Non-immunoglobulin Proteins Research Articles

A novel HER2 targeting nanoagent self-assembled from affibody-epothilone B conjugate for cancer therapy

Epothilone B (Epo B), a promising antitumor compound effective against various types of cancer cells in vitro. However, its poor selectivity for tumor cells and inadequate therapeutic windows significantly limit its potential clinical application. Affibody is a class of non-immunoglobulin affinity proteins with precise targeting capability to overexpressed molecular receptors on cancer cells, has been intensively investigated due to its exceptional affinity properties. In this study, we present a targeted nanoagent self-assembled from the precursor of an affibody conjugated with Epo B via a linker containing the thioketal (tk) group that is sensitive to reactive oxygen species (ROS). The core-shell structure of the ZHER2:342-Epo B Affibody-Drug Conjugate Nanoagent (Z-E ADCN), with the cytotoxin Epo B encapsulated within the ZHER2:342 affibody corona, leads to significantly reduced side effects on normal organs. Moreover, the abundant presence of ZHER2:342 on the surface effectively enhances the targeting capacity and tumor accumulation of the drug. Z-E ADCN can be internalized by cancer cells via HER2 receptor-mediated endocytosis followed by Epo B release in response to high levels of ROS, resulting in excellent anticancer efficacy in HER2-positive tumor models.

Comparison of approaches for increasing affinity of affibody molecules for imaging of B7-H3: dimerization and affinity maturation

BackgroundRadionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast.ResultsTwo dimeric variants of anti-B7-H3 Affibody molecules were produced (designated ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with 99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule.ConclusionThe improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.

Selection of Affibody Affinity Proteins from Phagemid Libraries.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.

Affibody-Based BCMA x CD16 Dual Engagers for Activation of NK Cells Towards Multiple Myeloma

The re-direction of natural killer (NK) cells is an emerging strategy for cancer immunotherapy. We describe the development and characterization of the so far smallest NK cell re-directing hBCMA x CD16 dual engagers for potential multiple myeloma treatment based on combinations of 7 kDa non-immunoglobulin affibody affinity proteins (Fig. 1). Affibodies to human CD16a protein (low affinity Fc-gamma receptor IIIa) were selected by phage display which resulted in the identification of binders with individual affinities (KD) for CD16a in the range of 100 nM to 3 µM, depending on the CD16 allotype (158V/F) and clone used in the analysis. Pairwise epitope binding experiments showed that binders with non-overlapping epitopes could be identified, allowing for the construction of bi-paratopic heterodimers with subnanomolar affinity for CD16a. In addition, a bivalent construct based on the variant with the highest monovalent affinity, denoted A10, showed subnanomolar apparent affinity (avidity) for both CD16a allotypes. The binding epitope of A10 was shown not to overlap with the Fc (hinge) binding activity of CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding affibody constructs containing one or two CD16a binding affibodies were genetically fused to a high-affinity (KD of ca. 7 nM) hBCMA-specific affibody, or a null-variant thereof. These 15-23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently induce synapse formation, activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines (Fig. 2), without evidence of NK cell fratricide. Hence, we report a novel class of versatile and uniquely small NK cell engagers with potent and specific binding properties and functional profiles. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Correlates with Vaccine Protective Capacity and COVID-19 Disease Symptoms Identified by Serum Proteomics in Vaccinated Individuals.

