Antibodies In Body Fluids Research Articles

A Promising Tool in Serological Diagnosis: Current Research Progress of Antigenic Epitopes in Infectious Diseases.

Infectious diseases, caused by various pathogens in the clinic, threaten the safety of human life, are harmful to physical and mental health, and also increase economic burdens on society. Infections are a complex mechanism of interaction between pathogenic microorganisms and their host. Identification of the causative agent of the infection is vital for the diagnosis and treatment of diseases. Etiological laboratory diagnostic tests are therefore essential to identify pathogens. However, due to its rapidity and automation, the serological diagnostic test is among the methods of great significance for the diagnosis of infections with the basis of detecting antigens or antibodies in body fluids clinically. Epitopes, as a special chemical group that determines the specificity of antigens and the basic unit of inducing immune responses, play an important role in the study of immune responses. Identifying the epitopes of a pathogen may contribute to the development of a vaccine to prevent disease, the diagnosis of the corresponding disease, and the determination of different stages of the disease. Moreover, both the preparation of neutralizing antibodies based on useful epitopes and the assembly of several associated epitopes can be used in the treatment of disease. Epitopes can be divided into B cell epitopes and T cell epitopes; B cell epitopes stimulate the body to produce antibodies and are therefore commonly used as targets for the design of serological diagnostic experiments. Meanwhile, epitopes can fall into two possible categories: linear and conformational. This article reviews the role of B cell epitopes in the clinical diagnosis of infectious diseases.

Nanotechnology: A Potential Weapon to Fight against COVID-19.

The COVID‐19 infections have posed an unprecedented global health emergency, with nearly three million deaths to date, and have caused substantial economic loss globally. Hence, an urgent exploration of effective and safe diagnostic/therapeutic approaches for minimizing the threat of this highly pathogenic coronavirus infection is needed. As an alternative to conventional diagnosis and antiviral agents, nanomaterials have a great potential to cope with the current or even future health emergency situation with a wide range of applications. Fundamentally, nanomaterials are physically and chemically tunable and can be employed for the next generation nanomaterial‐based detection of viral antigens and host antibodies in body fluids as antiviral agents, nanovaccine, suppressant of cytokine storm, nanocarrier for efficient delivery of antiviral drugs at infection site or inside the host cells, and can also be a significant tool for better understanding of the gut microbiome and SARS‐CoV‐2 interaction. The applicability of nanomaterial‐based therapeutic options to cope with the current and possible future pandemic is discussed here.

Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

AbstractEasy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.

Harnessing Effective Molarity to Design an Electrochemical DNA-based Platform for Clinically Relevant Antibody Detection.

Easy-to-use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi-step, time-consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity-driven electrochemical DNA-based detection of target antibodies. We show a highly selective, signal-on DNA-based sensor that takes advantage of antibody-binding-induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.

Serological Survey of Lyssaviruses in Polish Bats in the Frame of Passive Rabies Surveillance Using an Enzyme-Linked Immunosorbent Assay.

Background: Bats are known to host a number of nonpathogenic viruses, as well as highly pathogenic viruses causing fatal diseases like rabies. Serological surveys as part of active and passive bat rabies surveillance mainly use seroneutralization assays, demonstrating the presence of lyssavirus-specific antibodies in a variety of European bats, particularly against European bat lyssaviruses type 1 (EBLV-1). Here, we present the first serological survey in European bats of this kind during which European bats from Poland collected in the frame of passive rabies surveillance between 2012 and 2018, as well as Serotine bats (Eptesicus serotinus) and North American Big Brown bats (Eptesicus fuscus) from previous experimental studies, were tested using a commercial ELISA kit for the detection of anti-lyssavirus antibodies. Results: Lyssavirus-specific antibodies were detected in 35 (30.4%) out of 115 Polish bats of both sexes, representing nine out of 13 identified bat species endemic mainly to Central Southern Europe and Western Asia, i.e., Eptesicus serotinus, Nyctalus noctula, Myotis daubentonii, Plecotus auritus, Vespertillo murinus,Pipistrellus pipistrellus, Pipistrellus pipilstrellus/Pipistrellus pygmaeus, Myotis brandtii, and Barbastella barbastellus. Seroprevalence was highest in bat species of Nyctalus noctula, Eptesicus serotinus, Plecotus auritus, and Myotis daubentonii. More than 60% of the ELISA seropositive bats originated from the voivodeships of Silesia, Lower-Silesian, Warmian-Mazurian, and Mazowian. Rabies-specific antibodies were also found in Eptesicus fuscus bats from North America. Conclusions: The study demonstrates the principal application of the BioPro Rabies ELISA Ab Kit for the detection of anti-lyssavirus specific antibodies in body fluids and serum samples of bats. However, results may only be reliable for North American bats, whereas interpretation of results for European bats per se is difficult because proper validation of the test is hampered by the protected status of these species.

