Antibody Detection System Research Articles

Suppression of synaptic plasticity by cerebrospinal fluid from anti-NMDA receptor encephalitis patients

The functional effects of cerebrospinal fluid (CSF) from patients with anti-NMDA receptor (NMDAR) encephalitis on the NMDAR-mediated synaptic plasticity were evaluated by using mouse hippocampus slices. Anti-NMDAR antibody detection system was established by immunostaining recombinant NMDAR heteromers expressed in HEK cell culture as well as native NMDARs in cultured hippocampal neurons. Under a complete blind manner for the clinical information, CSF and sera collected from 36 pre-diagnosed patients were tested for anti-NMDAR antibodies. With this test, thirteen patients were diagnosed as anti-NMDAR encephalitis. CSF positive for anti-NMDAR antibodies suppressed induction of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in mouse hippocampal slices. LTP induction was not suppressed by CSF collected from herpes simplex virus (HSV) encephalitis or non-encephalitis control patients. Antibody absorption with NMDAR-expressing HEK cell culture reversed the suppression of LTP by anti-NMDAR encephalitis patients' CSF, confirming that anti-NMDAR antibodies suppressed LTP. The present experiments firmly support the proposal that the anti-NMDAR encephalitis autoantibody is responsible for cognitive disorders like amnesia accompanying this disease.

Abstract B4: Expression evaluation of TP53 in colorectal cancer

Abstract Background and Aim: Colorectal cancer (CRC) is the third most common type of cancer in men and women worldwide. CRC is thought to result from an interaction between environmental and genetic factors. Variation in the function of genes responsible for DNA repair mechanisms and cell cycle control in the presence of carcinogen-mediated cell damage is an attractive mechanism for explaining any inter-individual variation in CRC susceptibility. The TP53 tumor suppressor gene is one of the most commonly mutated genes in all types of cancer, playing a key role in the development and progression of disease. In addition to gene mutation, multiple single nucleotide polymorphisms (SNPs) have been identified in this gene, however the relevance of most of them is unclear. The p53 codon 72 SNP leads to a Proline to Arginine substitution, with different capacities for inducing gene transcription, interacting with other proteins or modulating apoptosis. Studies evaluating the association between this SNP and CRC showed inconsistent results, none evaluating the expression status. The aim of this study was to evaluate the expression status of TP53 expressed by tumor samples and their association with clinicopathological variables. Methods: 101 non-related patients with CRC were evaluated (52 women and 49 men) treated at ACCamargo Hospital in Brazil. Immunohistochemistry (IHC) was performed with monoclonal antibody and polymer-based detection system. RNA was isolated from frozen tumor tissue using phenol/chloroform protocol. The TP53 expression status was evaluated by RT-PCR followed by DNA Sanger sequencing. Associations were analyzed by Pearson Chi-Square or Fisher Exact tests and Multiple Logistic Regression. Results: TP53 mutation was found as a common event in this study. There were found different types of mutations among CRC patients. As expected, mutations were found more frequent in the positive than in negative IHC groups (P < .0001). Mutation profiles (missense, nonsense, silent and rearrangements types) were also different between p53 IHC positive and negative groups (P < .0001). The majority of mutations were concentrated in the TP53 DNA binding domain, mainly in codons considered as mutation hotspots. There were found changes in three samples that could be explained as putative splicing variants or splicing errors. The p53 codon 72 SNP was significantly associated with gender (P = .037), recurrence (P = .005), dirty tumor necrosis (P = .025), pattern border of tumor growth (P = .05), post chemoradiotherapy use (P = .002), p53 IHC expression (P = .041) and TP53 mutation (P = .004), suggesting that the expression of at least one Arg72 allele is associated with better prognosis. According to multiple logistic regression the only variable that remained independently associated with the presence of the Arginine allele is the absence of tumor necrosis factor (OD = 8.64-P = .047). Allele frequency of p53 Pro72 was 0.26. This is the first report describing the expression status of the p53 codon 72 alleles in CRC and indicates that this SNP is associated with CRC. Conclusions: The analysis of the whole transcript sequence of TP53 mRNA is important to determine its mutation pattern and the specific contribution of the p53 expressing allele. This approach is congruent with the tightly association of TP53 mutation presence with its protein accumulation pattern in cancer, permitting the appropriate identification of protein stabilization, which leads to the nuclear accumulation of p53. This strategy also facilitated the detection of possible splicing variants, which might have been underestimated in previous analyses. In relation of SNP analysis, the results suggest that p53 codon 72 SNP may play a role in CRC developing and progression, thus representing a possible genetic risk factor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B4.

