Extracellular 20S proteasome secreted via microvesicles can degrade poorly folded proteins and inhibit Galectin-3 agglutination activity.

Proteasomes are major non-lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular proteasome have also been evidenced in the plasma (p-proteasome) of patients with specific diseases. Here, we examined the process by which proteasomes are secreted, as well as their structural and functional features once in the extracellular space. We demonstrate that assembled 20S core particles are secreted by cells within microvesicles budding from the plasma membrane. Part of the extracellular proteasome pool is also free of membranes in the supernatant of cultured cells, and likely originates from microvesicles leakage. We further demonstrate that this free proteasome released by cells (cc-proteasome for cell culture proteasome) possesses latent proteolytic activity and can degrade various extracellular proteins. Both standard (no immune-subunits) and intermediate (containing some immune-subunits) forms of 20S are observed. Moreover, we show that galectin-3, which displays a highly disordered N-terminal region, is efficiently cleaved by purified cc-proteasome, without SDS activation, likely after its binding to PSMA3 (α7) subunit through its intrinsically disordered region. As a consequence, galectin-3 is unable to induce red blood cells agglutination when preincubated with cc-proteasome. These results highlight potential novel physio- and pathologic functions for the extracellular proteasome.

A multi-country comparative study of two treponemal tests for the serodiagnosis of syphilis amongst men who have sex with men (MSM): Chemo-luminescent assay vs Treponema pallidum particle agglutination assay.

INTRODUCTION: International guidelines recommend routine screening for syphilis (aetiological agent: Treponema pallidum subspecies pallidum) amongst key populations and vulnerable populations using tests detecting treponemal and non-treponemal antibodies. Whilst treponemal tests have high sensitivities and specificities, they differ regarding subjective or objective interpretation, throughput and workload. Chemiluminescence immunoassays (CLIAs) are cost- and time-effective automated methods for detecting treponemal antibodies. The Treponema pallidum particle agglutination assay (TPPA) has been considered the "gold standard" treponemal assay, however, this includes a highly manual procedure, low throughput and subjective interpretation. The present multi-country study evaluated the ADVIA Centaur® Syphilis CLIA (Siemens Healthcare) assay compared to the reference SERODIA-TP·PA® (Fujirebio Diagnostics) for the serodiagnosis of syphilis amongst men who have sex with men (MSM). METHOD: 1,485 MSM were enrolled in Brighton (UK), Malta, and Verona (Italy) as part of a larger WHO multi-country and multi-site ProSPeRo study. Ethical approval was obtained. Serum was tested with the ADVIA Centaur® Syphilis CLIA assay and SERODIA-TP·PA®, in accordance with the manufacturers' instructions, for a first round of validation. A second round of validation was carried out for discrepant results that were additionally tested with both Western Blot (Westernblot EUROIMMUN®) and an Immunoblot (INNO-LIA, Fujirebio Diagnostics). Sensitivity, specificity, positive and negative predictive value (PPV and NPV), likelihood ratios (positive/negative), and the Diagnostic Odds Ratio (DOR)/pre-post-test probability (Fagan's nomogram) were calculated. RESULTS: Out of 1,485 eligible samples analysed in the first phase, the SERODIA-TP·PA® identified 360 positive and 1,125 negative cases. The ADVIA Centaur® Syphilis CLIA assay (Siemens) identified 366 positives, missclassifying one TPPA-positive sample. In the second phase, the ADVIA Centaur® Syphilis CLIA resulted in 1 false negative and 4 false positive results. Considering the syphilis study prevalence of 24% (95% CI: 22-26.7), The sensitivity of the ADVIA Centaur® Syphilis CLIA assay was 99.7% (95% CI: 98.5-100), and the specificity was 99.4% (95% CI: 98.7-99.7). The ROC area values were 0.996 (95% CI: 0.992-0.999), and both the PPV and NPV values were above 98% (PPV 98.1%, 95% CI: 96.1-99.2; NPV 99.9%, 95% CI: 99.5-100). CONCLUSIONS: The ADVIA Centaur® Syphilis CLIA assay showed similar performance compared to the SERODIA-TP·PA®. Considering the study is based on QUADAS principles and with a homogeneous population, results are also likely to be generalisable to MSM population but potentially not applicable to lower prevalence populations routinely screened for syphilis. The automated CLIA treponemal assay confirmed to be accurate and appropriate for routine initial syphilis screening, i.e. when the reverse testing algorithm is applied.

