaccinated with human Ab2 [HAb2] were investigated for their lymphoproliferative responses before and after challenge infection with normal S. haematobium cercariae. HAb2 was purified from sera of chronically infected patients using protective rabbit antibodies [RAb1] isolated from sera of rabbits multiply immunized with UV-irradiated cercariae by affinity chromatography over soluble worm antigenic preparation [SWAP]. Vaccination of C57BL/6 [C57] mice with HAb2 resulted in - 31% and - 36% protection in two experiments of resistance to infection. Splenocytes were collected prior to challenge at week 6-post initial immunization and after challenge at days 6, 10, 28 and 90. Prior to challenge, in vitro splenic responses of HAb2-vaccinated animals [HAb2-group] to phytohaemagglutinin [PHA] declined while both SWAP and HAb2-driven responses increased, all compared to naive control. After challenge, PHA responses increased in the two test groups on day 6 then significantly decreased to lower levels. On the other hand, SWAP- and HAb2-driven responses of HAb2 group increased by day 6 then declined while the same responses in infected control mice increased on days 10 through 28 and decreased by day 90. Generally, proliferation obtained following in vitro stimulation with HAb2 was greater than that with SWAP in the HAb2-group after challenge. These results suggested that human anti-Id antibodies could mimic at the T cell level the properties of a protective antigenic epitopes of the irradiated-cercariae vaccine

Human, monoclonal and recombinant candidacidal, pneumocysticidal and mycobactericidal antibodies.

Antiidiotypic antibodies (antiIds) representing the internal image of some antigenic deteminants have been proposed as surrogate vaccines or, conjugated with toxins, in the immunotherapy of cancer. Experimental studies on antiidiotypic vaccination against fungal infections have been lacking. A conceptually new model of idiotypic vaccination against fungal infection has been recently developed. The vaccine used is monoclonal antibody that in vitro neutralizes the activity of killer toxin from the yeast, Pichia anomala (KT). This is effective against a wide range of fungal pathogens including Candida albicans and Pneumocystis carinii, and is also active against Mycobacterium tuberculosis. In an experimental mouse model, this vaccination was found to confer significant protection against systemic and mucosal infection with C. albicans. Human recombinant killer toxin antibodies and its synthetic derivatives hold promise of a potentially powerful tool against fungal and mycobacterial infections.

Immunotherapy of melanoma: peptide mimics of a human high molecular weight-melanoma associated antigen

The realization that tumor cells utilize multiple mechanisms to escape from immune recognition and destruction has stimulated interest in developing and applying immunotherapeutic strategies which target both humoral and cellular immunity to malignant cells. As a result, the tumor-associated antigens (TAA) used as targets have to be expressed on the cell surface membrane of malignant cells. Furthermore, since most of the TAA used for active specific immunotherapy are self-antigens, a challenge facing tumor immunologists is to develop strategies which are effective in breaking tolerance to self-antigens. This chapter describes one strategy which relies on the use of peptide mimics of the human high molecular weight-melanoma associated antigen (HMW-MAA) as immunogens to implement active specific immunotherapy in patients with malignant melanoma. These mimics, which are isolated from phage display peptide libraries by panning with anti-HMW-MAA monoclonal antibodies, are expected to induce both humoral and cellular anti-HMW-MAA immunity.

Anti Ro/SSA positive undifferentiated connective tissue disease in a mother with a newborn with complete congenital heart block: a case report.

An association between complete congenital heart block (CCHB) and anti-Ro/SSA antibody is well recognized but has never been reported in Thailand. We report here a 37-year-old female who was admitted because of massive epistaxis secondary to immune thrombocytopenia. She had given birth to a child with CCHB 2 years previously, when she was healthy. Antinuclear antibody and anti-Ro/SSA were positive in her sera, but were negative in her son. The relationship between anti-Ro/SSA antibody and outcome of mothers with infants with CCHB is reviewed.

In-vitro inhibition of antiplatelet autoantibodies by intravenous immunoglobulins and Rh immunoglobulins.

Autoimmune thrombocytopenia (AITP) is caused by autoantibodies to platelet glycoprotein antigens. Intravenous immunoglobulin (i.v.IgG) and Rh immunoglobulin infusions have found great significance in the treatment of AITP patients not responding to corticosteroids and other modes of therapy. In our study, it was observed that immunoglobulins (i.v.IgG & Rh), and their Fab fragments inhibited the binding of antiplatelet autoantibodies to normal platelets, from 15.8 to 90.7% and 25.6 to 90.08% respectively; whereas, their Fc portion did not show any inhibition. The presence of specific anti-idiotypic antibodies to antiplatelet autoantibodies was established by using monoclonal antibodies to Glycoprotein IIb/IIa and Glycoprotein Ib/IX, as the specific idiotype source. The i.v.IgG and Rh immunoglobulin products reacted with the monoclonal antibodies, only through their Fab and not through the Fc portions, thereby confirming its specific anti-idiotype activity.

Anti-IL-4 antibody inhibits antigen specific IgE response but fails to prevent chicken gamma globulin-induced active systemic anaphylaxis: evidence for the involvement of IgG antibodies

It has recently been reported that interleukin-4 (IL-4) is required for the production of IgE, and anti-IL-4 monoclonal antibody (mAb) inhibits in vivo IgE responses. These suggest that blocking of IL-4 activity may be useful for the prevention or treatment of immediate hypersensitivity disorders. In this study we investigated whether anti-IL-4 has a regulatory role in chicken-gamma globulin (CGG)-induced active systemic anaphylaxis. Multiple injections of anti-IL-4 (up to 40 mg/mouse) failed to protect the mice from fatal anaphylaxis. Anti-IL-4 strongly suppressed CGG-specific IgE response (>90%) without any suppressive effect on CGG-specific IgG (IgG1, IgG2a, IgG2b, and IgG3) responses. Because these data suggest the possibility that fatal anaphylaxis could be induced by IgG antibodies, we examined the possibility using anti-CGG polyclonal and the subclasses of IgG monoclonal antibodies. Passive sensitization of mice with polyclonal antibodies elicited severe and fatal anaphylactic shock; about 50% of the mice died. The activity of antibodies was not diminished by heat treatment (56 degrees C, 2h), suggesting that the anaphylaxis was not mediated by IgE. Shock was also elicited by each subclass of IgG mAb; of these, IgG1 was the most effective. Combination of the IgG subclasses elicited more exaggerated shock; about 30% of mice died. These data indicate that IgG antibodies are themselves sufficient to induce systemic anaphylaxis. Therefore, the failure of anti-IL-4 to prevent active anaphylaxis is probably due to the inability of anti-IL-4 to suppress the production of IgG antibodies.

A putative antiidiotype autoantibody regulates the antibromelain treated mouse red blood cell autoantibody secretion

Murine normal lymphoid tissues contain relatively high numbers of anti-BrMRBC RFC and PFC. In the bone marrow, these frequencies are inversely correlated probably due to different activation states of these anti-BrMRBC B cells. The sera from syngeneic, but not allogeneic, mice are able to inhibit the formation of these anti-BrMRBC plaques. This inhibition is enhanced when sera of LPS treated mice is employed. The inhibitory factor can be displaced from normal spleen cells by 10(-5) M phosphorylcholine (PC) hapten reversing the PFC inhibition. The dialyzed and concentrated supernatant from PC incubated splenocytes completely abolishes anti-BrMRBC PFC. Preliminary data suggest that the molecular weight of the inhibitory factor is higher than 600 KDa. We postulate an antiidiotype autoantibody nature for this inhibitory factor.