Detection of low-affinity anti-drug antibodies and improved drug tolerance in immunogenicity testing by Octet ® biolayer interferometry

We assessed the utility of the FortéBio Octet ® system for detection of anti-drug antibodies (ADAs) against an investigational therapeutic human IgG1 monoclonal antibody (mAb), CNTO X. To understand the relative merits of this technology, key performance requirements were compared with two popularly accepted ADA detection methods, a step-wise bridging ELISA and a Meso Scale Discovery (MSD) homogeneous (single step binding) bridging ECLIA. When used to detect 13 monoclonal ADAs of varying affinities and one polyclonal ADA, all three methods demonstrated their greatest apparent sensitivity to the polyclonal sample (1, 6, and 130 ng/mL, respectively for ECLIA, ELISA, and Octet). Sensitivity to monoclonal ADAs tended to vary in accordance with their affinities, however, the sensitivity of the Octet method varied much less between ADAs. As a result, the above ranking became reversed such that Octet was the most and ELISA least sensitive for detection of low-affinity ADAs. With regard to drug tolerance, the presence of CNTO X could lead to false-negative assay results, although each method was affected to a different degree, with the Octet method tolerating up to 10 times more drug than the ECLIA method, which in turn tolerated up to 10 times more than the ELISA. Finally, the ECLIA and Octet methods were applied to the bioanalysis of cynomolgus monkey sera from a pre-clinical multiple dose study of CNTO X. Octet indicated 3 positive animals developed ADA as early as day 15 of the dosing phase while drug was present at nearly 1 mg/mL. ECLIA detected only one of these, and only in a day 57 recovery sample after drug had cleared from circulation. We conclude that the Octet is a promising platform for detection of lower affinity ADAs and is particularly suitable for ADA detection when drug persists at levels that negatively impact bridging immunoassays.

Impact of cytomegalovirus (CMV) antibody reflex testing in the transfusion service on management of CMV‐seronegative blood inventory

Our goal is to minimize unnecessary cytomegalovirus (CMV)-seronegative blood transfusion to preserve the CMV-seronegative blood inventory for patients who are identified as CMV seronegative. We implemented a CMV antibody reflex testing protocol for patients who require CMV-compatible blood but in whom a CMV serostatus is unknown (coded as CMVT in our computer system). A solid-phase red blood cell (RBC) adherence antibody detection system was validated to detect CMV antibodies in plasma samples (received for ABO/Rh type and RBC antibody screen) with acceptable sensitivity and specificity. We evaluated the impact of this CMV antibody reflex testing on the management of RBC and platelet (PLT) inventory for patients requiring CMV-compatible blood. Over a 16-month period, implementation of CMV antibody reflex testing identified 361 (34%) of 1063 previously CMV-untested patients who required CMV-compatible blood and who were CMV seronegative. We observed a 75% decrease in the number of CMVT patients in our data base from 190 per month before implementation to 57 at 16 months postimplementation. Consequently we reevaluated the percentage in our blood inventory of CMV-seronegative units required while potentially saving 1234 CMV-seronegative blood products (835 RBCs and 399 PLTs) each month. A strategy of performing CMV antibody reflex testing in the transfusion service allows more effective blood inventory management and control in maintaining a CMV-seronegative blood inventory dedicated for patients who truly require it.

The research of MPT64 antibodies aptamer of Mycobacterium tuberculosis in the serological diagnosis

Objective To establish mixed-sandwich ELISA detection system by screening aptam-ers of MPT64 antibodies with SELEX to detect clinical serum samples, and explore the potential laboratory diagnosis value of this method. Methods To detect the affinity of the final round ssDNA library to MPT64 antibodies inhibited by MPT64 antigen with the competitive ELISA method, optimize the mixed-sandwich ELISA detection method that was aptamer-serum-horseradish peroxidase labeled goat anti-human IgG anti-body detection system to detect 230 cases of clinical serum samples as well as the lowest concentration of MPT64 antibodies and the linear range. Results In competitive ELISA test results, the percentage of inhi-bition effect of MPT64 antigen to final round ssDNA library is from 0.25% to 80% when the MPT64 antigen concentration rised from 2 μg/ml to 256 μg/ml. The Optimized detection system of mixed-sandwich ELISA was constitute of the concentration of ssDNA coated with 0.1μg/hole, serum dilution of 1/200, horseradish peroxidase labeled goat anti-human IgG antibody concentration of 1/40 000. The lowest concentration of MPT64 antibody is 3 mg/L and the linear range is between 10 mg/L and 1000 mg/L. The serum samples of 100 cases of tuberculosis patients, 100 healthy individuals and 30 cases of non-tuberculesis were tested in this system and the test result was analyzed with Graphpad Prism, the difference of tuberculosis group and healthy group was statistically significant (P<0.001 ), the difference of TB group and non-TB control group was also statistically significant (P<0.001). The specificity and the sensitivity was 96.1% and 31.0% re-spectively. Conclusion The aptamer mixed-sandwich ELISA method will play an important role in the sero-logical diagnosis of tuberculosis. Key words: MPT64 antibodies; SELEX; Aptamer; ssDNA; Serological detection

