IgA Type Research Articles

Detection of Hepatitis Antibodies using ELISA Technique in Basrah Province

The possibility of detecting acute infection and immunity using body fluids those are easier to collect than blood, mainly in children, would facilitate the investigation and follow-up of outbreaks of hepatitis A (HAV). So to evaluate the detection of anti-HAV type IgM, IgA and total antibodies in saliva using serum samples as reference. Forty three paired serum and saliva samples were analyzed. From this total, 24 samples were obtained from children and 1 from one adult during the course of acute hepatitis A; an additional 18 samples were obtained from health professionals from Basrah hospital. The sensitivity to detect anti-HAV IgM was 100% (95%CI: 79.1 to 100.0%), employing saliva as clinical samples. In detecting anti-HAV IgA, the sensitivity was 80.8% (95%CI: 60.0 to 92.7%) and for the total antibodies was 82.1% (95%CI: 62.4 to 93.2%). The specificity was 100% for each. The rate of agreement was high comparing the results of serum and saliva samples for detecting HAV antibodies. We conclude that saliva is an acceptable alternative specimen for diagnosing acute hepatitis A infection, and for screening individuals to receive hepatitis A vaccine or immunoglobulin.

Effect of human papillomavirus and Chlamydia trachomatis co‐infection on sperm quality in young heterosexual men with chronic prostatitis‐related symptoms

To investigate the effect of human papillomavirus (HPV) and Chlamydia trachomatis (Ct) co-infection on sperm concentration, motility and morphology, in a large cohort of young heterosexual male patients with chronic prostatitis-related symptoms. Patients with chronic prostatitis-related symptoms, attending the same centre for sexually transmitted diseases from January 2005 and December 2010, were consecutively enrolled in this cross-sectional study. All patients underwent clinical and instrumental examination, microbiological cultures for common bacteria, DNA extraction, mucosal and serum antibodies evaluation for Ct, specific tests for HPV and semen analysis. The semen variables analysed were: volume; pH; sperm concentration; motility; and morphology. Subjects were subdivided in two groups: group A, patients with Ct infection alone and group B, patients with Ct and HPV co-infection. The main outcome measurement was the effect of Ct and HPV co-infection on the semen variables examined. Of 3050 screened patients, 1003 were enrolled (32.9%) in the study. A total of 716 (71.3%) patients were allocated to group A, and 287 (28.7%) to group B. Significant differences between the two groups were reported in terms of percentage of motile sperm (degrees of freedom [df] = 1001; t-test = 11.85; P < 0.001) and percentage of normal morphological forms (df = 1001; t-test = 7.18; P < 0.001), while no differences were reported in terms of semen volume or pH. According to World Health Organization thresholds for normal semen, 364 (50.8%) men in group A and 192 (66.8%) men in group B were subfertile (odds ratio = 1.95; 95% confidence interval 1.46-2.60; P < 0.001). No correlation between HPV genotype, mucosal IgA type and semen variables was found. In a population of prostatitis-related symptoms attributable to Ct infection, co-infection with HPV has a significant role in decreasing male fertility, in particular with regard to sperm motility and morphology.

High prevalence of IgA class anti-neutrophil cytoplasmic antibodies (ANCA) is associated with increased risk of bacterial infection in patients with cirrhosis

Anti-neutrophil cytoplasmic antibodies (ANCA) are a non-uniform family of antibodies recognizing diverse components of neutrophil granulocytes. ANCA formation might be induced by protracted bacterial infections or probably reflect an abnormal immune response to commensal microorganisms. Bacterial infections are common complications in cirrhosis with high incidence of episodes caused by enteric organisms, therefore, we sought to study the presence and clinical importance of ANCA in cirrhosis. Sera of 385 patients with cirrhosis of different etiologies were assayed for ANCA of IgG, IgA, IgA1, IgA2, and secretory IgA subtypes by indirect immunofluorescence and ELISAs. The control group comprised 202 patients with chronic liver diseases without cirrhosis and 100 healthy subjects. In cirrhosis, a 2-year follow-up, observational study was conducted to assess a possible association between the presence of ANCA and clinically significant bacterial infections. Prevalence of ANCA IgA was significantly higher in cirrhosis (52.2%) compared to chronic liver diseases (18.6%) or healthy controls (0%, p<0.001 for both). ANCA IgA subtyping assays revealed marked increase in the proportion of IgA2 subtype (46% of total ANCA IgA) and presence of the secretory component concurrently. Presence of ANCA IgA was associated with disease-specific clinical characteristics (Child-Pugh stage and presence of ascites, p<0.001). During a 2-year follow-up period, risk of infections was higher among patients with ANCA IgA compared to those without (41.8% vs. 23.4%, p<0.001). ANCA IgA positivity was associated with a shorter time to the first infectious complication (pLogRank <0.001) in Kaplan-Meier analysis and was identified as an independent predictor in multivariate Cox-regression analysis (HR:1.74, 95% CI: 1.18-2.56, p=0.006). Presence of IgA type ANCA is common in cirrhosis. Involvement of gut mucosal immune system is in center of their formation and probably reflects sustained exposure to bacterial constituents.