In the last two years, the coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a scientific and social challenge worldwide. Vaccines have been the most effective intervention for reducing virus transmission and disease severity. However, genetic virus variants are still circulating among vaccinated individuals with different disease symptomatology. Understanding the protective- or disease-associated mechanisms in vaccinated individuals is relevant to advances in vaccine development and implementation. To address this objective, serum-protein profiles were characterized by quantitative proteomics and data-analysis algorithms in four cohorts of uninfected and SARS-CoV-2-infected vaccinated individuals with asymptomatic, non-severe, and severe disease symptomatology. The results show that immunoglobulins were the most overrepresented proteins in infected cohorts when compared to PCR-negative individuals. The immunoglobulin profile varied between different infected cohorts and correlated with protective- or disease-associated capacity. Overrepresented immunoglobulins in PCR-positive individuals correlated with protective response against SARS-CoV-2, other viruses, and thrombosis in asymptomatic cases. In non-severe cases, correlates of protection against SARS-CoV-2 and HBV together with risk of myasthenia gravis and allergy and autoantibodies were observed. Patients with severe symptoms presented risk for allergy, chronic idiopathic thrombocytopenic purpura, and autoantibodies. The analysis of underrepresented immunoglobulins in PCR-positive compared to PCR-negative individuals identified vaccine-induced protective epitopes in various coronavirus proteins, including the spike receptor-binding domain RBD. Non-immunoglobulin proteins were associated with COVID-19 symptoms and biological processes. These results evidence host-associated differences in response to vaccination and the possibility of improving vaccine efficacy against SARS-CoV-2.

Differentially Represented Proteins in Response to Infection with Mycobacterium tuberculosis Identified by Quantitative Serum Proteomics in Asian Elephants.

Tuberculosis is a major global concern. Tuberculosis in wildlife is a risk for zoonotic transmission and becoming one of the challenges for conservation globally. In elephants, the number of cases is likely rising. The aim of this study was to identify proteins related to tuberculosis infection in elephants, which could then be used for the development of diagnostic tools and/or vaccines. A serum proteomics approach was used to characterize differentially represented proteins in response to Mycobacterium tuberculosis in Asian elephants (Elaphas maximus). Blood samples were collected from eight elephants, four of which were antibody positive for tuberculosis and four were antibody negative. Proteomics analysis identified 26 significantly dysregulated proteins in response to tuberculosis. Of these, 10 (38%) were identified as immunoglobulin and 16 (62%) as non-immunoglobulin proteins. The results provided new information on the antibody response to mycobacterial infection and biomarkers associated with tuberculosis and protective response to mycobacteria in Asian elephants. Protective mechanisms included defense against infection (Alpha-1-B glycoprotein A1BG, Serpin family A member 1 SERPINA1, Transthyretin TTR), neuroprotection (TTR), and reduced risks of inflammation, infections, and cancer (SERPINA1, Keratin 10 KRT10). Using a translational biotechnology approach, the results provided information for the identification of candidate diagnostic, prognostic, and protective antigens for monitoring and control of tuberculosis in Asian elephants.

Engineering non-antibody human proteins as efficient scaffolds for selective, receptor-targeted drug delivery

Self-assembling non-immunoglobulin scaffold proteins are a promising class of nanoscale carriers for drug delivery and interesting alternatives to antibody-based carriers that are not sufficiently efficient in systemic administration. To exploit their potentialities in clinics, protein scaffolds need to be further tailored to confer appropriate targeting and to overcome their potential immunogenicity, short half-life in plasma and proteolytic degradation. We have here engineered three human scaffold proteins as drug carrier nanoparticles to target the cytokine receptor CXCR4, a tumoral cell surface marker of high clinical relevance. The capability of these scaffolds for the selective delivery of Monomethyl auristatin E has been comparatively evaluated in a disseminated mouse model of human, CXCR4+ acute myeloid leukemia. Monomethyl auristatin E is an ultra-potent anti-mitotic drug used against a range of hematological neoplasias, which because of its high toxicity is not currently administered as a free drug but as payload in antibody-drug conjugates. The protein nanoconjugates generated here offer a collective strength of simple manufacturing process, high proteolytic and structural stability and multivalent ligand receptor interactions that result in a highly efficient and selective delivery of the payload drug and in a potent anticancer effect. The approach shown here stresses this class of human scaffold proteins as promising alternatives to antibodies for targeted drug delivery in the rapidly evolving drug development landscape.

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches.