Easy identification of antibodies to high‐prevalence Scianna antigens and detection of admixed alloantibodies using soluble recombinant Scianna protein

Identification of antibodies against high-prevalence Scianna (Sc; ERMAP) antigens, like Sc1 and Sc5, is difficult and may incur delays in blood procurement and costs. The detection of additional clinically significant alloantibodies is hampered in the presence of anti-Scianna. Soluble recombinant Scianna protein is demonstrated to facilitate antibody diagnostics in both cases. Soluble recombinant Scianna protein (Sc:1,-2,3,-4,5,6,7) was produced comprising the antigenic extracellular domain fused to a V5-His tag. The protein was isolated from eukaryotic cell culture supernatants of stably transfected HEK293 cells. Seven serum samples with anti-Sc1, anti-Sc2, and anti-Sc5 and 30 serum samples with antibodies to other blood group antigens were evaluated in hemagglutination inhibition assays. Antisera with mixed antibody specificities and autoantibodies were also tested. Soluble Scianna protein inhibited specifically antibodies to the high-prevalence Scianna antigens Sc1 and Sc5. No antibodies were neutralized that were directed to the low-prevalence Sc2 antigen or to a large representative set of antigens from other blood group systems. Clinically relevant antibodies could be identified despite being masked by anti-Sc1 and anti-Sc5. A mixture of Scianna and JMH proteins allowed detecting a common antibody despite the presence of antibodies to high-prevalence antigens of the Scianna or JMH blood group systems. Antibody detection systems comprising soluble recombinant Scianna protein provide an easy single-step method for detection and identification of antibodies to high-prevalence Scianna antigens. Reagents with Scianna and other recombinant blood group proteins and mixtures of such proteins would be useful routine reagents in immunohematology.

Value of Candida serum markers in patients with invasive candidiasis after myeloablative chemotherapy

Invasive Candida infections are associated with a significant morbidity and mortality. Detection of circulating biomarkers has been shown to precede conventional diagnostic methods, which is important in improving outcome. We investigated the performance of multiple biomarkers using Candida antigen and anti- Candida antibody detection systems of Platelia and Serion and β- d-glucan detection in serial serum samples from patients, treated for leukemia, with invasive candidiasis. The performance of single assays and combined detection appeared different for patients with 1 or more episodes of neutropenia and is therefore related to the phase of therapy for the underlying leukemia of the patient. These new insights may help to optimize the diagnostic strategies for the diagnosis of invasive candidiasis.

Concentration of antibodies to extractable nuclear antigens and disease activity in systemic lupus erythematosus

A hallmark of systemic lupus erythematosus (SLE) is the production of autoantibodies directed against intracellular antigens. Antibodies to double stranded DNA (dsDNA) are most closely associated with the clinical manifestations of the condition and appear to have a direct role in pathogenesis. On the contrary, the relationship between disease activity in SLE and anti-extractable nuclear antigen (ENA) antibodies has not been well demonstrated. Despite this, commercial assays for the quantification of anti-ENA antibodies are now widely available, although their usefulness in clinical practice is not known. The aim of this study was to investigate whether there is an association between disease activity in SLE and concentrations of individual anti-ENA antibodies. A prospective 2-year study of 45 patients with SLE, known to be positive for at least one anti-ENA antibody, was performed. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Anti-ENA antibodies were quantified using a commercial antibody detection system. A total of 45 patients were studied over a 2-year period (median number of assessments 5, range 2-9). Of them 29 patients were positive for Ro, 8 for La, 9 for Sm and 27 for RNP antibodies. In the population as a whole, there was a weak relationship between peak SLEDAI score and anti-Sm concentration (r = 0.57, NS), but no relation with the other anti-ENA antibodies. In a small number of patients, there appeared to be either a positive (Ro, Sm) or negative (La, Sm, RNP) association between ENA antibody concentration and disease activity over time; however, this was not apparent for the majority of individuals. These results show that in SLE, clinically significant changes in disease activity do not correlate well with concentrations of anti-ENA antibodies, either within the population as a whole or on an individual basis. Repeated quantitative measurement of anti-ENA antibodies does not therefore appear to provide useful additional information in assessing disease activity in SLE. The widespread application of commercial quantitative assays to routine clinical practice is not recommended.

