Antibody Cocktail Research Articles

Flow cytometry lyophilised-reagent tube for quantifying peripheral blood neutrophil myeloperoxidase expression in myelodysplastic syndromes (MPO-MDS-Develop): protocol for a diagnostic accuracy study

IntroductionSuspicion of myelodysplastic syndromes (MDS) is the most common reason for bone marrow aspirate in elderly patients. Peripheral blood neutrophil myeloperoxidase expression quantified by flow cytometric analysis might rule out...

An antibody cocktail with broadened mutational resistance and effective protection against SARS-CoV-2.

Neutralizing antibodies have been proven to be highly effective in treating mild and moderate COVID-19 patients, but continuous emergence of SARS-CoV-2 variants poses significant challenges. Antibody cocktail treatments reduce the risk of escape mutants and resistance. In this study, a new cocktail composed of two highly potent neutralizing antibodies (HB27 and H89Y) was developed, whose binding epitope is different from those cocktails that received emergency use authorization. This cocktail showed more potent and balanced neutralizing activities (IC50 0.9–11.3 ng mL−1) against a broad spectrum of SARS-CoV-2 variants over individual HB27 or H89Y antibodies. Furthermore, the cocktail conferred more effective protection against the SARS-CoV-2 Beta variant in an aged murine model than monotherapy. It was shown to prevent SARS-CoV-2 mutational escape in vitro and effectively neutralize 61 types of pseudoviruses harbouring single amino acid mutation originated from variants and escape strains of Bamlanivimab, Casirivimab and Imdevimab with IC50 of 0.6–65 ng mL−1. Despite its breadth of variant neutralization, the HB27+H89Y combo and EUA cocktails lost their potencies against Omicron variant. Our results provide important insights that new antibody cocktails covering different epitopes are valuable tools to counter virus mutation and escape, highlighting the need to search for more conserved epitopes to combat Omicron.Supporting InformationThe supporting information is available online at 10.1007/s11427-022-2166-y. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Targeting of palpable B16-F10 melanoma tumors with polyclonal antibodies on white blood cells

BackgroundAntibodies and other recognition molecules direct cancer cell death by multiple types of immune cells. Therapy directed at only one target typically results in tumor regrowth because of tumor heterogeneity. Our goal is to direct therapy to multiple targets simultaneously. Our previous studies showed that multiple antibodies targeting mutated tumor proteins inhibited tumor growth when injected subcutaneously near the time of cancer cell implantation. MethodsA cocktail of rabbit antibodies against B16-F10 cell surface related mutated proteins were generated. Implanted B16-F10 cells were allowed to grow to palpable size before treatment. Antibodies were administered using different routes of exposure. Free antibody was compared to antibody armed on mouse splenic white blood cells (WBCs). Binding of the antibody cocktail was determined for mouse and human WBCs. ResultsThe antibody cocktail inhibited tumor growth and prolonged survival when administered as free antibody or armed on WBCs. The antibody cocktail armed on WBCs achieved similar tumor inhibition as free antibody but at a dose 1000-fold less. Armed WBCs achieved tumor inhibition by intravenous and subcutaneous administration. The antibody cocktail bound well to human WBCs and saturation dose was defined. Binding was stable under simulated in vivo condition in human plasma at 37 °C. ConclusionsAntibodies targeting multiple tumor mutated proteins inhibited tumor growth and prolonged survival. Effective antibody dose was reduced 1000-fold by arming WBCs. Rabbit antibodies saturated human WBCs using <1 mg per billion cells. A phase I trial in cancer patients using this strategy has been approved by the FDA.

Therapeutic Role of Neutralizing Antibody for the Treatment against SARS-CoV-2 and Its Emerging Variants: A Clinical and Pre-Clinical Perspective.