Long-term kinetics of Zika virus RNA and antibodies in body fluids of a vasectomized traveller returning from Martinique: a case report

BackgroundThe magnitude of the current Zika virus (ZIKV) epidemic has led to a declaration of a Public Health Emergency of International Concern by the WHO. Findings of viable viral particles in semen for several weeks are corroborating reports of sexual transmission of ZIKV. Serious consequences of a positive diagnostic result particularly in the pregnant patient are calling for precise diagnostic tools also at later time points after infection. Currently, recommendations suggest a diagnostic period of direct viral detection of 5 to 7 days after onset of symptoms in serum or plasma, and up to 3 weeks in urine samples.Case presentationA vasectomized 41-year-old German returning from Martinique presented at the outpatient clinic of the Department for Infectious Diseases and Tropical Medicine, Munich, with subfebrile temperature, rash, malaise, severe retro-orbital pain and occipital lymphadenopathy. The main complaints resolved after ten days without specific treatment. We are reporting on clinical course and results of direct and indirect detection methods of ZIKV in different sample types including whole blood, ejaculate, urine, serum, plasma and saliva samples up to 119 days post symptom onset. Ejaculate samples remained PCR positive for ZIKV until day 77, whole blood samples until day 101.ConclusionsThe case presentation adds to the still limited knowledge of kinetics of detection of ZIKV by direct as well as indirect methods. Here, a complete data set including results from PCR, serology and cell culture is provided allowing an improved evaluation of optimum diagnostic periods for testing a variety of sample types. Moreover, a high viral load of ZIKV RNA was detected in ejaculate of the vasectomized patient. This finding sheds new light on the possible localizations of ZIKV replication in the human male reproductive tract.

Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry

Antibacterial antibody responses that target surfaces of live bacteria or secreted toxins are likely to be relevant in controlling bacterial pathogenesis. The ability to specifically quantify bacterial-surface-binding antibodies is therefore highly attractive as a quantitative correlate of immune protection. Here, binding of antibodies from various body fluids to pure-cultured live bacteria is made visible with fluorophore-conjugated secondary antibodies and measured by flow cytometry. We indicate the necessary controls for excluding nonspecific binding and also demonstrate a cross-adsorption technique for determining the extent of cross-reactivity. This technique has numerous advantages over standard ELISA and western blotting techniques because of its independence from scaffold binding, exclusion of cross-reactive elements from lysed bacteria and ability to visualize bacterial subpopulations. In addition, less than 10(5) bacteria and less than 10 μg of antibody are required per sample. The technique requires 3-4 h of hands-on experimentation and analysis. Moreover, it can be combined with automation and mutliplexing for high-throughput applications.

Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections.

Development of an Enzyme-Linked Immunosorbent Assay System to Determine the Presence of Antibodies Specific for Taxane Structures