Anaplastic large cell lymphoma with cardiac metastasis. A case report

We present a case of an anaplastic large cell lymphoma (ALCL) in a 61 years old patient with multiple metastases including cardiac metastasis (left posterior ventricular wall). Anaplastic large cell lymphoma, ALCL is a rare condition in adult and cardiac metastasis are extremely rare. Most probably the metastatic spread was a hematogenous invasion via the coronary arteries. Initially a pulmonary small cell carcinoma was diagnosed and the patient underwent a lung partial resection. However, H&E histopathology demonstrated a specific pattern for the tumoral cells (“hallmark” cells); further immune histochemistry (RTU Peroxidase Detection System for Novocastra RTU Primary Antibodies, RE7100-CE), proved to be positive for CD 30, identifying an anaplastic large cell lymphoma, ALCL. Other markers were searched on using IHC techniques in order to confirm the affiliation to the lymphoma family (CD30, CD 3, CD 20 positive) and to exclude the affiliation to the pulmonary carcinoma and neuroendocrine tumors (Pan CK AE1-AE3, TTF1, CK 7, CD56 negative) or pluripotential hemopoietic stem cells or vascular-associated tissue including monocytes/macrophages (CD 34, CD 68 negative).

A new automatic system for Cytomegalovirus antibodies screening: preliminary evaluation

The aim of our study was to evaluate the automated enhanced-chemiluminescence VITROS EciQ (Ortho) system compared to the EIA ALISEI (Radim) system for the first level detection of CMV IgG and IgM antibodies. Samples derived from 95 immunocompetent patients were examined. Both systems showed similar results for CMV IgG: 63 samples were positive, 28 negative and 4 samples were weakly positive. The four weakly positive samples were subsequently confirmed with the LIAISON (DiaSorin) system. Agreement was 91% concerning CMV IgM detection: 79 samples were negative, 7 positive and 9 samples releved discrepancy between the two systems.When these 9 cases were tested with LIAISON system for CMV IgM and IgG and the CMV IgG avidity test, data revealed complete agreement between VITROS EciQ and LIAISON systems.VITROS EciQ system seems to be a valuable system for the first level diagnosis of CMV infection.

24-P: Anti-latex antibodies and sera with high backgrounds do not appear to interfere with DynaChip™ hla antibody detection system

24-P: Anti-latex antibodies and sera with high backgrounds do not appear to interfere with DynaChip™ hla antibody detection system

Helicobacter pylori infection‐negative gastric cancer in Japanese hospital patients: Incidence and pathological characteristics

We used Helicobacter pylori sero-positivity and mucosal atrophy as detected by the serum pepsinogen method to identify H. pylori infection-negative gastric cancer patients with or without atrophy. One hundred and six of 748 (14.2%) primary gastric cancer patients were infection-negative by a serum antibody detection system. Further, 121 (16.2%) of the 748 were negative for gastric mucosal atrophy by the pepsinogen method, of whom 15/748 (2.0%) were H. pylori-negative by pepsinogen I level (>70 ng/mL) and pepsinogen I/II ratio (>3.0). Twenty-seven of 782 (3.6%) gastric cancer patients were H. pylori-negative by antibodies and severe atrophy as determined by pepsinogen I level (<30 ng/mL) and pepsinogen I/II ratio (<2.0). H. pylori-negative gastric cancer patients with severe atrophy likely had a previous infection. These results indicate that the actual number of H. pylori-negative patients is 2.0% at minimum and 10.6% (14.2% minus 3.6%) at maximum in the general Japanese population. Five of 15 (33%) cases displaying neither anti-H. pylori antibodies nor atrophy were intestinal-type and 10 (67%) were diffuse-type adenocarcinomas. Thirteen surgical patients with primary gastric cancer displaying neither antibodies nor mucosal atrophy were further analyzed for pathological and phenotypic characteristics. The mucin phenotype was divided into four gastric, five gastric and intestinal, two intestinal and two null types, independent of histological classification. Intestinal phenotype elements were detected by Cdx2 immunohistochemical methods in nine of 13 (70%) cases examined. We conclude that a small fraction of gastric cancer patients displayed multifactorial carcinogenesis without H. pylori infection, indicating that gastric cancer risk still exists in the absence of H. pylori infection, at an incidence of 2.0% at minimum and 10.6% at maximum in the general Japanese population.