Since early 2020, the entire world has been facing a disastrous outbreak of the SARS-CoV-2 virus, with massive reporting of death and infections per day. Medical practitioners adopted certain measures such as convalescent plasma therapy, antibody treatment, and injecting vaccines to eradicate the pandemic. In this review, we have primarily focused on the neutralizing antibodies presently under pre-clinical and clinical trials, focusing on their structures, binding affinity, mechanism of neutralization, and advantages over other therapeutics. We have also enlisted all the nAbs against SARS-CoV-2 and its emerging variants in different phases of clinical trials (phase-1, phase-II, and phase-III). The efficacy of administering antibody cocktails over the normal antibodies and their efficacy for the mutant variants of the SARS-CoV-2 virus in minimizing viral virulence is discussed. The potent neutralizing antibodies have eliminated many of the common problems posed by several other therapeutics. A common mechanism of the antibodies and their relevant sources have also been listed in this review.

Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) shows increased antigen-binding and virus-neutralizing activities against multiple SARS-CoV-2 variants as well as increased breadth of neutralizing activity compared to the cocktail. X-ray crystallography and cryo-EM reveal distinct binding models for individual cocktail antibodies, and computational simulations suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and multiple variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

CLL-393 Outcomes of COVID-19 Infections in Patients With Hematological Malignancies in Republic of North Macedonia

Context: Patients with hematological malignancies are at increased risk of complications and adverse outcomes associated with COVID-19, according to current available studies. These patients are particularly vulnerable due to impaired or compromised immune responses caused by treatment or the disease itself. Objective: Our aim was to examine the severity and outcomes of COVID-19 infections among patients with hematological malignancies in Republic of North Macedonia. Design: This is the first retrospective descriptive study on patients with COVID-19 and underlying hematological malignancies in Republic of North Macedonia. We focused on clinical characteristics and COVID-19 infection outcomes in different malignancy settings. Patients: We collected data on clinical characteristics, diagnosis, treatment, and outcomes in adult patients with hematologic malignancies and confirmed COVID-19 infections between June 2020 and April 2022. Parametric data were compared using Fisher's exact test. Results: Of 206 patients, 153 (74%) had lymphoid, and 53 (26%) had myeloid malignancies. Median age was 61 years (IQR 47–70 years), 118 (57%) patients were male, and 86 (74%) patients were older than 60 years and had ≥2 comorbidities, with only 16 (17%) patients younger than 60 years. Severe/critical clinical severity was identified in 77 (37%). Overall, 42 patients (20%) died, including 33 patients (78%) older than 60 years. Patients older than 60 years had significantly higher mortality (p=0.0015) than younger patients. The majority of patients (58%) became COVID-19–positive while on active treatment. Significantly worse outcomes were observed among patients who were on active treatment compared with patients who were in hematological remission (p=0.0028). Among lethal cases, the majority of patients were diagnosed with CLL and NHL. We found no significant differences in mortality between treated and untreated CLL and NHL patients. Among 37 patients treated with a cocktail of monoclonal antibodies (casirivimab/imdevimab), no significant difference in outcome was observed, although 86% of them survived. Conclusions: Patients with hematological malignancies and COVID-19 infection were associated with higher mortality associated with older age, more comorbidities, and active disease. Patients with B-cell malignancy were the most affected, but no significant difference in mortality rate was observed across different subgroups, which may suggest that B-cell impairment is a key factor leading to severe infection.

Malignant T cells induce skin barrier defects through cytokine-mediated JAK/STAT signaling in cutaneous T-cell lymphoma

AbstractCutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA–mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier.

Clinical Study of Antibody Cocktail Therapy for COVID-19

Clinical Study of Antibody Cocktail Therapy for COVID-19

Preclinical characterization of amubarvimab and romlusevimab, a pair of non-competing neutralizing monoclonal antibody cocktail, against SARS-CoV-2.