Short Prewarming at 41°C to Correct the Interference in Samples with the Presence of Cold Agglutination.

BACKGROUND: Cold agglutinins (CAs) in blood samples can cause a reversible agglutination of red blood cell (RBC) which result in an incorrect complete blood count (CBC). So, it is important to explore new simple and feasible treatment conditions for clinical work. METHODS: The CAs group included 32 samples with CAs. The parameters of CBC at room temperature or after prewarming at 37°C or 41°C for different time periods were compared. The consistency and correlation of those parameters were analyzed. The morphology of erythrocytes in the CAs group was observed manually. The control group included 45 samples without CAs and prewarmed at 37°C or 41°C for different time periods. The differences were also analyzed. RESULTS: CAs have a significant effect on CBC. After prewarming at 37°C or 41°C the interferences are all corrected. Consider prewarming at 37°C for 120 minutes as the standard procedure. The consistency and correlation analysis showed there was no statistical difference between the results of each subgroup and standard group, except the MCHC of group 41°C 10 minutes. The correlation of parameters between all subgroups and the standard group is satisfied. Microscopic examination showed no RBC aggregation or fragmentation after prewarming at 41°C or 37°C. According to the maximum bias requirements for expert performance in Validation, Verification, and Quality Assurance of Automated Hematology Analyzers, 2nd Edition (CLSI H26-A2), the differences in overall results in control group are negligible. CONCLUSIONS: The 41°C 2 minutes prewarming method is a rapid and effective way for treating samples with CAs. It is an efficient way to obtain more reliable CBC results, without specific instruments.

Dissociation Phenomenon of Erythrocyte Agglutination and Its Application to Assay of Functional Activity of the Complement System in Clinical Laboratory.

OBJECTIVE: The objective of the study was to validate the dissociation phenomenon of erythrocyte agglutination which is based on erythrocyte fragments and to apply it in the functional activity assay of the complement system. METHODS: The dissociation-agglutination effect of erythrocyte fragments was validated by detecting the number of free erythrocytes after the action of erythrocyte fragments on agglutinated erythrocytes. The number of free erythrocytes produced after hemolysis of agglutinated erythrocytes caused by complements and complement activators(CAs) was detected by auto hematology analyzer and the results were indicated by mean hemoglobin concentration of erythrocytes (MCHC). We optimized the test conditions and validated the inter-batch stability, explored the resolution of the assay method, and assayed for the total complement activity (AC) and the CAs activated complement activity (ACA) in serum from patients and healthy individual groups. RESULTS: Erythrocyte fragments have a dissociative effect on agglutinated erythrocytes. The auto hematology analyzer was able to detect AC and ACA, where AC showed an inverse correlation with MCHC, and ACA demonstrated a positive correlation with MCHC. The inter-batch CV of AC, ACA, and ACA/AC was found to be 5%, 9%, and 11.7%, respectively, with good stability. The study found that serum samples from acute phase reaction patients showed significant differences in ACA compared with healthy individuals, with a p value of 0.018; serum samples from patients with nephrotic syndrome showed significant differences in AC, ACA, and ACA/AC compared with healthy individuals, with p values of 0.014, 0.002, and 0.041, respectively. CONCLUSION: Erythrocyte fragments have dissociation-agglutination effect. The complement system immunological functional detection method, based on this effect, has potential clinical application value due to its sensitivity and accuracy.

A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors.

Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was the hemagglutination of red blood cells. Although it is insightful, this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study, we aimed to validate a more precise and quantitative method to enable the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat-bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer, a more comparable effect was observed between blood group B and O, but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration responses and expected selectivity profiles for a set of small-molecule glycomimetics, confirming the historical profiles obtained in biochemical binding and functional cellular assays.

Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance.