Whole IgG antivenoms are prepared from hyperimmune animal plasma by various refinement strategies. The ones most commonly used at industrial scale are precipitation by sodium or ammonium sulphate (ASP), and caprylic acid precipitation (CAP) of non-immunoglobulin proteins. The additional procedures, which have so far been used for experimental purposes only, are anion-exchange (AEX) and cation-exchange chromatography (CEX), as well as affinity chromatography (AC) using IgG’s Fc-binding ligands. These protocols extract the whole IgG fraction from plasma, which contains both venom-specific and therapeutically irrelevant antibodies. Such preparations represent a complex mixture of various IgG subclasses whose functional and/or structural properties, as well as relative distribution, might be affected differently, depending on employed purification procedure. The aim of this work was to compare the influence of aforementioned refinement strategies on the IgG subclass distribution, venom-specific protective efficacy, thermal stability, aggregate formation and retained impurity profile of the final products. A unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma was used as a starting material, enabling direct comparison of five purification approaches. The highest purity was achieved by CAP and AC (above 90% in a single step), while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated IgG sample from AEX processing. Alpha-1B-glycoprotein was present in CAP IgG fraction, as well as in those from ASP- and AEX-based procedures. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparation as measured in vivo. AC and CEX remarkably affected drug’s venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. Presented data might improve the process of designing and establishing novel downstream processing strategies and give guidance for optimization of the current ones by providing information on potency-protecting and purity-increasing properties of each purification principle.

Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts

Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with 111In and characterized in vitro. Tumor-targeting properties of [111In]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [99mTc]Tc(CO)3-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [111In]In-DTPA-G250(Fab’)2, in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the 99mTc-labeled parental variant, [111In]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [111In]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [111In]In-G250(Fab’)2. In conclusion, [111In]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

Lectin populations of NK cells against tumors coupled to viral infections (review of literature).

Analysis of the human NK (natural killers) cells and their functionally different populations in connection to tumor processes accompanied with viral infections is presented. Receptor lectins (non-immunoglobulin proteins and their complexes recognizing polysaccharides, glycoconjugates and glycopattern-containing molecules) play important role in regulation of innate immunity. Communicative diversity of NK-cell populations (in which lectins cofunction to other receptors) is directed against tumors and viruses. Effectiveness and selectivity of action of NK cell populations can be increased in cooperation together with adaptive immunity. Evaluations of occurrence, redistribution (also under influence of cytokines) and contribution of NK-populations (depending on lectin receptors recognition coupled to multifunctions of receptors) in respect of increasing antitumor and antiviral immune responces are given. The data indicate extended prospects of lectin receptors (coupled to other type receptors) containing NK populations of the network compartment of innate immunity upon realization of different variants of organism protection in cooperation with cellular and humoral immunity. Such NK populations are the basis for further intercellular interactions. Innate immunity Cross-Talk, involving the leader NK cell populations acting according to humoral immunity mechanisms, acts on duty regime (importance for therapy of chronic pathology) that results in providing optimal combined antitumor and antiviral cytokine and cytotoxic responses according to the principle of action as «network-in-network». The influence network of lectin, Ig-like, cytotoxic, other regulator NK populations (also throuph redistribution of production of cytokines by immunocompetent cells) is perspective for forming early prolongated antitumor and antiviral processes of different types in organism. It is of importance to consider CD diversity of receptor repertuar of lectin, Ig-like and other NK populations revealing different ontogenesis as well as to seach patient key NK-populations to select and construct personally (or for contingents in cases of epidemiological significance) optimal therapeutic/prophylactic NK populations (like variants of CAR-T). Aforementioned data indicate perspectiveness of NK cell populations in development of new antitumor/antiviral effective and selective vaccine strategies, preparations and regimes of their applications. Probiotic lectins reveal features of perspective ligands cofunctioning to network of NK cell populations.

Affimer-Enzyme-Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins.

Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an "active" enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme-inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-β-lactamase and its inhibitor protein, such that target binding disrupts the enzyme-inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics.

Structural differences between the ectodomains of murine and human CD98hc.

The CD98 heavy chain (CD98hc) constitutes both a promising cell surface target for the treatment of cancers and a transcytosis receptor potentially useful for the brain delivery of therapeutics. However, pharmacokinetic studies and safety assessment of cognate antibodies or nonimmunoglobulin binding proteins in rodents is hampered by cross-species variability of both amino acid sequence and glycosylation pattern. Here, we report the crystal structure of the murine CD98hc extracellular domain and a comprehensive comparison with its human ortholog, revealing only one conserved surface patch that is neither shielded by glycosylation nor by the cell membrane with an accessible surface area typical for an antibody epitope. Our results imply the necessity of a surrogate approach for CD98hc-specific binding proteins with predictive power for clinical investigations.

