Variety Of Immunoassays Research Articles

Advances in D-dimer testing: progress in harmonization of clinical assays and innovative detection methods.

The D-dimer is a sensitive indicator of coagulation and fibrinolysis activation, especially valuable as a biomarker of intravascular thrombosis. Measurement of plasma D-dimer levels plays a crucial role in the diagnosis and monitoring of conditions such as deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. A variety of immunoassays, including enzyme-linked immunosorbent assays, latex-enhanced immunoturbidimetric assays, whole-blood aggregation analysis, and immunochromatography assays, are widely used in clinical settings to determine D-dimer levels. However, the results obtained from different D-dimer assays vary significantly. These assays exhibit intra-method coefficients of variation ranging from 6.4% to 17.7%, and the measurement discrepancies among different assays can be as high as 20-fold. The accuracy and reliability of D-dimer testing cannot be guaranteed due to the lack of an internationally endorsed reference measurement system (including reference materials and reference measurement procedures), which may lead to misdiagnosis and underdiagnosis, limiting its full clinical application. In this review, we present an in-depth analysis of clinical D-dimer testing, summarizing the existing challenges, the current state of metrology, and progress towards harmonization. We also review the latest advancements in D-dimer detection techniques, which include mass spectrometry and electrochemical and optical immunoassays. By comparing the basic principles, the definition of the measurand, and analytical performance of these methods, we provide an outlook on the potential improvements in D-dimer clinical testing.

B-087 The Antifouling Properties of BSA on Magnetic Beads are Affected by BSA Grade, Bead Type, and pH

Abstract Background BSA (bovine serum albumin) is a protein that is widely used for its antifouling properties in a variety of immunoassays. One such immunoassay is a bead-based ELISA (enzyme-linked immunosorbent assay), which can provide quantitative detection of many diverse analytes. BSA increases the sensitivity of a bead-based ELISA by coating the beads and blocking nonspecific interactions, thereby lowering background noise. However, the BSA grade chosen for the assay is frequently based on experience, as there is a lack of understanding of how BSA grades interact with different bead surfaces under various assay conditions. The objective of this study is to examine if different BSA grades work better with particular bead chemistries and how pH influences those interactions. Specifically, the aim is to quantify the amount of BSA on ELISA beads and assess the blocking of nonspecific antibody signal with different bead types, BSA grades, and pH. Methods The mass of BSA binding to beads was measured by heating BSA-coated beads in 1x SDS loading dye, then running on a denaturing PAGE gel. Total protein was measured by densitometry against a standard curve using ImageJ. The amount of blocking was measured using a modified bead-based ELISA, comparing the amount of antibody-HRP binding to the beads in the presence and absence of BSA. The amount of antibody-HRP was measured using a SpectraMax Plus 384 plate reader against a standard curve. All data were analyzed using GraphPad Prism and presented as the average of at least three independent replicates with error bars representing standard deviation. Conditions tested include different Dynabeads (hydrophilic vs. hydrophobic - Dynabeads M-270 Carboxylic Acid and M-280 Tosylactivated), BSA grade (fatty acid (FA) vs. fatty acid-free (FAF) - Proliant SKUs 68100 and 68700), and pH (7 vs. 5.2). Results The choice of pH is more influential than the BSA grade on bead surface coating and blocking, regardless of bead type. Lowering the pH from 7 to 5.2 increases the mass of BSA on the beads ∼1.7-fold for hydrophobic beads and ∼10-fold for hydrophilic beads. However, the mass of BSA on the beads is reduced after the pH returns to 7, indicating the increased BSA binding at pH 5.2 is reversible. We also observed BSA decreases nonspecific antibody-bead interactions by up to 90%, depending on assay conditions. Though the amount of BSA coating the beads between FA and FAF grade BSA is not significant, FAF grade BSA blocks the antibody-bead interactions ∼10% more than FA grade BSA, suggesting FAF grade BSA has better antifouling properties. Conclusion Herein, we show the presence of BSA reduces the noise within bead-based ELISAs by binding to the bead and blocking nonspecific antibody-bead interactions, regardless of BSA grade, bead type, and pH. FAF grade BSA demonstrates slightly better blocking than FA grade BSA, but both BSA grades decreased nonspecific antibody-bead interactions. Further, BSA shows antifouling properties at the typical assay pH of 7, but lowering the pH to 5.2 can significantly increase BSA bead coating.

Nets of biotin-derived gold nanoparticles as a label for the C-reactive protein immunoassay.