Relationship between renal pathology and glomerular podocyte injury in childhood Henoch-Schönlein purpura nephritis

Objective To explore the relationship between renal pathology and glomerular podocyte injury in childhood Henoch-Schonlein purpura nephritis(HSPN). Methods Renal pathological types of 72 children suffering from HSPN were reviewed during the period of Jan.2008 to Jan.2011 from Nanjing General Hospital of Nanjing Command.In those cases, grade in pathology and immunofluorescence types were acquired.Podocyte injury was classified by electron microscope.The relationship between podocyte injury and grade in pathology was analyzed. Results Pathological findings were classified, grade Ⅱ in 34 cases(47.2%) and grade Ⅲ in 38 cases(52.8%). Glomerular podocyte injury was classified by electron microscope as food processes of podocyte fused extensively in 21 cases; food processes of podocyte fused segmental in 35 cases, food processes of podocyte without fusion in 11 cases.There was without renal glamorous in nephridial tissue by electron microscope in 5 cases.Cases with foot process of podocyte fused extensively were more grade Ⅲ than those cases with foot process fused segmental and without fusion.According to renal immunofluorescence pathology, IgA+ IgM+ IgG type was mostly in cases with foot process of podocyte fused extensively, IgA+ IgG type was mostly in cases with food processes of podocyte fused segmental and IgA type was mostly in cases with food processes of podocyte without fusion. Conclusions In childhood HSPN, renal pathology changed not only expressing on mesangial cell hyperplasia but also podocyte injury.The more seriously on the degree of podocyte injury, more obviously in pathologic change. Key words: Henoch-Schonlein purpura nephritis; Renal pathology; Glomerular podocyte injury; Child

An attempt to identify antigen for IgA pemphigus accidentally showed the presence of transmembrane secretory component in keratinocytes

In this study, to identify unknown autoantigen for intraepidermal neutrophilic IgA dermatosis type IgA pemphigus (IEN), we performed a new immunoprecipitation (IP), in which biotinylated KU-8 cell extract was precipitated by peptide M agarose, that has specific binding ability to human IgA. By the IP combined with mass spectrometry, we identified transmembrane secretory component (TSC) as a candidate. TSC was attractive as an antigen for IEN, because TSC delivers IgA in the mucosae. Polyclonal antibody to TSC reacted with the epidermis in immunofluorescence (IF).

Novel Agents and Autologous Stem Cell Transplantation Improve Survival of Multiple Myeloma Patients with Ages 65–70 Years: A Multicenter Retrospective Collaborative Study Between Japanese Society of Myeloma and European Myeloma Network