Monoclonal antibodies (mAbs) targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have demonstrated clinical efficacy in preventing or treating coronavirus disease 2019 (COVID-19), resulting in the emergency use authorization (EUA) for several SARS-CoV-2 targeting mAb by regulatory authority. However, the continuous virus evolution requires diverse mAb options to combat variants. Here we describe two fully human mAbs, amubarvimab (BRII-196) and romlusevimab (BRII-198) that bind to non-competing epitopes on the receptor binding domain (RBD) of spike protein and effectively neutralize SARS-CoV-2 variants. A YTE modification was introduced to the fragment crystallizable (Fc) region of both mAbs to prolong serum half-life and reduce effector function. The amubarvimab and romlusevimab combination retained activity against most mutations associated with reduced susceptibility to previously authorized mAbs and against variants containing amino acid substitutions in their epitope regions. Consistently, the combination of amubarvimab and romlusevimab effectively neutralized a wide range of viruses including most variants of concern and interest in vitro. In a Syrian golden hamster model of SARS-CoV-2 infection, animals receiving combination of amubarvimab and romlusevimab either pre- or post-infection demonstrated less weight loss, significantly decreased viral load in the lungs, and reduced lung pathology compared to controls. These preclinical findings support their development as an antibody cocktail therapeutic option against COVID-19 in the clinic.

A Critical Analysis of the Use of Cilgavimab plus Tixagevimab Monoclonal Antibody Cocktail (Evusheld™) for COVID-19 Prophylaxis and Treatment.

Evusheld® (tixagevimab + cilgavimab; AZD7442) was the first anti-Spike monoclonal antibody (mAb) cocktail designed not only for treatment but also with pre-exposure prophylaxis in mind. The immunoglobulins were engineered for prolonged half-life by modifying the Fc fragment, thus creating a long-acting antibody (LAAB). We review here preclinical development, baseline and treatment-emergent resistance, clinical efficacy from registration trials, and real-world post-marketing evidence. The combination was initially approved for pre-exposure prophylaxis at the time of the SARS-CoV-2 Delta VOC wave based on a trial conducted in unvaccinated subjects when the Alpha VOC was dominant. Another trial also conducted at the time of the Alpha VOC wave proved efficacy as early treatment in unvaccinated patients and led to authorization at the time of the BA.4/5 VOC wave. Tixagevimab was ineffective against any Omicron sublineage, so cilgavimab has so far been the ingredient which has made a difference. Antibody monotherapy has a high risk of selecting for immune escape variants in immunocompromised patients with high viral loads, which nowadays represent the main therapeutic indication for antibody therapies. Among Omicron sublineages, cilgavimab was ineffective against BA.1, recovered efficacy against BA.2 and BA.2.12.1, but lost efficacy again against BA.4/BA.5 and BA.2.75. Our analysis indicated that Evusheld® has been used during the Omicron VOC phase without robust clinical data of efficacy against this variant and suggested that several regulatory decisions regarding its use lacked consistency. There is an urgent need for new randomized controlled trials in vaccinated, immunocompromised subjects, using COVID-19 convalescent plasma as a control arm.

A counterintuitive antibody cocktail disrupts coxsackievirus

Utilizing monoclonal antibodies to prevent and treat infectious diseases has been accelerated by the COVID-19 pandemic. In this issue of Cell Host & Microbe, Zheng et al. show how a three-monoclonal-antibody cocktail, that defies conventions of “rational design” for a therapeutic agent, functions cooperatively to disrupt coxsackievirus virions.

Recombinant Decoy Exhibits Broad Protection against Omicron and Resistance Potential to Future Variants.

The Omicron variant has swept through most countries and become a dominant circulating strain, replacing the Delta variant. The evolutionary history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggests that the onset of another variant (possibly another variant of concern (VOC) is inevitable. Therefore, the development of therapeutics that enable treatments for all Omicron-included VOCs/variants of interest (VOIs) and future variants is desired. Recently, the recombinant receptor decoy therapeutic angiotensin-converting enzyme 2 (ACE2)-Fc has exhibited good safety in a phase 1 clinical trial; therefore, its variant-resistant profile needs to be understood. Here, we conducted a comprehensive evaluation of its neutralization breadth against the Omicron variant and other VOCs/VOIs. Furthermore, to evaluate its resistance to future variants, we investigated its ability to neutralize various single-residue mutated variants. Next, we demonstrated its resistance to evasion via an experiment that rapidly and effectively stimulates virus evolution with a replication-competent virus model. In addition, we evaluated its efficacy for cocktail therapy. The combination of ACE2-Fc and neutralizing antibodies showed both efficacy and breadth in the simulation experiment. The underlying mechanism was revealed to be a synergistic effect in the cocktails. Collectively, this study deepens the understanding of the resistance profile of recombinant receptor decoy therapeutics and highlights the potential value of ACE2-Fc and neutralizing antibody cocktails in the subsequent anti-SARS-CoV-2 campaign. Furthermore, we also provide an effective method to study the resistance profile of antiviral agents and rapidly screen for potential cocktails to combat future variants.