This study presents a promising approach for the one-pot generation of the biotin-derived gold nanoparticles (GNPs@biotin). We report a direct method for the reduction of tetrachloroauric acid with biotin and generation of the labels due to nets formed via biotin-streptavidin interactions. The synthesized GNPs@biotin have a characteristic plasmon maximum, excellent colloidal stability, and streptavidin coupling efficiency. The size of the GNPs@biotin:streptavidin nets and the efficiency of interaction with specific antibodies can be easily customized by the component concentrations and time of their interaction. Moreover, the proposed labels require no additional reagents or manipulations for the synthesis, separation, or purification. The developed labels were applied for the detection of the model antigen of C-reactive protein (CRP) as a major inflammation biomarker. The assembling labels demonstrated a competitive advantage limit of CRP detection (LOD) of 1.2ng/mL and a limit of quantification (LOQ) of 3.9ng/mL in human plasma comparable to classical immunoassays. Moreover, the proposed approach is universal and can be potentially applied for the quantitative determination of other biomarkers in a variety of immunoassays in a combination with specific biotinylated antibodies.

Recreation of Fetal Neonatal Alloimmune Thrombocytopenia in CRISPR/Cas9 Gene-Edited Mice Expressing the Human Platelet Alloantigen, HPA-1a

Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening alloimmune disorder caused by maternal antibodies directed against fetal platelet alloantigens. The human platelet alloantigens, (HPA)-1a/HPA-1b (also known as the PlA1 alloantigen system), residing on the integrin β3 subunit (= platelet membrane glycoprotein IIIa), is the most frequently implicated HPA for causing FNAIT in Caucasians, and a single Leu33Pro amino acid polymorphism is responsible for generating the HPA-1a and HPA-1b epitopes. Neither human nor mouse anti-HPA-1a antibodies recognize murine GPIIIa due to amino acid differences both within and surrounding the polymorphic amino acid, which lies within a long flexible loop in PSI domain of the molecule. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically-important platelet antigen. We employed CRISPR/Cas9 gene editing technology to generate mice expressing the human HPA-1a allogeneic epitope. Four critical amino acid substitutions (A30P32L33D39) were introduced into the PSI domain of murine GPIIIa. The resulting humanized form of murine GPIIIa, termed APLD, reacted in a variety of immunoassays with both murine monoclonal (mAb) and human maternal polyclonal antibodies specific for the human HPA-1a epitope. Intraperitoneal injection of an anti-HPA-1a mAb induced thrombocytopenia in APLD, but not wild-type, mice. Importantly, wild-type mice immunized with murine APLD transgenic platelets generated a high-titer HPA-1a-specific antibody response, demonstrating that the humanized form of murine GPIIIa is immunogenic in mice. Finally, pre-immunized female wild-type mice bred with APLD males gave birth to neonatal mice that exhibited profound thrombocytopenia, with the maternal titer directly related to the degree of thrombocytopenia in the neonates. Recapitulating the essential features of FNAIT in mice will facilitate the investigation of a wide-range of clinically-important issues in the diagnosis, treatment, and management of platelet alloimmune disorders. DisclosuresNo relevant conflicts of interest to declare.

Genetically functionalized ferritin nanoparticles with a high-affinity protein binder for immunoassay and imaging

Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas.

Next generation vaccines

Next generation vaccines

Identification of porcine reproductive and respiratory syndrome virus ORF1a-encoded non-structural proteins in virus-infected cells

The porcine reproductive and respiratory syndrome virus (PRRSV) replicase gene consists of two large ORFs, ORF1a and ORF1b, the latter of which is expressed by ribosomal frameshifting. The ORF1a-encoded part of the resulting replicase polyproteins (pp1a and pp1ab) is predicted to be processed proteolytically into ten non-structural proteins (nsps), known as nsp1-8, with both the nsp1 and nsp7 regions being cleaved internally (yielding nsp1α and nsp1β, and nsp7α and nsp7β, respectively). The experimental verification of these predictions depends strongly on the ability to identify individual cleavage products with specific antibodies. In this study, a panel of monoclonal and polyclonal antibodies was generated, which together were able to recognize eight ORF1a-encoded PRRSV nsps. Using these reagents, replicase cleavage products were detected in PRRSV-infected MARC-145 cells using a variety of immunoassays. By immunofluorescence microscopy, most nsps could be detected by 6 h post-infection. During the early stages of infection, nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8 co-localized in distinct punctate foci in the perinuclear region of the cell, which were determined to be the site of viral RNA synthesis by in situ labelling. Western blot and immunoprecipitation analysis identified most individual nsps and several long-lived processing intermediates (nsp3-4, nsp5-7, nsp5-8 and nsp3-8). The identification and subcellular localization of PRRSV nsps in virus-infected cells documented here provides a basis for the further structure-function studies. Thus, this PRRSV antibody panel will be an important tool for future studies on the replication and pathogenesis of this major swine pathogen.

