Changes in dynamic and static structures of the HIV-1 p24 capsid protein N-domain caused by amino-acid substitution are associated with its viral viability.

HIV-1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N-terminal domain protein were examined: G60W (α3-4 loop), M68T (helix 4), and P90T (α4-5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N-domain mutants, examined by NMR, was the long-range effect of more ß-structures at the ß2-strand in the N-terminal region compared with the wild-type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C-terminal part of helix 4. This suggests that both N-terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis-conformation or trans-conformation of wild-type adopted by isomerization at G89-P90. Since P90T mutant adopts only a trans-conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild-type correspond to trans-conformation. Furthermore, it was indicated that the region in cis-conformation (minor; 15%) was more stabilized than that observed in trans-conformation, based on the analyses of heteronuclear Overhauser effect as well as the order-parameter. Therefore, it was concluded that the cis-conformation is more favorable than the trans-conformation for the interaction between the p24 N-terminal domain and cyclophilin-A. This is because HIV-1 with a P90T protein, which adopts only a trans-conformation, is associated with non-viability of biological activity.

Direct single-molecule imaging for diagnostic and blood screening assays.

Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10-18 M) for model assay systems and to the low femtomolar (10-16 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10-18 to 10-5 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.

Amplifying immunogenicity of prospective Covid-19 vaccines by glycoengineering the coronavirus glycan-shield to present α-gal epitopes.

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galß1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10-200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC.

Increasing the Assembly Efficacy of Peptidic ß-Sheets for a Highly-Sensitive HIV Detection.

Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.

Composition and Orientation of the Core Region of Novel HIV-1 Entry Inhibitors Influences Metabolic Stability.

Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage therapies only. As such, the development of a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy (cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore, in this short communication, we assess the metabolic stabilities of five of our novel chemotype entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor with more than twice the metabolic stability of temsavir and demonstrated that the orientation of the core replacement is critical for this increase. This work further demonstrates the feasibility of our long-term goal-to design an entry inhibitor with improved drug-like qualities-and warrants expanded studies to achieve this.

TRIM5α self-assembly and compartmentalization of the HIV-1 viral capsid.

The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α.

Direct single-molecule counting for immunoassay applications.

Single-molecule methods offer specificity in studying complex systems and dynamics, but they also offer high sensitivity for basic enumeration. We apply single-molecule TIRF to immunoassays by counting the number of target molecules captured on a streptavidin surface. We demonstrate the utility of using single-molecule counting on eluted detection conjugate, following the capture and sandwich formation portions of the assay having been completed on microparticles. This approach is simple and effective, and creates the opportunity for a universal detection platform that can be used to perform a variety of diagnostic and blood screening assays. We take advantage of the low volume requirements of single-molecule detection and apply a sample reloading approach to concentrate sample onto the detection surface. Due to the high affinity of the streptavidin-biotin reaction, concentration through reloading is both quick and robust. These findings are demonstrated on a model system and in an HIV p24 antigen assay. Single-molecule detection techniques do not need to be complex to exhibit power and flexibility, and so can become valuable in the field of immunoassay diagnostics.

Self-Assembly of Antigen Proteins into Nanowires Greatly Enhances the Binding Affinity for High-Efficiency Target Capture.

High-efficiency target capture is an essential prerequisite for sensitive immunoassays. However, the current available immunoassay approaches are subject to deficient binding affinities between predator-prey molecules that greatly restrict the target capture efficiency and immunoassay sensitivity. Herein, we present a new strategy through the self-assembly of antigen proteins into nanowires to enhance the binding affinity between an antigen and antibody. Through the genetic fusion of antigen proteins (e.g., HIV p24) with the yeast amyloid protein Sup35 self-assembly domain, specific antigen nanowires (Ag nanowires) were constructed and demonstrated a remarkable enhancement in binding affinity compared with that of the monomeric antigen molecule. The Ag nanowires were further combined with magnetic beads to form a 3D magnetic probe based on a seed-induced self-assembly strategy. Taking advantage of both the strong binding affinity and the rapid magnetic separation and enrichment capacity, the specific 3D magnetic probe achieved a 100-fold improvement in detection sensitivity within a significantly shorter period of 20 min over that of the conventional enzyme-linked immunosorbent assay method.