Taxanes, which are widely used in treatment of numerous cancer types, are well-known to induce hypersensitivity reactions (HSR), especially in the case of paclitaxel. Although the cause of the HSR is commonly thought to be a non-immunological direct effect of the diluent which is used to dissolve paclitaxel, some reports suggest the possibility of the presence of an immunological reaction to the common taxane structure. The aim of this study was to establish a method to determine the presence of anti-taxane antibodies in body fluids of patients who have previously received paclitaxel, in order to estimate the risk of the occurrence of HSR to other taxane compounds, such as docetaxel. To prepare an enzyme-linked immunosorbent assay (ELISA) plate for determining taxanes, 10-deacetylbaccatin III (DAB) was first succinylated by use of dimethylaminopyridine and succinic anhydride in dried pyridine. After the succinylation reaction, three different products were obtained, and these were confirmed as 7-succinoyl DAB (7-DAB), 10-succinoyl DAB (10-DAB), and 7,10-disuccinoyl DAB (7,10-DAB) by (1)H-NMR analysis. Each of these three products was conjugated with bovine serum albumin (BSA), and adsorbed on an ELISA plate. By using a commercially available anti-taxane monoclonal antibody as a model antibody, the detection limit of the anti-taxane antibodies on the 7-DAB-BSA-, 10-DAB-BSA-, and 7,10-DAB-BSA-conjugated ELISA plate was estimated as 0.3, 1 and 10 ng/ml, respectively. The ELISA system established in this study may therefore be useful for estimating the risk of HSR to taxanes in a patient prior to the use of these drugs.

Decoding Serological Response to Candida Cell Wall Immunome into Novel Diagnostic, Prognostic, and Therapeutic Candidates for Systemic Candidiasis by Proteomic and Bioinformatic Analyses

In an effort to bring novel diagnostic and prognostic biomarkers or even potential targets for vaccine design for systemic candidiasis (SC) into the open, a systematic proteomic approach coupled with bioinformatic analysis was used to decode the serological response to Candida wall immunome in SC patients. Serum levels of IgG antibodies against Candida wall-associated proteins (proteins secreted from protoplasts in active wall regeneration, separated by two-dimensional gel electrophoresis, and identified by mass spectrometry) were measured in 45 SC patients, 57 non-SC patients, and 61 healthy subjects by Western blotting. Two-way hierarchical clustering and principal component analysis of their serum anti-Candida wall antibody expression patterns discriminated SC patients from controls and highlighted the heterogeneity of their expression profiles. Multivariate logistic regression models demonstrated that high levels of antibodies against glucan 1,3-beta-glucosidase (Bgl2p) and the anti-wall phosphoglycerate kinase antibody seropositivity were the only independent predictors of SC. Receiver operating characteristic curve analysis revealed no difference between their combined evaluation and measurement of anti-Bgl2p antibodies alone. In a logistic regression model adjusted for known prognostic factors for mortality, SC patients with high anti-Bgl2p antibody levels or a positive anti-wall enolase antibody status, which correlated with each other, had a reduced 2-month risk of death. After controlling for each other, only the seropositivity for anti-wall enolase antibodies was an independent predictor of a lower risk of fatality, supporting that these mediated the protective effect. No association between serum anti-cytoplasmic enolase antibody levels and outcomes was established, suggesting a specific mechanism of enolase processing during wall biogenesis. We conclude that serum anti-Bgl2p antibodies are a novel accurate diagnostic biomarker for SC and that, at high levels, they may provide protection by modulating the anti-wall enolase antibody response. Furthermore serum anti-wall enolase antibodies are a new prognostic indicator for SC and confer protection against it. Bgl2p and wall-associated enolase could be valuable candidates for future vaccine development.

Principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibodies in body fluids

The sensitivity and specificity of enzyme immunoassay for antibodies in body fluids have been improved considerably by transferring the complex of labelled antigen and antibody to be detected from one solid phase to another to eliminate interfering substance(s) in the samples (immune complex transfer enzyme immunoassay). Usefulness of the new method has been tested for antibodies in serum as well as in urine. Anti-thyroglobulin IgG could be measured not only in serum of all patients with autoimmune thyroid diseases and almost all healthy subjects but also in the urine of most of the patients. Anti-HTLV-I IgG was unequivocally demonstrated in some of sera, which were indeterminate or negative by Western blotting, and diagnosis of HIV infection by detecting anti-HIV IgG in urine and saliva would be possible with higher reliability than by conventional methods.

Immunoglobulin G antibody capture enzyme-linked immunosorbent assay: a versatile assay for detection of anti-human immunodeficiency virus type 1 and 2 antibodies in body fluids.