Diagnosis of histoplasmosis

Endemic mycoses can be challenging to diagnose and accurate interpretation of laboratory data is important to ensure the most appropriate treatment for the patients. Although the definitive diagnosis of histoplasmosis (HP), one of the most frequent endemic mycoses in the world, is achieved by direct diagnosis performed by micro and/or macroscopic observation of Histoplasma capsulatum (H. capsulatum), serologic evidence of this fungal infection is important since the isolation of the etiologic agents is time-consuming and insensitive. A variety of immunoassays have been used to detect specific antibodies to H. capsulatum. The most applied technique for antibody detection is immunodiffusion with sensitivity between 70 to 100 % and specificity of 100%, depending on the clinical form. The complement fixation (CF) test, a methodology extensively used on the past, is less specific (60 to 90%). Detecting fungal antigens by immunoassays is valuable in immunocompromised individuals where such assays achieve positive predictive values of 96-98%. Most current tests in diagnostic laboratories still utilize unpurified antigenic complexes from either whole fungal cells or their culture filtrates. Emphasis has shifted, however, to clinical immunoassays using highly purified and well-characterized antigens including recombinant antigens. In this paper, we review the current conventional diagnostic tools, such as complement fixation and immunodiffusion, outline the development of novel diagnostic reagents and methods, and discuss their relative merits and disadvantages to the immunodiagnostic of this mycosis.

Immunoassays: their history, development and current place in food science and technology

AbstractThe purpose of this paper is to introduce the reader to immunoassays. This paper is the first in a themed issue of the Journal in which a number of papers have been brought together in order to demonstrate the types and variety of immunoassays, which are currently available. Indeed it might be said that all an analyst needs to do is to name a molecule and somewhere there will now be an immunoassay for the detection of that molecule. This obviously is not entirely accurate, however immunoassays do provide a powerful tool, which can be used in the analysis and quality control of food materials. For both the novice and the experienced worker the specialist terminology of a subject presents an initial barrier, which must be overcome before full understanding is achieved. In this paper an attempt is made to introduce the important terms with which the reader should be familiar and to try to set the various technologies in context. The various basic methods are described and the theoretical and practical basis of more sophisticated assays now being devised are introduced.

Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains

Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl- S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (SCH 2CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP–antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP–antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP–antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.

A combinatorial phage display library for the generation of specific Fab fragments recognizing human spermatozoa and inhibiting fertilizing capacity in vitro.

To select a source of lymphocytes for the generation of an anti-sperm-biased combinatorial phage display library, venous blood was obtained from 34 vasovasostomy (vasectomy reversal) patients approximately 3 mo after surgery. Using a variety of immunoassays, serum was analyzed for antibodies against human spermatozoa, and a patient was selected on the basis of high titer of antibodies that recognized the equatorial region of the sperm head and inhibited sperm fertilizing capacity in vitro. Total RNA isolated from the stored lymphocytes of this individual was reversed transcribed, and gamma1 (Fd) region and kappa chains were amplified by polymerase chain reaction for the successful construction of an antibody phage display library. The library was panned against human spermatozoa to isolate sperm-specific phage that recognized the equatorial region of the sperm head. Three preparations of Fab were tested via the hamster egg penetration test. Each preparation significantly (p < 0. 005) inhibited sperm-egg binding and fusion, with one preparation (designated Fab-G) causing complete inhibition. Sequence analysis of the kappa light gene encoding Fab-G revealed a 93% homology with the light chain of human anti-human immunodeficiency virus gp120 p35 variable region. This technology may have a practical application in characterization of the immune response to spermatozoa and for the design of sperm-based contraceptive vaccines.

Fluorescence Assays: Fluorescence Polarization and Time-Resolved Pulse

In recent years techniques and instrumentation have been developed that allow fluorescence to be applied to a wide variety of immunoassays, with c goal of replacing radioimmunoassay. This article presents two of these—fluorescence polarization immunoassay and time-resolved pulse fluorescence. Fluorescence polarization assays ave already become the standard method for several tests. The high sensitivity and low interference of the newer time-resolved pulse fluorescence assays will make them increasingly popular.