AbstractAbstract 5030With the emergence of novel agents, the treatment paradigm of multiple myeloma (MM) has changed dramatically in the last decade. Although there have been many attempts to improve the outcome by using novel agents as first-line and/or salvage therapies, it is not clear whether the combined use of novel agents with autologous stem cell transplantation (SCT) has translated into better outcome for elderly patients with MM. To clarify this issue, we retrospectively analyzed the clinical features and treatment outcome of patients with ages 65–70 (median 67) years. From January 2004 to December 2009, a total of 318 patients (167 male and 151 female) were treated in 38 centers in Japan and Italy. The monoclonal immunoglobulin (Ig) was IgG in 169 patients, IgA in 80, light chain in 56, IgD in 7, and others in 6, respectively. Twenty-two patients were classified as Durie & Salmon stage I, 77 were stage II, and 212 were stage III, respectively. As for the International Staging System (ISS), 86 patients were in ISS stage I, 107 were in ISS stage II, and 102 were in ISS stage III, respectively. Treatment for each patient was determined by a physician-in-charge. As initial therapy, 192 patients were treated with conventional chemotherapy; melphalan and prednisone (MP) in 78, vincristine, adriamycin, and dexamethasone (VAD) in 56, and other regimens in 58, respectively. Eighty-eight patients were treated with novel agent-containing regimens composed of bortezomib in 62, lenalidomide in 17, and thalidomide in 9, respectively. Twenty-one patients were treated with conventional chemotherapy (VAD) plus SCT, and 17 were with novel agents (bortezomib and dexamethasone) plus SCT. The median progression-free survivals (PFS) of the 4 different treatment groups were 19. 1 months, 24. 5 months, 26. 8 months, and 35. 2 months, respectively, and there was a significant improvement in the groups with novel agents (p=0. 006) and novel agents plus SCT (p=0. 032) compared with the conventional chemotherapy group. The median overall survival (OS) of the conventional chemotherapy group was 46. 0 months, while those of other groups were not reached. When compared with the outcome of the conventional chemotherapy group, there was a significant improvement in OS in the groups with novel agents (p=0. 001), chemotherapy plus SCT (p=0. 02), and novel agents plus SCT (p=0. 02), and novel agents plus SCT resulted in the best OS (88% at 6 years). Best responses to treatment of 318 patients were as follows: complete response (CR) in 26 patients (8. 2%), very good partial response (VGPR) in 56 (17. 6%), partial response (PR) in 127 (39. 9%), stable disease (SD) in 92 (28. 9%), and disease progression (PD) in 17 (5. 3%), respectively. Significant differences in OS were found between CR and PR groups (p=0. 002) and between VGPR and PR groups (p=0. 045). In 168 relapsed patients after initial treatment, 148 patients were treated with novel agent-containing regimens and the median OS from the time of relapse was 45. 5 months compared with 15. 0 months for 20 patients treated with conventional chemotherapy alone (p=0. 03). In a multivariate analysis using stepwise selection, IgG or IgA type (p=0. 013), normal serum albumin (p<0. 0003), ISS 1–2 (p=0. 004), and the use of novel agents (p=0. 002) were independent prognostic factors significantly associated with better OS. These results indicate that first-line autologous SCT prolongs OS compared with conventional chemotherapy alone and induction therapy with novel agents has a major impact on OS with or without autologous SCT. Thus, combination with novel agents and autologous SCT may improve outcome even in this subgroup of patients with MM. Disclosures:No relevant conflicts of interest to declare.

Assessment of the diagnostic performances of IgA heavy and light chain pairs in patients with IgA monoclonal gammopathy

ObjectivesPreliminary results of the IgA Hevylite™ assay including the establishment of the 95% reference interval and assessment of the specificity and sensitivity in different populations are reported. Design and methodsThe concentrations of IgA heavy and light chains (HLC) enabling to determine an IgAκ/IgAλ ratio were quantified in 119 apparently healthy individuals to generate 95% reference intervals. The specificity of this assay was assessed in 48 patients with an isolated polyclonal IgA increase. In a retrospective analysis of 68 patients with a monoclonal component type IgA (MC-IgA) identified by serum immunofixation (IFE), IgA HLC ratio values were compared with known results for serum protein electrophoresis (SPE) and free light chain (FLC) ratios. ResultsThe 95% reference range obtained in 119 controls (0.91–2.04) was close to that quoted by the manufacturer (0.80–2.04). Eight of the 48 patients (16.7%) with a polyclonal IgA increase had an IgA HLC ratio above the upper limit of the 95% reference interval.The IgA HLC ratio identified 65 (95.6%) among 68 patients with MC-IgA identified on the basis of IFE. For 34 of these patients (50%), MC-IgA was not detected by SPE due to its co-migration with alpha-2 or beta-globulins. ConclusionsCompared with serum IFE, the IgA HLC ratio has a sensitivity of 95.6%. Further studies are needed to assess the specificity of the IgA HLC ratio in patients with an isolated polyclonal increase of serum IgA.