Pathenogenesis-related (PR) proteins are extensively used as molecular markers to dissect the signaling cascades leading to plant defense responses. However, studies focusing on the biochemical or biological properties of these proteins remain rare. Here, we identify and characterize a class of apple (Malus domestica) PR proteins, named M. domestica AGGLUTININS (MdAGGs), belonging to the amaranthin-like lectin family. By combining molecular and biochemical approaches, we show that abundant production of MdAGGs in leaf tissues corresponds with enhanced resistance to the bacterium Erwinia amylovora, the causal agent of the disease fire blight. We also show that E. amylovora represses the expression of MdAGG genes by injecting the type 3 effector DspA/E into host cells and by secreting bacterial exopolysaccharides. Using a purified recombinant MdAGG, we show that the protein agglutinates E. amylovora cells in vitro and binds bacterial lipopolysaccharides at low pH, conditions reminiscent of the intercellular pH occurring in planta upon E. amylovora infection. We finally provide evidence that negatively charged polysaccharides, such as the free exopolysaccharide amylovoran progressively released by the bacteria, act as decoys relying on charge-charge interaction with the MdAGG to inhibit agglutination. Overall, our results suggest that the production of this particular class of PR proteins may contribute to apple innate immunity mechanisms active against E. amylovora.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination.

BACKGROUND AIMS: Cold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3-4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. CASE REPORTS: Four cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. CONCLUSIONS: While uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

In situ characterization of the agglutination of lectins via cross-linking of carbohydrates by time-resolved measurement of forward static light scattering.

We present real-time observations of a structurally variable process for cross-linking agglutination between multivalent lectins and glycoclusters using a small-angle forward static light scattering (F-SLS) technique. In this study, a cross-linking agglutination reaction was carried out using a tetravalent Neu5Acα2,6LacNAc-glycocluster and Sambucus sieboldiana agglutinin (SSA). The scattering intensity of time-resolved F-SLS increased with formation of the Neu5Acα2,6LacNAc-glycocluster-SSA cross-linked complex. Using this approach, fine sequential cross-linking agglutination between glycoclusters and lectins was observed in real-time. The rate of increase in the intensity of time-resolved F-SLS increased with the concentration of sialo-glycoclusters and SSA. Structural analysis based on the fractal dimension using time-resolved F-SLS patterns revealed that the density of the aggregates changed with progression of the cross-linking reaction until equilibrium was reached. This is the first report to evaluate the cross-linking agglutination reaction between glycoclusters and lectins and analysis of the subsequent structure of the obtained aggregates using time-resolved measurements of F-SLS.

Fluorescent Janus emulsions for biosensing of Listeria monocytogenes.

Here we report a sensing method for Listeria monocytogenes based on the agglutination of all-liquid Janus emulsions. This two-dye assay enables the rapid detection of trace Listeria in less than 2 h via an emissive signal produced in response to Listeria binding. The biorecognition interface between the Janus emulsions is assembled by attaching antibodies to a functional surfactant polymer with a tetrazine/transcyclooctene click reaction. The strong binding between Listeria and the Listeria antibody located at the hydrocarbon surface of the emulsions results in the tilting of the Janus structure from its equilibrium position to produce emission that would ordinarily be obscured by a blocking dye. This method provides rapid and inexpensive Listeria detection with high sensitivity (<100 CFU/mL in 2 h) that can be paired with many antibody or related recognition elements to create a new class of biosensors.

Comparison of solid-phase red cell adherence and microcolumn agglutination technology using untreated and enzyme-treated red blood cells.

Screening for clinically significant antibodies is crucial in transfusion medicine and is a routine part of pre-transfusion testing. The indirect antiglobulin test (IAT) is the most reliable and effective test for detecting clinically significant alloantibodies reacting at the antihuman globulin phase. Two of the main methods used for antibody detection and identification are solid-phase red cell adherence (SPRCA) and microcolumn agglutination technology (CAT), with or without enzyme-treated red blood cells (RBCs). This study was undertaken to detect and identify alloantibodies by performing antibody screen (ABS) and antibody identification (ABID) testing using SPRCA and CAT, with and without ficin-treated RBCs. Residual patient samples collected between 1 December 2020 and 19 May 2021 were saved, de-identified, and frozen at ≤-30°C before testing for alloantibodies. Seventy antibodies were detected in 53 samples among the 203 samples that underwent an ABS. Of those samples, 150 (73.0%) were nonreactive, 47 (23.1%) yielded positive results with both CAT and SPRCA, and six (3.0%) yielded positive ABS results with SPRCA only. Fifty-three samples that underwent ABID by both methods yielded eight samples with antibodies identified by SPRCA only. Additional enhancement of the CAT method by the use of ficin-treated RBCs was required to detect seven of the eight SPRCA-only antibodies; one sample remained nonreactive regardless. SPRCA testing detected clinically significant antibodies without the addition of enzyme-treated RBCs that was necessary in the CAT testing.