DARPins: Promising Scaffolds for Theranostics.

Monoclonal antibodies are the classical basis for targeted therapy, but the development of alternative binding proteins has made it possible to use non-immunoglobulin proteins as targeting modules. The advantages of DARPins, scaffold proteins based on ankyrin repeats, over antibodies are as follows: small size, stability over a wide range of temperatures and pH values, low aggregation tendency, and ease of production in heterologous expression systems. The differences in the structure of the paratope of DARPin and antibodies broaden the spectrum of target molecules, while the ease of creating hybrid fusion proteins allows one to obtain bispecific and multivalent constructs. In this article, we summarize recent data on the development of therapeutic and imaging compounds based on DARPins.

Identification and In Silico Analysis of Lectins in Gray Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) Based on the Transcriptomes

Lectins, one of the most important bioactive compounds, are nonimmunoglobulin proteins that can bind carbohydrates specifically. However, few reports have been published on Pleurotus ostreatus lectin at the molecular level. Hence, in this study, seven lectins were identified based on transcriptomes in four developmental stages, i.e., mycelium, primordium, young fruiting body, and mature fruiting body. The expression profiles of the lectin genes were verified by quantitative real-time PCR. Lectin2-lectin6 had the highest expression in mycelium, while lectin1 was rich in mature fruiting body, and lectin7 was in primordium. We inferred that lectin2-lectin6 may take part in cell flocculation, lectin7 was the critical gene for primordium formation, and lectinl may be involved in fruiting body maturation, respectively. By in silico analysis, all lectins were divided into three distinct groups. Lectin1-Lectin5 were about 38.5-40.7 kDa as extracellular protein and belonged to the PCL-like lectins. Lectin6 (15.4 kDa) was predicted in nucleus and belonged to fungal fruit body lectins. Lectin7 (38.5 kDa) was a member of legume-like lectins and located in the plasma membrane. This study will help us understand how lectins mediate mushroom development.

Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours

ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)2- (DiADAPT6L2), and -(SSSG)3- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with 111In and 125I, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

Autotransporter-Mediated Display of a Naïve Affibody Library on the Outer Membrane of Escherichia coli.

Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naïve affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 109 variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.

Preclinical Evaluation of [68Ga]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors.

Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide 68Ga. Stability, specificity of binding to EGFR-expressing cells, and processing of [68Ga]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [68Ga]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [89Zr]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 ± 3%) and stably labeled with 68Ga. Binding of [68Ga]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [68Ga]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [89Zr]Zr-DFO-ZEGFR:2377, [68Ga]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [68Ga]Ga-DFO-ZEGFR:2377. In conclusion, [68Ga]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.

Optimization of purification procedure for horse F(ab´)2 antivenom against &lt;i&gt;Androctonus crassicauda&lt;/i&gt; (Scorpion) venom

Purpose : To immunize antiserum of horse with Androctonus crassicauda scorpion venom in order to achieve an antivenom with higher purity by combined caprylic acid/ammonium sulfate. Methods : The optimum pH to terminate enzymatic digestion was evaluated. Purification was performed by various combinations of caprylic acid (0 to 2.5 mL %) and ammonium sulfate (0 to 20 g %) at 25, 30 and 37 oC. The effects of three factors (caprylic acid, ammonium sulfate and temperature) were evaluated based on precipitation of non-immunoglobulin proteins. Antivenom purity was evaluated by determining the concentration of desired soluble protein and undesired albumin, as well as by turbidity and titration. Results : The results showed that the optimum pH for inhibition of enzyme activity and precipitation of impurities was 4.8. SDS-PAGE revealed that the highest impurity precipitation and lowest protein aggregation was occurred at the combination of 1.5 mL % caprylic acid and 10 g % ammonium sulfate at 37 oC. Conclusion : The modified method of purification significantly decreases turbidity, albumin impurity concentration and processing time but increased antibody titer and purity of antivenom. Therefore, it is a potentially suitable method for purifying antivenom in commercial production. Keywords : Antivenom, Scorpion, Androctonus crassicauda , Purification, Albumin, Antiserum

A Novel Approach to Estimating M-Protein Concentration: Capillary Electrophoresis Quantitative Immunosubtraction.