Isolated Abducens Nerve Palsy in Multiple Myeloma With Hyperviscosity of the Serum

A 40-year-old woman without contributory medical history was diagnosed as having IgA type multiple myeloma, based on the immunological and pathological investigation. Blood pathology demonstrated the rouleaux formation. Then, sudden onset of left-sided complete abducens nerve palsy (6NP) without pain was preceded to initiation of chemotherapy. Cranial and orbital magnetic resonance imaging, and cerebrospinal fluid examination demonstrated no abnormalities. Under chemotherapy, 6NP was resolved within 5 weeks. This is a first reported case of multiple myeloma presenting with 6NP without intracranial lesion. We speculated that 6NP might be caused by peripheral ischemia due to hyperviscosity of the serum. J Med Cases. 2012;3(6):373-374 doi: https://doi.org/10.4021/jmc855w

Multiplexed electrochemical immunosensor for detection of celiac disease serological markers

Celiac disease (CD) is a gluten-induced autoimmune enteropathy characterized by the presence of antibodies against gliadin (AGA) and anti-tissue transglutaminase (anti-tTG) antibodies. A disposable electrochemical dual immunosensor for the simultaneous detection of IgA and IgG type AGA and anti-tTG antibodies in real patient's samples is presented. The proposed immunosensor is based on a dual screen-printed carbon electrode, with two working electrodes, nanostructured with a carbon–metal hybrid system that worked as the transducer surface. The immunosensing strategy consisted of the immobilization of gliadin and tTG (i.e. CD specific antigens) on the nanostructured electrode surface. The electrochemical detection of the human antibodies present in the assayed serum samples was carried out through the antigen–antibody interaction and recorded using alkaline phosphatase labelled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions was used as the substrate. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with commercial ELISA kits indicating that the developed sensor can be a good alternative to the traditional methods allowing a decentralization of the analyses towards a point-of-care strategy.

Clinical Variability of Family Members with the C104R Mutation in Transmembrane Activator and Calcium Modulator and Cyclophilin Ligand Interactor (TACI)

Common Variable Immunodeficiency Disorder (CVID) is a complex disorder that predisposes patients to recurrent and severe infections. The C104R mutation in the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) is the most frequent mutation identified in patients with CVID. We carried out a detailed immunological and molecular study in a family with a C104R mutation. We have undertaken segregation analysis of a kindred with C104R mutations of the TACI gene. Detailed immunological and molecular investigations were carried out for this kindred and the clinical phenotype was compared to the genotype. Segregation analysis of our kindred showed that inheriting single or double copy of the C104R mutation does not consign an individual to CVID. All heterozygotes in the family were phenotypically different, ranging from asymptomatic to ill-health. A family member with a wild type TACI variant had CVID-related phenotype including IgA deficiency and type 1 diabetes. Interestingly, a family member with the homozygous C104R/C104R variant did not meet the criteria for CVID because he had excellent, albeit unsustained, vaccine responses to T cell dependent and T cell independent vaccine antigens despite profound hypogammaglobulinemia. The C104R mutation does not correlate with the clinical phenotypes in this family.

Screening for Celiac Disease in Children with Dental Enamel Defects

Background. Dental enamel defects (DEDs) are seen in celiac disease (CD). Aim was to detect frequency of CD among such patients. Methods. This study included 140 children with DED. They were tested for CD. Gluten-free diet (GFD) was instituted for CD patients. A cohort of 720, age and sex-matched, normal children represented a control group. Both groups were evaluated clinically. Serum calcium, phosphorus, alkaline phosphatase, serum IgA, and tissue transglutaminase (tTG) IgG and IgA types were measured. Results. CD was more diagnosed in patients with DEDs (17.86%) compared to controls (0.97%) (P < 0.0001). Majority of nonceliac patients showed grade 1 DED compared to grades 1, 2, and 3 DED in CD. Five children had DED of deciduous teeth and remaining in permanent ones. After 1 year on GFD, DED improved better in CD compared to nonceliac patients. Gastrointestinal symptoms did not vary between celiac and nonceliac DED patients. Lower serum calcium significantly predicted CD in this cohort. Conclusion. CD is more prevalent among children with DED than in the general population. These DEDs might be the only manifestation of CD; therefore, screening for CD is highly recommended among those patients especially in presence of underweight and hypocalcemia.

Evaluation of Circulating Antisperm Antibody in Infertile Women with Polycystic Ovary Syndrome

Objective To evaluate levels of the circulating antisperm antibody (ASA) in infertile women with polycystic ovary syndrome (PCOS). Methods Forty-six infertile women with PCOS were enrolled into this study. The serum was screened by the indirect immunobead test for ASA type IgG and IgA according to the WHO laboratory manual. Results Of the 46 patients assessed for ASA-IgG, 3 cases had immunobead binding which were 20%, 27% and 35%, respectively, and the sub-positive rate was 6.5%. No case had the clinical positive level according to the WHO criteria (50% or more of the motile sperm with immunobead binding). ASA-IgA was not detected in all cases. Conclusions The circulating ASA is not associated with the pathogenesis of infertile women with PCOS.