Outcomes After Lysis of Adhesions and Dilator Placement for Treatment of Vulvovaginal Agglutination Due to Lichen Planus.

OBJECTIVE: The aim of the study is to determine intraoperative and postoperative surgical outcomes for the treatment of vulvovaginal agglutination secondary to lichen planus (LP) following a standard protocol using intraoperative dilator placement and postoperative intravaginal steroid use. MATERIALS AND METHODS: This was a retrospective chart review of patients who underwent surgical management of vulvovaginal agglutination due to LP following a protocol that included surgical lysis of vulvovaginal adhesions, intraoperative dilator placement and removal 48 hours later, and high-potency intravaginal corticosteroid and regular dilator use thereafter. Demographic and clinical data were abstracted from the medical record and analyzed using descriptive statistics. RESULTS: Thirty-four patients, with mean age 51.2 ± 11 years and body mass index 32.8 ± 8.5 kg/m 2 , underwent lysis of vulvovaginal adhesions between 1999 and 2021 with 8 different surgeons at a single institution. The mean preoperative, immediate postoperative, and 6-week postoperative vaginal lengths were 2.8 ± 1.8 cm ( n = 18), 8.0 ± 1.9 cm ( n = 21), and 7.9 ± 2.2 cm ( n = 16), respectively. The mean estimated blood loss intraoperatively was 16 ± 15 mL. No patients had a documented surgical site infection or reoperation within 30 days after surgery. Of patients who had it documented ( n = 26), 70% (18/26) reported postoperative sexual activity. Where documented, 100% (18/18) reported preoperative dyspareunia, while 17% (3/18) did postoperatively. Six percent (2/34) had recurrent severe agglutination and 3% (1/34) underwent reoperation. CONCLUSIONS: Lysis of vulvovaginal adhesions, intraoperative dilator placement, and postoperative intravaginal corticosteroids with dilator use is a safe and effective treatment option to restore vaginal length for those with vulvovaginal LP.

Agglutination of Borreliella burgdorferi by Transmission-Blocking OspA Monoclonal Antibodies and Monovalent Fab Fragments.

Lyme disease vaccines based on recombinant Outer surface protein A (OspA) elicit protective antibodies that interfere with tick-to-host transmission of the disease-causing spirochete Borreliella burgdorferi. Another hallmark of OspA antisera and certain OspA monoclonal antibodies (MAbs) is their capacity to induce B. burgdorferi agglutination in vitro, a phenomenon first reported more than 30 years ago but never studied in molecular detail. In this report, we demonstrate that transmission-blocking OspA MAbs, individually and in combination, promote dose-dependent and epitope-specific agglutination of B. burgdorferi. Agglutination occurred within minutes and persisted for hours. Spirochetes in the core of the aggregates exhibited evidence of outer membrane (OM) stress, revealed by propidium iodide uptake. The most potent agglutinator was the mouse MAb LA-2, which targets the OspA C terminus (ß-strands 18 to 20). Human MAb 319-44, which also targets the OspA C terminus (ß-strand 20), and 857-2, which targets the OspA central ß-sheet (strands 8 to 10), were less potent agglutinators, while MAb 221-7, which targets ß-strands 10 to 11, had little to no measurable agglutinating activity, even though its affinity for OspA exceeded that of LA-2. Remarkably, monovalent Fab fragments derived from LA-2, and to a lesser degree 319-44, retained the capacity to induce B. burgdorferi aggregation and OM stress, a particularly intriguing observation considering that "LA-2-like" Fabs have been shown to experimentally entrap B. burgdorferi within infected ticks and prevent transmission during feeding to a mammalian host. It is therefore tempting to speculate that B. burgdorferi aggregation triggered by OspA-specific antibodies in vitro may in fact reflect an important biological activity in vivo.

Comparison between tube test and automated column agglutination technology on VISION Max for anti-A/B isoagglutinin titres: A multidimensional analysis.