M-protein quantification is routinely performed by demarcating serum protein electrophoresis (SPE) regions. However, quantification of β-migrating M-protein is hindered by overlapping nonimmunoglobulin protein. Immunosubtraction (ISUB) on capillary electrophoresis is a method currently used qualitatively to subtract out (and therefore highlight) immunoglobulin isotypes in serum, thus reducing the masking effect of normal serum proteins. This study expands on traditional ISUB by developing a quantitative immunosubtraction (qIS) methodology. qIS is achieved by estimating the monoclonal class-specific immunoglobulin contribution to the SPE region containing the M-protein. We conducted a recovery study by use of serial dilutions from 3 patients with β-region M-proteins (n = 22), performing SPE and ISUB on each dilution. We visualized the difference between the ISUB electrophoresis trace and the involved ISUB isotype-subtracted trace to distinguish M-protein and background polyclonal immunoglobulins, which was demarcated independently by 3 pathologists. The M-protein contribution to the β-region was calculated and applied to the β-region protein concentration producing the quantitative M-protein concentration, while minimizing contamination by nonimmunoglobulin or polyclonal immunoglobulin proteins. Using a quality target of 25% error, we determined that our analytical measurable range spanned the maximum concentration tested (0.81 g/dL) to 0.05 g/dL. Passing-Bablok regression between qIS and the expected M-protein produced a slope of 1.04 (95% CI, 0.94-1.09), r = 0.99. Total CV was 4.8% and intraclass correlation between pathologists was 0.998. qIS promises quantification of β-migrating M-proteins at concentrations an order of magnitude lower than traditional SPE methodology, allowing earlier detection of increasing or decreasing M-protein.

Abstract 547: High-density Lipoprotein Particle Diversity From Selective Combinatorial Assembly of Particle Constituents Result in a Dynamic Particle Population

Introduction: Mass spectrometry is starting to elucidate the extent of lipoprotein diversity. It is believed that HDL is comprised of proteins encoded by &gt;100 genes and ~200 distinct lipid species in an undetermined number of combinations. Multiple proteoforms derived from a single gene contribute to particle complexity, confound proteome identification and makes differentiation of distinct subpopulations a challenge. Subtle proteoform variants can account for alterations in particle physicochemical properties and critical functions. Refining high-definition constituent identification within a structured atlas may reveal mechanism-based pathophysiology of a variety of diseases. Hypothesis: The ongoing identification of HDL constituents necessitates an effort to catalogue, categorize, map and relate entities in a framework that organizes this knowledge in a form of testable observations, explanations and predictions. Defining constituent interactions will advance knowledge and guide inquiry that enables HDL research, clinical diagnostics and subsequent therapeutic strategies. Methods: Informatic analysis of the literature produced an HDL proteome reference set. Proteome specific analysis of gene isoforms, proteolytic products, amino acid modifications and cSNPs were used to prepare a proteoform index. Theoretical tryptic peptide maps were generated and referenced to the PeptideAtlas and available published mass spec peptide lists. Results: Over 380 non-immunoglobulin proteins were identified in high-density lipoprotein fractions from human samples. A consensus-based scoring produced an “unofficial” list of 122 genes. UniProtKB was used to expand an index of potential proteoforms and derive a theoretical peptide mass library used to map empirically defined peptide spectral lists. Conclusions: Utilizing a reductionism model to HDL measurement has proven insufficient and undermines the application of Precision Medicine for CAD. Understanding the health benefits of HDL requires clinical diagnostics that captures particle diversity and population heterogeneity. Constructing a conceptual framework that unifies a constituent map is the initial step to resolving their relational context and functional consequences.