Evaluation of Circulating ASA in Women with Secondary Infertility

Objective To evaluate levels of the circulating antisperm antibody (ASA) in women with secondary infertility. Methods Eighty-three women defined as secondary infertility were enrolled into this study. Based on their outcomes of previous pregnancy, these women were divided into two groups: group HC (having at least one child, n=45), group NC (no child but one to three abortions, n=35). The serum was screened by the indirect immunobead test for ASA type IgG and IgA according to the WHO laboratory manual. Results Of the 83 patients assessed for ASA-IgG, 14 had immunobead binding rate ranged from 15% to 43%, and the sub-positive rate was 16.9%. Five cases showed ASA-IgA sub-positive, and had binding rate between 10% and 20%. No case had the clinical positive level according to the WHO criteria. No differences were found on ASA-IgG and ASA-IgA between HC and NC groups (P>0.05). Conclusion The circulating ASA may be a possible cause to decrease fertility in women with secondary infertility.

Multiple Myeloma Variant with Ascites and Cytoplasmic Crystalline Granules in Plasma Cells

Abstract 5088CASE REPORT: CLINICAL HISTORY:A 62-year-old male patient who presented with anemia and findings of monoclonal gammopathy of IgA type was referred to the Hematology clinic for work-up and management of possible multiple myeloma. At the same time, the patient was being managed by the Gastroenterology service with diuretics and recurrent paracentesis for an ongoing significant ascites of unclear etiology. These interventions had been performed for a year on a biweekly basis. CLINICAL WORK-UP:The patient had IgA gammopathy with a monoclonal protein level of 1.2 Gm/dL and findings on skeletal survey consistent with lytic lesions in the pelvic bones. PROCEDURE:A bone marrow biopsy obtained from the patient revealed a plasmacytosis of 15% with atypical crystalline cytoplasmic inclusions seen in the majority of plasma cells and some histiocytes as well. The transudative ascitic fluid was analyzed for crystalline deposits, but these could not be conclusively detected even though there were inclusions seen in some macrophages that may represent remnants of the crystals. TREATMENT:The patient was diagnosed with IgA myeloma given the above findings and was treated with a combination of Revlemid, Velcade and Dexamethazone with good response in terms of his IgA monoclonal protein as well as in significant improvement in his ascites, which essentially resolved. He no longer required therapeutic paracentesis upon initiation of chemotherapy and was able to get weaned off of diuretics as part of his treatment. CONCLUSION:This case represents what we believe to be a rare variant of multiple myeloma that presented with monoclonal gammopathy in the blood as well as concomitant ascites. The bone marrow showed plasma cells containing crystalline inclusions. The precise nature of these inclusions is presently unknown, however it is felt most likely to be precipitated immunoglobulins. We plan to investigate this further. For reasons that are not entirely clear, this particular biology appears to induce a reactive process in the peritoneum causing a development of ascites. The resolution of such ascites with treatment directed toward the myeloma seems to indicate that the two are related. A case similar to this was reported by Doctors Martin, et al in 1987 with a patient presenting with similar type of crystalline inclusions in plasma cells with clinically associated ascites.1 There was no information available on treatment effect and so it was not possible to tie the clinical presentation of the myeloma and ascites together. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon–metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.

Linear immunoglobulin A/immunoglobulin G bullous dermatosis associated with Vogt-Koyanagi-Harada disease

Vogt-Koyanagi-Harada disease is characterized by marked bilateral uveitis associated with symmetric vitiligo, alopecia, poliosis and dysacousia. Linear immunoglobulin (Ig)A bullous dermatosis (LABD) is characterized by small, tense, subepidermal bullae caused by IgA type autoantibody targeting the basal lamina. LABD patients sometimes show coexistence of IgG type autoantibody, termed linear IgA/IgG bullous dermatosis (LAGBD). We reported a 35-year-old Japanese male case of combined LAGBD and Vogt-Koyanagi-Harada disease. His human leukocyte antigen typing was -A24, B52, C*1202, DR*1502, DQ*0601. Immunoblot revealed that patient sera reacted to both 180- and 230-kDa proteins at the IgA and IgG level. Because Vogt-Koyanagi-Harada disease and LABD are reported to be associated with other autoimmune diseases, it is probable that Vogt-Koyanagi-Harada disease and LAGBD in our case may be associated with each other in the pathomechanism. However, we cannot exclude the possibility of this being mere coincidence.