The Presence of PDL-1 on CD8+ Lymphocytes Is Linked to Survival in Neonatal Sepsis.

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. Neonatal sepsis is the main cause of death in newborns, especially preterm infants. The pathogenesis of sepsis is based on a hyper-inflammatory syndrome combined with an immunosuppressive mechanism in sepsis. This study aimed to find critical parameters that are associated with the outcome of newborns with suspected sepsis. Understanding the association might have clinical relevance for immuno-monitoring, outcome prediction, and targeted therapy. Methods: A total of 210 newborn infants no older than 4 days with suspected sepsis at admission in Karaganda (Kazakhstan) were prospectively enrolled. Blood cultures were incubated, and pathogens in positive cultures were determined by MALDI-TOF. An immunological assay for blood cell components was conducted by flow cytometry with antibody cocktails. The diagnostic criteria for neonatal sepsis were identified by qualified neonatologists and included both clinical sepsis and/or positive blood culture. The analyzed infants were grouped into non-septic infants, surviving septic infants, and deceased septic infants. The results showed that deceased septic newborns had a lower level of CD8+ lymphocytes and higher PDL-1 expression in comparison with surviving septic newborns. PDL-1 expression on CD8+ T cells might play an immunosuppressive role during neonatal sepsis and might be used as a laboratory biomarker in the future.

Increased frequency of TIGIT+ CD4 T Cell subset in autoantibody-positive first-degree relatives of patients with rheumatoid arthritis.

BackgroundDespite immune cell dysregulation being an important event preceding the onset of rheumatoid arthritis (RA), the phenotype of T and B cells in preclinical RA is less understood. The aim of this study was to characterize T and B cell populations in RA patients and their autoantibody (aAb) negative and positive first-degree relatives (FDR).MethodsCryopreserved peripheral blood mononuclear cells (PBMCs) collected at scheduled visits from aAb-(n=25), and aAb+ FDR (n=10) and RA patients (n=13) were thawed and stained using optimized antibody cocktails as per a specific 13-color T or B cell panel. Immunophenotyping was performed using a Cytoflex LX (Beckman-Coulter) flow cytometer and FlowJo software was used for analyzing the frequency of immune cell populations.ResultsMulticolor flow cytometry experiments identified an increased TIGIT expression in circulating lymphocytes of aAb+ FDR and RA patients, relative to aAb- FDR (P<0.01). These TIGIT+ T cells exhibited a memory phenotype and expressed high levels of PD-1, ICOS, HLA-DR, CXCR3 and CXCR5. Moreover, increased TIGIT+ CD4 T cell frequency correlated with the frequency of PD-1+ CD4 T cells (r = 0.4705: P = 0.0043) and circulating levels of ACPA and RF. We also identified a decreased frequency of CD27+IgD- switched memory B cells in RA patients (P < 0.01), while increased frequency of TIGIT+ CD4 T cells in FDR correlated with the frequency of PD1+PTEN+ B cells (r = 0.6838, P = 0.0004) and autoantibody positivity (P = 0.01).ConclusionWe demonstrate TIGIT as a distinct CD4 T cell marker for differentiating aAb- FDR from aAb+FDR and might play a critical role in regulating T and B cell crosstalk in preclinical RA.

Post-vaccination antibody evaluation for nosocomial SARS-CoV-2 delta variant breakthrough infection.