Pathophysiology and diagnosis of allograft rejection in pediatric heart transplantation

Attempts at improving diagnostic monitoring of allograft rejection continue at a steady pace. The main issue remains whether these methods will replace the standard method of tissue histology from endomyocardial biopsy. The aim in the development of novel techniques to diagnose rejection is the application of noninvasive methods. These range from echocardiography, biomarkers, and genomic profiling to more sensitive antibody detection systems. No single method has the accuracy to be a stand-alone test. Methods of assessing graft dysfunction alone may not be accurate enough in this population. Nonetheless, these and other clinical descriptive studies help us better understand the rejection process in pediatric recipients. Solid organ transplantation creates the ideal medium where basic science meets clinical science. Clinical cardiology continues to improve on ways to assess organ dysfunction, but to correlate these methods to early graft rejection, immunobiologic techniques will probably need to be incorporated.

Detection of trypanosome‐specific antibodies in saliva, towards non‐invasive serological diagnosis of sleeping sickness

The detection of trypanosome-specific antibodies in saliva is technically feasible, and, if clinically validated, could become an attractive option for non-invasive diagnosis of sleeping sickness. We wanted to optimize the test format of an enzyme-linked immunosorbent assay (ELISA)-based antibody detection system. Different ELISA formats for antibody detection in serum and saliva were developed and standardized. Saliva and serum samples were collected from 78 patient and 128 endemic control samples, and sensitivity and specificity of saliva ELISAs, serum ELISAs and the card agglutination test for trypanosomiasis (CATT), were evaluated. All ELISA formats showed sensitivity and specificity above 90%. Saliva ELISAs showed a similar test performance as serum ELISAs and the CATT on whole blood or serum. This study confirms the potential of trypanosome-specific antibody detection in saliva.

Simultaneous five cell‐lineage flow cytometric analysis system for detection of leucocyte antibodies

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive.

Non-capsid proteins to identify foot-and-mouth disease viral circulation in cattle irrespective of vaccination

The ability of foot-and-mouth disease virus (FMDV) to establish subclinical and even persistent infection, the so called carrier state, imposes the need to reliably demonstrate absence of viral circulation, to monitor the progress of control measures, either during eradication programs or after reintroduction of virus in free areas. This demonstration becomes critical in immunized populations, because of the concern that silent viral circulation could be hidden by immunization. This concern originates from the fact that vaccination against foot-and-mouth disease (FMD) protects against clinical disease, but not necessarily against subclinical infection or establishment of the carrier state in cattle. A novel approach, developed and validated at PANAFTOSA during the 1990s, based on an immunoenzymatic system for detection of antibodies against non-capsid proteins (NCP) has proven valuable for monitoring viral circulation within and between herds, irrespective of the vaccination status. Antibodies against NCP are induced during infection but, in principle, not upon vaccination. The validation of this system led to its international recognition as the OIE index test. The fitness of this serosurvey tool to assess viral circulation in systematically vaccinated populations was demonstrated through its extensive application in most regions in South America. The experience attained in these regions supported the incorporation of the “free of FMD with vaccination” provisions into the OIE code. Likewise, it opened the way to alternatives to the “stamping out” policy. The results gave input to an old controversy related to the real epidemiological significance, if any, of carrier animals under the vaccination conditions in South America, and supported the development of recommendations and guidelines that are being implemented for serosurveys that go with control measures in vaccinated populations.

Standardization of an indirect PTA-ELISA for detection ofFusarium spp. in infected grains

Plant pathogenic fungi of the genusFusarium cause agriculturally important diseases of small grain cereals and maize. Especially the contamination of grains with the mycotoxin deoxynivalenol (DON) is harmful for animals and humans. For quantification of the severity ofFusarium head blight (FHB) in resistance evaluation and selection of cereals a fast, economical and reliable method is essential. Immunological methods appear to be particularly suitable for such an assessment. The results demonstrate that a selected antiserum was appropriate for the detection ofFusarium-exoantigens (ExoAg) in cereal grains by an indirect ELISA-format, although a discrimination between variousFusarium-species was not possible. The polyclonal antibody detection system was optimized for different parameters and a standard test protocol was elaborated.

Humoral immune response to recombinant viral NS3 protein in patients with hepatitis C

Full-length recombinant NS3 protein was used in a test system for detection of specific antibodies in the sera from patients with hepatitis C. Possible antigenic determinants in NS structure were predicted. It was demonstrated that serological analysis requires enzyme immunoassay with full-length NS3.

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service