Use of a fully automated immunoassay for celiac disease screening in a pediatric population

Celiac disease (CD) is an immune-mediated disorder of the small bowel in which gluten and related cereal ingestion leads to chronic inflammation and damage to the small bowel mucosa in genetically susceptible individuals. The presence of IgA type tissue-transglutaminase antibodies (IgA-anti-tTG) in individuals without IgA deficiency is a highly-specific indicator of CD. We compared the accuracy of IgA-anti-tTG detection using a fully automated fluoroenzyme immunoassay (FEIA) with that of the standard enzyme-linked immunosorbent assay (ELISA) in a clinical setting. We tested 1227 serum samples obtained from three groups of subjects: 1160 randomly selected schoolchildren, 36 pediatric patients with biopsy confirmed CD and 31 control children with biopsy confirmed normal small bowel mucosa. IgA-anti-tTG detection was compared using the FEIA method and the ELISA method, both of which use baculovirus-expressed recombinant human tissue-transglutaminase (tTG) as the antigen. Both tests identified pediatric patients with CD equally well in the random population (0.34% with CI: 0.09%-0.88%). Furthermore, both tests gave similar results for the CD group and the control group with normal small bowel mucosa (sensitivity: 90% and specificity: 100% for both assays). The FEIA method is an equivalent substitute for the traditional ELISA for IgA-anti-tTG detection associated with CD in a random pediatric population.

Immunoglobulin Heavy/Light Chain Ratios as An Alternative to Immunofixation In the Identification of Monoclonal Gammopathies

AbstractAbstract 5018 IntroductionHistorically, serum protein electrophoresis (SPE), urine protein electrophoresis (UPE) and immunofixation (IFE) have been used to identify and quantify monoclonal proteins (M-proteins). Whilst this approach is adequate for the identification of intact immunoglobulin multiple myeloma (MM), it is not sensitive enough to detect free light chain MM (LCMM). Therefore, an algorithm which utilises SPE, serum free light chain (FLC) immunoassays and IFE for the identification of M-proteins has been suggested. Assays have now been developed which utilise polyclonal antisera raised against the kappa and lambda light chain types of IgG, IgA and IgM immunoglobulins (HLC). Here we report the use of these assays as an alternative to IFE and propose a screening algorithm which utilises SPE, FLC and HLC Materials and MethodsSerum FLC measurement was added to 1063 requests for SPE, from primary care or from a hospital source. Samples from patients with previously diagnosed MM, Waldenstrom's Macroglobulinemia and lymphoma were, where possible, removed. Sera showing monoclonal proteins or hypogammaglobulinemia (by SPE) or an abnormal FLC ratio were tested further by IFE and IgG, IgA, IgM HLC assays. SPE and IFE were performed on a SEBIA Hydrasys system, and gels were interpreted by experienced clinical chemists. FLC and HLC measurements were performed on a Siemens BN™II nephelometer. HLC results were compared with IFE results and clinical diagnoses. The study was approved by the Wolverhampton, New Cross, NHS Trust Review Board Results80/1063 patients were identified as having abnormal SPE or abnormal FLC results. 42/80 patients had positive IFE results. 24/42 of these patients were positive by HLC (Table 1), 11/42 had light chain only myeloma/MGUS, the remaining 7/42 were MGUS patients. The 7 MGUS patients (6 IgG and 1 IgM) with normal HLC ratios and positive IFE all had less than 2g/L monoclonal protein measured by SPE densitometry and a normal FLC ratio. Of the 38/80 with normal IFE's all had been investigated further because of abnormal FLC results. 9/38 had abnormal HLC ratios of which 3/9 had confirmed hematological malignancies (1× chronic lymphocytic leukemia (CLL), 1× small lymphocytic lymphoma (SLL) and 1× asymptomatic MM (ASMM)).The use of FLC immunoassays alongside SPE as part of the primary screening protocol identified 10 additional hematological malignancies (1× ASMM, 6×CLL, 2× non-Hodgkin lymphoma, 1× SLL). DiscussionHLC ratio analysis matched IFE for the identification of all symptomatic haematological malignancies. Abnormal FLC ratios identified 10 additional haematological malignancies of which 3 also had abnormal HLC ratios, which would have been missed using SPE/ IFE. In 7/19 MGUS (6×IgG, 1×IgM) patients there were normal HLC ratios. In all cases the monoclonal protein load was below 2g/L and the FLC ratio was normal; identifying the IgG patients as having a low risk and the IgM patient as having a low/intermediate risk of progression. It may be beneficial not to identify these patients, who do not require therapeutic intervention or justify close monitoring. Another advantage of using HLC analysis instead of IFE is that the HLC ratio has been found to be a prognostic indicator in myeloma and MGUS. It would also form a useful “baseline” comparison if HLC assays were used in monitoring or for the detection of residual disease. ConclusionsHLC analysis identified all symptomatic patients who were IFE positive and in an additional 3 hematological malignancies. Low risk MGUS patients may not be identified using these tests but this might be beneficial to patients and physicians alikeTable 1IFE positive patients with SPE densitometry and heavy/light chain immunoglobulin results.DiseasenRange SPE(g/L)HLCr abnormalIgA MM20–122/2IgA MGUS505/5IgGMM40–274/4IgG MGUS110–65/11IgG Amyloid101/1IgG Plasmacytoma1231/1Waldenstrom's101/1IgM MGUS32–202/3IgM lymphoma30–123/3 Disclosures:Harding:Binding Site Group Ltd: Employment.