Waning humoral immunity after mRNA vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a significant problem for public health. Breakthrough infection in hospitals over several months after vaccination has not been fully characterized, especially against the delta (B.1.617.2) variant. Here, we describe an outbreak in our hospital in September of 2021, mainly through serological evaluation of the breakthrough infection. This retrospective observational study was done at an emergency and acute care hospital with 204 beds and 486 staff members where most staff members (92.6%) had had their second BNT162b2 vaccination by May of 2021. The peri-infection anti-spike RBD protein IgG (anti-S IgG) titers (lowest values between 11 days before and 7 days after onset or diagnosis) of serum samples from the breakthrough-infected persons were quantified. We also logarithmically estimated the anti-S IgG titers during the exposure period in September of uninfected staff members from their samples collected in May and December 2021. Whole-genome sequencing was done on obtained samples. In this outbreak, twelve persons (ten inpatients and two staff members) were diagnosed with SARS-CoV-2 infection by Loop-Mediated Isothermal Amplification (LAMP) or RT-PCR, eight of whom had been vaccinated twice. Peri-infection anti-S IgG titers could be determined in seven of the eight breakthrough cases, with a geometric mean titer (GMT) of 1,034 AU/ml (95% confidence interval [CI], 398 to 2,686). Among 289 uninfected staff members with data from the two sampling points, the GMT of the estimated anti-S IgG titers during the exposure period in 51 staff members, who were working at the outbreak ward and potentially exposed but uninfected, and 238 other unexposed staff members were 1,458 AU/ml (95% CI, 1,196 to 1,777) and 1,628 AU/ml (95% CI, 1,500 to 1,766), respectively. All viruses from the eight samples for which whole-genome sequencing was available were identified as delta variants. Of the infected persons, one remained asymptomatic throughout the course of treatment, and eleven had an illness of mild to moderate severity, including ten who received monoclonal antibody cocktail (Casirivimab/imdevimab) therapy. Measurement and estimation of anti-spike antibody levels after SARS-CoV-2 vaccination would be helpful for evaluating the risk of breakthrough infection and for determining the necessity of booster vaccination.

Prescription of Anti-Spike Monoclonal Antibodies in COVID-19 Patients with Resistant SARS-CoV-2 Variants in Italy.

Anti-Spike monoclonal antibodies have been considered a promising approach to COVID-19 therapy. Unfortunately, the advent of resistant lineages jeopardized their effectiveness and prompted limitations in their clinical use. Change in the dominant variant can be fast to such an extent that, in the absence of timely medical education, prescribers can keep using these drugs for relatively long periods even in patients with resistant variants. Therefore, many patients could have been exposed to drugs with unlikely benefits and probable risks. We show here that about 20% of bamlanivimab+etesevimab, 30% of casirivimab+imdevimab, and 30% of sotrovimab courses were administered in Italy during periods in which a fully resistant variant was dominant. Additionally, for monoclonal antibody cocktails, the vast majority of usage occurred against variants for which one of the mAbs within the cocktail was ineffective. Given the high costs of these drugs and their potential side effects, it would be important to consider a frequent review of the appropriateness of these drugs and timely communication when the benefit/risk balance is no longer favorable.

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).

Establishment of an efficient ex vivo expansion strategy for human natural killer cells stimulated by defined cytokine cocktail and antibodies against natural killer cell activating receptors

IntroductionCell-based immunotherapy is categorized as a regenerative therapy under the Regenerative Medicine Safety Act in Japan. Natural killer (NK) cell-based immunotherapy is considered a promising strategy for treating cancer, including glioblastoma (GBM). We previously reported an expansion method for highly purified human peripheral blood-derived NK cells using a cytokine cocktail. Here, we aimed to establish a more efficient NK cell expansion method as compared to our previously reported method.MethodsT cell-depleted human peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers. The depleted PBMCs were cultured in the presence of recombinant human interleukin (rhIL)-18 and high-dose rhIL-2 in anti-NKp46 and/or anti-CD16 antibody immobilization settings. After 14 days of expansion, the purity and expansion ratio of CD3-CD56+ NK cells were determined. The cytotoxicity-mediated growth inhibition of T98G cells (an NK activity-sensitive GBM cell line) was evaluated using a non-labeling, impedance-based real-time cell analyzer.ResultsAnti-NKp46 stimulation increased the NK cell purity and expansion ratio as compared to the non-antibody-stimulated population. Anti-CD16 stimulation weakly enhanced the NK cell expansion ratio of the non-antibody-stimulated population and enhanced the NK cell purity and expansion ratio of anti-NKp46-stimulated populations. All NK cell-containing populations tested distinctly inhibited T98G cell growth. These effects tended to be enhanced in an NK cell purity-dependent manner. In some cases, anti-CD16 stimulation decreased growth inhibition of T98G cell compared to other conditions despite the comparable NK cell purity.ConclusionsWe established a robust large-scale feeder-free expansion system for highly purified human NK cells using a defined cytokine cocktail and anti-NK cell activating receptor antibodies. The expansion system could be feasible for autologous or allogeneic NK cell-based immunotherapy of GBM. Moreover, it is easily controlled under Japanese law on regenerative medicine.