BACKGROUND AND OBJECTIVES: VISION Max (Ortho Clinical Diagnostics, Raritan, NJ) measures anti-A/B isoagglutinin titres using automated column agglutination technology (CAT). We compared tube test (TT) and CAT of VISION Max comprehensively, including failure mode and effect analysis (FMEA), turnaround time (TAT) and cost, and suggested modified CAT (MCAT). MATERIALS AND METHODS: For 100 samples (each 25 for blood type A, B and O with anti-A and anti-B), anti-A/B isoagglutinin titres were measured by TT and CAT (1:2-1:1024 dilution), as well as by MCAT (with agglutination at 1:32 dilution, then perform additional testing from 1:64 to 1:1024). We assessed the agreement and correlation between TT and CAT and compared FMEA (risk priority number [RPN] score), TAT (h:min:sec) and cost (US dollar, US $) among TT, CAT and MCAT. RESULTS: TT and CAT showed overall substantial agreement (k = 0.73) and high correlation (ρ ≥ 0.75) except blood type O with anti-A (ρ = 0.68). Compared with TT, CAT showed lower RPN scores in FMEA and similar TAT and cost (FMEA, 33,700 vs. 184,300; TAT, 15:23:00 vs. 14:26:40; cost, 1377.4 vs. 1312.4, respectively). Regarding FMEA, TAT and cost, MCAT was superior to CAT or TT (43,810; 13:28:00; 899.2, respectively). CONCLUSION: This is the first multidimensional analysis on VISION Max CAT for measuring anti-A/B isoagglutinin titres. The results of anti-A/B isoagglutinin titres by CAT were comparable with those of TT. MCAT would be a safe, time-saving and cost-effective alternative to TT and CAT in high-volume blood bank laboratories.

Amyloid peptides with antimicrobial and/or microbial agglutination activity.

Microbe (including bacteria, fungi, and virus) infection in brains is associated with amyloid fibril deposit and neurodegeneration. Increasing findings suggest that amyloid proteins, like Abeta (Aß), are important innate immune effectors in preventing infections. In some previous studies, amyloid peptides have been linked to antimicrobial peptides due to their common mechanisms in membrane-disruption ability, while the other mechanisms of bactericidal protein aggregation and protein function knockdown are less discussed. Besides, another important function of amyloid peptides in pathogen agglutination is rarely illustrated. In this review, we summarized and divided the different roles and mechanisms of amyloid peptides against microbes in antimicrobial activity and microbe agglutination activity. Besides, the range of amyloids' antimicrobial spectrum, the effectiveness of amyloid peptide states (monomers, oligomers, and fibrils), and cytotoxicity are discussed. The good properties of amyloid peptides against microbes might provide implications for the development of novel antimicrobial drug. KEY POINTS: • Antimicrobial and/or microbial agglutination is a characteristic of amyloid peptides. • Various mechanisms of amyloid peptides against microbes are discovered recently. • Amyloid peptides might be developed into novel antimicrobial drugs.

Pathogen clearance and immune adherence "revisited": Immuno-regulatory roles for CRIg.

Rapid elimination of microbes from the bloodstream, along with the ability to mount an adaptive immune response, are essential for optimal host-defense. Kupffer cells are strategically positioned in the liver sinusoids and efficiently capture circulating microbes from the hepatic artery and portal vein, thus preventing bacterial dissemination. In vivo and in vitro studies have probed how complement receptor of the immunoglobulin superfamily (CRIg), also referred to as Z39Ig and V-set and Ig domain-containing 4 (VSIG4), acts as a critical player in pathogen recognition and clearance. While recent data suggested that CRIg may bind bacterial cell wall components directly, the single transmembrane receptor is best known for its interaction with complement C3 opsonization products on the microbial surface. On Kupffer cells, CRIg must capture opsonized microbes against the shear forces of the blood flow. In vivo work reveals how immune adherence (IA), a process in which blood platelets or erythrocytes associate with circulating bacteria, plays a critical role in regulating pathogen capture by CRIg under flow conditions. In addition to its typical innate immune functions, CRIg was shown to directly and indirectly influence adaptive immune responses. Here, we review our current understanding of the diverse roles of CRIg in pathogen elimination, anti-microbial immunity and autoimmunity. In particular, we will explore how, through selective capturing by CRIg, an important balance is achieved between the immunological and clearance functions of liver and spleen.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

Characterization of agglutinating antibodies detected by the direct agglutination test for visceral leishmaniasis diagnosis.