In tests on specimens of dried blood, saliva, and urine from 55 human immunodeficiency virus (HIV)-seropositive and 55 HIV-seronegative patients, an immunoglobulin G capture enzyme immunoassay for the detection of antibodies to HIV types 1 and 2, GACELISA, gave 109 of 110, 109 of 109, and 109 of 110 correct results, respectively. This performance, achieved in a laboratory previously unfamiliar with the assay, suggests that GACELISA is a useful new epidemiological tool for the study of HIV infection, equally applicable to all three kinds of specimen.

In vitro synthesis of antibodies to myelin antigens by Epstein-Barr virus-transformed B lymphocytes from patients with neurologic disorders

Anti-myelin antibodies can be found in sera from patients with neurologic disorders of suspected immune-mediated pathogenesis such as multiple sclerosis and inflammatory polyneuropathies. However, the specificity of these findings is controversial. In the present study, in vitro synthesis of antibodies to myelin components was compared to their presence in sera in diverse neurological disorders. Epstein-Barr virus was used to activate B lymphocytes for in vitro antibody production. Anti-myelin basic protein and anti-galactocerebroside antibodies were secreted in vitro by B lymphocytes derived from patients with neurological disorders of various etiologies and pathogenetic mechanisms. Anti-myelin basic protein antibodies were detected in many more cell culture supernatants than in sera from the same patients. In vitro secretion of antibodies to myelin antigens, as well as the presence of these antibodies in body fluids, are apparently non-specific for disease type and may be secondary to neural tissue damage.

Diagnosis of transplacentally induced toxoplasmosis in pigs

SUMMARY Seventeen sows were fed 1,000 Toxoplasma gondii oocysts of isolates GT-1 or PT-1 at 32 to 92 days of gestation, and the products of conception were examined for T gondii anibodies and parasites. Twelve of these sows were euthanatized near term between 21 and 62 days after being fed T gondii; fetal body fluids or fetal sera were examined for agglutinating T gondii antibodies, and tissues were bioassayed in mice for T gondii parasites. Six sows produced pigs that had been transplacentally infected with T gondii; one of them aborted a T gondii-infected fetus 17 days after ingesting oocytes. Agglutinating antibodies were detected in fetuses infected in utero, but transplacental transfer of T gondii antibodies was not observed in noninfected fetuses. Transcolostrally acquired T gondii antibodies disappeared by 3 months of age. Diagnosis of transplacental toxoplasmosis was confirmed on the basis of detection of T gondii organisms in fetal tissues by use of histologic examination and bioassay in mice. In conclusion, finding of T gondii antibodies in body fluids could serve as a rapid screening test for transplacental T gondii infection in pigs.

Class-specific antibodies to Streptococcus mutans in human serum, saliva and breast milk

Previous techniques used for the detection and quantitation of antibodies in body fluids may be inappropriate where only small volumes are available, or may not be sensitive enough to detect low levels of specific antibodies. An indirect ELISA technique has successfully been employed to estimate class-specific antibody levels to Streptococcus mutans in serum and secretions in a group of mothers and their neonates, and an attempt has been made to relate such levels to the presence or absence of active caries in the mothers. A high maternal serum IgG antibody level appears to exert a protective effect against dental caries. Antibody levels in maternal saliva and colostrum/breast milk showed no differences between the 2 groups. The presence of active caries in mothers was associated with an elevated IgA antibody level in neonatal saliva. Although ELISA permitted the detection of low levels of antibody in the small volumes of neonatal saliva collected, a further increase in sensitivity and specificity of the assay would be advantageous.

Solid phase antigen luminescent immunoassays (SPALT) for the determination of insulin, insulin antibodies and gentamicin levels in human serum.

We describe an interesting and novel alternative to conventional immunoassay techniques for the measurement of antigens and antibodies in body fluids. The label used for all assays is a pyruvate kinase-IgG conjugate of the relevant second (species-specific) antibody. All assays follow the same principle in which a solid phase antigen is used to adsorb unreacted first (substance-specific) antibody following a conventional antibody-antigen reaction in a liquid phase. After washing, the solid phase antigen-first antibody is allowed to react with the labelled second antibody. The solid phase is then washed and the pyruvate kinase bound to the solid phase is used to generate ATP which is measured kinetically in a luminometer. Assays are described for insulin, insulin antibodies and gentamicin to demonstrate both the versatility and sensitivity of this type of assay. The insulin assay had a lower detection limit of under 0.25 microU per tube and was comparable with the radioimmunoassay used for routine purposes both in sensitivity and reproducibility. The insulin antibody assay correlated well with the radiometric determination used routinely in the laboratory. The gentamicin assay correlated well with the routine commercial radioimmunoassay and also had comparable coefficients of variation. In all cases, the inter- and intra-assay variation was under 10% in the range of interest.