Development of a lateral flow immunoassay using antibody cocktail for the detection of the various epidemic subtypes of group A Streptococcus in clinical samples

Group A Streptococcus (GAS), a major pathogen, causes various respiratory diseases with substantial morbidity and mortality worldwide. In this study, we develop an immunoassay for the early screening and diagnosis of GAS infections. A latex beads-based lateral flow immunoassay (LBs-LFIA) and a fluorescent beads-based LFIA (FBs-LFIA) using an antibody cocktail that was coated on the test line (T line), the limit of detection (LoD) of LBs-LFIA was 105 CFU mL−1 and FBs-LFIA was 103 CFU mL−1. Furthermore, the use of the antibody cocktail led to the detection of the various epidemic clinical types of GAS with high sensitivity (100%) and specificity (＞95%). To devise a point-of-care testing (POCT) platform for mass screening in low-resource areas, a mobile phone-based portable strip reader capable of visually interpreting the results was developed and the LoD of LBs-LFIA and FBs-LFIA were calculated as 104 CFU mL−1 and 102 CFU mL−1, respectively. Taken together, we suggest that the developed LBs-LFIA and FBs-LFIA using the antibody cocktail can be used for the clinical emergency detection of GAS infections.

Development of a 27-color panel for the detection of measurable residual disease in patients diagnosed with acute myeloid leukemia.

Acute myeloid leukemia (AML) measurable residual disease (MRD) evaluated by multiparametric flow cytometry (MFC) is a surrogate for progression-free and overall survival in clinical trials and patient management. Due to the limited number of detection channels available in conventional flow cytometers, panels used for assessing AML MRD are typically split into multiple tubes. This cripples the simultaneous and correlated assessment of all myeloblast measurements. In response, we prototyped a single-tube 27-color MFC assay for the evaluation of AML MRD, incorporating all recommended markers. Marrow aspirates from 22 patients were processed for analysis using full spectrum flow cytometry (FSFC). The signal resolution of each marker was compared between samples stained with single antibody vs. the fully stained panel. The analytical accuracy for quantifying hematopoietic cells between our established 8-color assay and the new 27-color method were compared. Variations within an operator and between separate operators were assessed to evaluate the assays reproducibility. The limited of blank (LOB), limit of detection (LOD), and lower limit of quantification (LLOQ) of the 27-color method were empirically determined using limiting dilution experiments. The stability of antibody cocktails over a period of 120 h was also studied using cryopreserved marrow cells. The stain indices for all antibodies were lower in the fully stained panel compared to cells stained with one antibody but clear separations between negative and positive signals were achieved for all antibodies. Our results demonstrated a high concordance between the established 8-color method and the new 27-color assay for enumerating myeloblasts and MRD interpretation within and between operators. The data further showed that the single-tube 27-color assay easily achieved the minimum required detection sensitivity of 0.1%. When antibodies were combined, however, expression intensity of some antigens deteriorated significantly when stored. Our single-tube 27-color panel is a suitable, high sensitivity flow cytometric approach that can be used for AML MRD testing, which improves the correlation of aberrant antigens and detection of asynchronous differentiation patterns. Based on the stability study, we recommend the full panel be made prior to staining.