This study aimed to characterize agglutinating antibodies detected by the direct agglutination test (DAT-LPC) for the diagnosis of visceral leishmaniasis (VL). The DAT-LPC antigen/antibodies complex was recovered, washed, and used as antigenic substrate in a modified enzyme-linked immunosorbent assay (modified ELISA), revealed with anti-human IgM, IgG, and IgG subtype conjugates, and in the immunofluorescent antibodies test (IFAT), revealed with anti-human IgG and IgG1 conjugates. IgM antibodies were detected in 50%, IgG and IgG1 in 100%, and IgG3 in 52.8% of the 36 samples from VL patients. IFAT showed that agglutinating IgG and IgG1 antibodies recognized more intensely antigens located in the membrane and kinetoplast of the parasite. No antibodies were detected in the 15 samples from healthy individuals. This study shows for the first time that the antibodies responsible for agglutination in DAT-LPC are mostly of the IgG1 subtype.

Use of thiol reagents to disperse cold autoagglutination.

Thiol reagents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) are sulfhydryl reagents that can be used to disperse cold autoagglutinins coating red blood cells (RBCs). DTT and 2-ME are primarily used when warm washing of the coated RBCs fails to successfully disperse the cold autoantibody. Using a weak concentration of DTT or 2-ME, the cold IgM agglutinin can be removed from the coated RBCs without disrupting the IgG or complement coating the RBCs. The treated RBCs can be used for ABO typing, antigen typing, or the direct antiglobulin test.

A pilot study to assess the performance of a rapid ultrasound particle agglutination method for the detection of HIV antibodies.

Anti-HIV antibody screening and confirmatory tests include rapid diagnostics tests (RDT), which have limited sensitivity, and high-sensitivity ELISA and western blot tests, which are laborious and require technical proficiency. Thus, there is an unmet need for novel rapid, simple, and highly sensitive tests. A pilot study was conducted to assess the performance of a recently developed ultrasound particle agglutination (UPA) method for high-sensitivity HIV antibody detection using 51 confirmed positive and 310 presumably negative plasma samples, and 6 commercially available anti-HIV-1 seroconversion panels (total 56 members). Optimal cutoff value of the UPA method was determined by receiver operating characteristics (ROC) analysis, providing clinical sensitivity and specificity of 100% and 98.1%, respectively. The performance characteristics of UPA, compared with those of some established RDT's and ELISA tests using HIV seroconversion panels, showed 2 days earlier HIV antibody detection than other RDT's and 2nd-generation ELISA, and at approximately the same time as 3rd-generation ELISA. The preliminary analysis of the UPA method performance characteristics showed that it meets the minimum requirements of the WHO guidelines for RDTs as first-line assays. This pilot study paves the way for more detailed validation studies of the UPA method for HIV antibody detection in clinical practice.

Effective Optical Image Assessment of Cellulose Paper Immunostrips for Blood Typing.

Novel high-performance biosensing devices, based on a microporous cellulose matrix, have been of great interest due to their high sensitivity, low cost, and simple operation. Herein, we report on the design and testing of portable paper-based immunostrips (IMS) for in-field blood typing in emergencies requiring blood transfusion. Cellulose fibrils of a paper membrane were functionalized with antibodies via supramolecular interactions. The formation of hydrogen bonds between IgM pentamer and cellulose fibers was corroborated using quantum mechanical calculations with a model cellulose chain and a representative amino acid sequence. In the proposed immunostrips, paper with a pore size of 3 µm dia. was used to enable functionalization of its channels with antibody molecules while blocking the red blood cells (RBC) from channel entering. Under the optimized test conditions, all blood types of AB0 and Rh system could be determined by naked eye examination, requiring only a small blood sample (3.5 µL). The durability of IgM immunostrips against storing has been tested. A new method of statistical evaluation of digitized blood agglutination images, compatible with a clinical five-level system, has been proposed. Critical parameters of the agglutination process have been established to enable future development of automatic blood typing with machine vision and digital data processing.