37 MORPHOLOGICAL FEATURES OF SPERMATOZOA (LIKE “HEAD-IN-TAIL” ANOMALY) AND “COMET ASSAY” FROM PATIENTS WITH DECLINED QUALITY OF SEMEN

the mean age was 33.29 years. The clinical trial was conducted in Ivanovo clinic-diagnostic center of municipal hospital No. 8. Statistical analysis was performed with Microsoft Excel v.12.0 and Statistica v6.1. Men applied to the hospital with infertility and diagnosed the autoimmune infertility (MAR%IgG and/or MAR%IgA 50%) were included in this trial. Spermogram results: the mean spermatozoa with adhesive antisperm antibodies (ASA) type IgG was 54%, and type IgA 60%; mean spermatozoa A 9%, mean spermatozoa B 16%; total amount of spermatozoa in ejaculate A 22.46mil, total amount of spermatozoa in ejaculate B 32.98 mil; mean spermatozoid speed was 8.83mm/s, mean viscosity 3.63 cm. All patients were treated with Longidaza. All the same indicators were evaluated after 3 months of treatment. The mean spermatozoa with ASA type IgG significantly decreased to 15% (p = 0.0016), type IgA to 14% (p = 0.001). Mean spermatozoa A was 11%, B 21%, A+B 32%, total amount of spermatozoa in ejaculate A 30.08mil, total amount of spermatozoa in ejaculate B 30.35mil; mean spermatozoid speed was 9.67mm/s, mean viscosity 3.18 cm. No side or adverse effects were registered by the patients during the treatment. Therefore, statisticallyvalid decrease of spermatozoa with ASA type IgG mean 51%, and type IgA 37%. Qualitative and quantitative spermogram indicators improved, but not statistically-valid, apparently, this is connected with small sample size.

Production and Characterization of Monoclonal Antibodies Against NS1 Protein of H5N1 Avian Influenza Virus

A panel of 11 murine monoclonal antibodies (MAbs) was generated against NS1 (non-structural) protein of highly pathogenic avian influenza H5N1 virus using whole viral antigen as immunogen and as antigen in the preliminary screening of clones followed by a more specific screening through recombinant NS1 protein-based ELISA. Isotyping showed two clones 10E2 (IgG2b) and 5H8 (IgG3) to be IgG type and 6B9 and 4C4 to be IgA type, while the rest could not be isotyped due to weak reactivity with NS1 antigen. The subclone 10E2E7G3 was selected for subsequent work, which showed comparatively higher OD in NS1-based ELISA. The reactivity of 10E2E7G3 with H5N1 virus as well as recombinant NS1 protein was confirmed in Western blot analysis. The anti-NS1 MAbs produced in the present work may be valuable in developing an immunodiagnostic assay as a DIVA test for differentiating infected from vaccinated birds in AIV.