Enzyme-linked immunosorbent assays in murine cryptococcosis.

In this investigation, enzyme-linked immunosorbent assay (ElISA) procedures were used to study the time of appearance and the duration of demonstrable antigen and antibody in body fluids of mice with disseminated cryptococcosis. The ELISA antigen procedure detected cryptococcal capsular polysaccharide (CCP) in the serum and urine of infected mice 3 days after infection--4 days before it could be demonstrated by the latex agglutination procedure. ELISA-reactive antibody was present throughout the course of infection (mean death time, 32 days), whereas antibody was not detected by whole cell agglutination after day 20. High serum concentrations of CCP (titers to 64,000) persisted throughout the course of infection, while antibody declined to low levels with progression of disease. ELISA provides a sensitive system for quantitation and monitoring of antigen (CCP) processing and clearance (or storage), and for cryptococcal antibody formation in progressive cryptococcosis.

The two-cross immunodiffusion technique: Diffusion coefficients and precipitating titers of IgG in human serum and rabbit serum antibodies

The two-cross technique, a new two-dimensional double-diffusion technique in gelplates, has been applied for simultaneous determination of precipitating titers and diffusion coeffients of antigen and antibody in body fluids. The advantage of this technique is that it works without using any standard solution and ensures conditions of “time-invariant sink”. The theory of the technique has been verified by experimental results on the precipitating system human serum-rabbit anti-human IgG in phosphate-buffered saline solution at pH 7.4. The results obtained using several modes of calculations from experimental parameters have been compared and found satisfactory. The accuracy and reproducibility of the results have been confirmed. It has been found that at 20°C the diffusion coefficient of human IgG in 10-times-diluted serum is (4.4 ± 0.2) × 10 −7 cm 2 s −1, while the diffusion coefficient of rabbit anti-human IgG in a purified preparation is (2.9 ± 0.2) × 10 −7 cm 2 s −1. The critical precipitating concentration of human IgG against rabbit anti-human IgG is invariable to concentration and amounts to 0.174 ± 0.03 mg/100 ml at pH 7.4.

Immunofluorescence as a diagnostic aid in cryptococcal meningitis

This paper concerns a patient who had long standing rheumatoid arthritis for which she received prednisone and chlorambucil. Steroid induced diabetes mellitus and subsequent cryptococcal meningitis caused her death. Circulating cryptococcal antigen was demonstrated in the patient's cerebrospinal fluid and blood by the latex cryptococcal antigen test. A new rapid method utilizing immunofluorescence to identify cryptococcal antigen and antibody in body fluids was utilized. The indirect fluorescent antibody test for detection of Cryptococcus was positive, utilizing the patient's serum antibody and the patient's cryptococcal organisms as the antigen. This test is a more specific and more sensitive diagnostic test than current serologic tests.

Immunofluorescence as a Diagnostic Aid in Cryptococcal Meningitis

To the Editor.— Crytpococcosis involves the lungs and the central nervous system. The causative organism,Cryptococcus neoformans, is frequently an opportunistic, secondary invader, associated with severe debilitating illness and drug treatments. The diagnosis depends on culture procedures and biochemical techniques performed on the positive cultures, as well as serological tests, tube agglutination, complement-fixation, immunodiffusion, and latex cryptococcal antigen agglutination tests.1-3 A patient with long-standing rheumatoid arthritis had received prednisone and chlorambucil and developed steroid-induced diabetes mellitus and subsequentCryptococcusmeningitis, which caused her death. A new rapid method utilizing indirect immunofluorescence to identify cryptococcal antigen and antibody in body fluids is described here. Report of a Case.— A 61-year-old white woman with a long history of rheumatoid arthritis since age 14 had been treated with prednisone and for a short period with chlorambucil. Adult onset diabetes mellitus developed that was probably steroid induced. Chlorambucil therapy was begun in early