Computational prediction of antifungal peptides via Chou's PseAAC and SVM.

With the increase in immunocompromised patients in the recent years, fungal infections have emerged as new and serious threat in hospitals. This, and the insufficiency of current antifungal therapies alongside their toxic effects on patients, has led to the increased interest in seeking new antifungal peptides. In the present study, we have developed a prediction method for screening of antifungal peptides. For this, we have chosen Chou's pseudo amino acid composition (PseAAC) to translate peptide sequences into numeric values. Thus, the SVM classifier was performed for binomial classification of antifungal peptides. The performance of the classifier was evaluated via ten-fold cross-validation and an independent dataset. For further validation of the model developed, 22 P24-derived peptides were predicted using the classifier and in vitro assays were performed on the three peptides with the highest prediction score. The results showed that the PseAAC + SVM method is able to predict AFPs with ACC of 94.76%. In vitro results also validate the SEN and SPC of the classifier. The results suggest that the computational approach used in this study is highly efficient for prediction of antifungal peptides, which can save time and money in AFP screening and synthesis of novel peptides.

Comparative genetic variability in HIV-1 subtype C p24 Gene in early age groups of infants.

It is important to study the molecular properties of vertically transmitted viruses in early infancy to understand disease progression. P24 having an important role in virus assembly and maturation was selected to explore the genotypic characteristics. Blood samples, obtained from 82 HIV-1 positive infants, were categorized into acute (≤ 6 months) and early (> 6-18 months) age groups. Of the 82 samples, 79 gave amplification results for p24, which were then sequenced and analysed. Amino acid heterogeneity analysis showed that substitutions were more frequent. Several substitution mutations were present in some of the sequences of both the age groups in the functional motifs of the gene namely Beta hairpin, CyPA binding loop, residues L136 and L190, linker region and major homology region. In the acute age group, an insertion of Asparagine residue (N5NL6) was observed in the ß hairpin region in one of the sequences. This insertion was accompanied with analogous substitutions of N5Q, Q7L and G8R. In the early age group, a deletion of two residues; VK181-182, was observed at the C-terminal end in one of the sequences. These mutations may impair the structure of the protein leading to defective virus assembly. Protein variation effect analyzer software showed that deleterious mutations were more in the acute than the early age group. Variability analysis revealed that the amino acid heterogeneity was comparatively higher in the acute than the early age group. Variability in the virus was decreasing with the increasing age of the infants indicating that the virus is gradually evolving under positive selection pressure. HLA class 1 binding peptide analysis showed that the epitopes TPQDLNTML and RMYSPVSIL may be helpful in designing epitope based vaccine.

Rhodamine-Derived Fluorescent Dye with Inherent Blinking Behavior for Super-Resolution Imaging.

Super-resolution microscopy enables imaging of structures smaller than the diffraction limit. Single-molecule localization microscopy methods, such as photoactivation localization microscopy and stochastic optical reconstruction microscopy, reconstruct images by plotting the centroids of fluorescent point sources from a series of frames in which only a few molecules are fluorescing at a time. These approaches require simpler instrumentation than methods that depend on structured illumination and thus are becoming widespread. The functionalized rhodamine derivative reported in this paper spontaneously converts between a bright and dark state due to pH-dependent cyclization. At pH 7, less than 0.5% of the dye molecules are fluorescent at any given time. Blinking occurs on time scales of seconds to minutes and can therefore be used for single-molecule localization microscopy without sample treatment or activation. The dye is bright and straightforward to use, and it is easy to synthesize and functionalize. Thus, it has potential to become a new and powerful addition to the toolset for super-resolution imaging.

Graph-based optimization of epitope coverage for vaccine antigen design.

Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapid characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. We also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications. Published 2017. This article is a U.S. Government work and is in the public domain in the USA. Statistics in Medicine published by John Wiley & Sons Ltd.

Improving bioassay sensitivity through immobilization of bio-probes onto reactive micelles.

An antigen probe (HIV-1 p24) immobilized onto N-succinimidyl ester based micelles was used as a solid phase coating in ELISA test, and induced a significant improvement in antibody detection sensitivity as compared to the standard free antigen coating. The relevance of this straightforward approach to improve the bioassay sensitivity was confirmed by using biotin as a generic probe.

Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights.

Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators. A crystal structure of the lead nanobody and p24 was obtained and used alongside molecular dynamics simulations to elucidate the molecular basis of these enhanced performance characteristics. They indicate that binding occurs at C-terminal helices 10 and 11 of p24, a negatively charged region of p24 complemented by the positive surface of the nanobody binding interface involving CDR1, CDR2, and CDR3 loops. Our findings have broad implications on the design of novel antibodies and a wide range of advanced biomedical applications.

A multi-walled carbon nanotubes based molecularly imprinted polymers electrochemical sensor for the sensitive determination of HIV-p24.

To develop a rapid, simple and sensitive method for the determination of human immunodeficiency virus p24 (HIV-p24), a novel molecularly imprinted polymers (MIPs) electrochemical sensor was constructed on the surface of a multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE) by surface polymerization using acrylamide (AAM) as functional monomer, N,N'-methylenebisacrylamide (MBA) as cross-linking agent and ammonium persulphate (APS) as initiator. Each modification step was carefully examined by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and scanning electron microscope (SEM). The proposed MIPs electrochemical biosensor exhibited specific recognition to HIV-p24 and displayed a broad linear detection range from 1.0×10-4 to 2ngcm-3 with a low detection limit of 0.083pgcm-3 (S/N=3). This performance is superior to most HIV-p24 sensors based on other methods. Meanwhile, this sensor possessed of good selectivity, repeatability, reproducibility, stability and was successfully applied for the determination of HIV-p24 in real human serum samples, giving satisfactory results. The accuracy and reliability of the sensor is further confirmed by enzyme-linked immunosorbent assay (ELISA).

Array-in-well binding assay for multiparameter screening of phage displayed antibodies.

Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10×10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.

Effect of Several HIV Antigens Simultaneously Loaded with G2-NN16 Carbosilane Dendrimer in the Cell Uptake and Functionality of Human Dendritic Cells.

Dendrimers are highly branched, star-shaped, and nanosized polymers that have been proposed as new carriers for specific HIV-1 peptides. Dendritic cells (DCs) are the most-potent antigen-presenting cells that play a major role in the development of cell-mediated immunotherapy due to the generation and regulation of adaptive immune responses against HIV-1. This article reports on the associated behavior of two or three HIV-derived peptides simultaneously (p24/gp160 or p24/gp160/NEF) with cationic carbosilane dendrimer G2-NN16. We have found that (i) immature DCs (iDCs) and mature (mDCs) did not capture efficiently HIV peptides regarding the uptake level when cells were treated with G2-NN16-peptide complex alone; (ii) the ability of DCs to migrate was not depending on the peptides presence; and (iii) with the use of molecular dynamic simulation, a mixture of peptides decreased the cell uptake of the other peptides (in particular, NEF hinders the binding of more peptides and is especially obstructing of the binding of gp160 to G2-NN16). The results suggest that G2-NN16 cannot be considered as an alternative carrier for delivering two or more HIV-derived peptides to DCs.

A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study.

There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.

A Novel Electrochemiluminescence Immunosensor for the Analysis of HIV-1 p24 Antigen Based on P-RGO@Au@Ru-SiO2 Composite.

Ru(bpy)3(2+)-doped silica (Ru-SiO2) nanoparticles and gold-nanoparticle-decorated graphene (P-RGO@Au) were combined to form a P-RGO@Au@Ru-SiO2 composite. The composite was used to develop a novel sandwich-type electrochemiluminescence immunosensor for the analysis of HIV-1 p24 antigen. The composite worked as carrier to immobilize target antibody and to build a sandwich-type electrochemiluminescence immunosensor through an interaction between antigen and antibody. Importantly, high ECL signal could be obtained due to the large amounts of Ru(bpy)3(2+) molecules per Ru-SiO2 nanoparticle. The P-RGO@Au composite with good conductivity and high surface area not only accelerated the electron transfer rate but also improved the loading of both ECL molecules and capture antibody, which could further increase the ECL response and result in high sensitivity. Taking advantage of both Ru-SiO2 nanoparticles and the P-RGO@Au composite, the proposed immunosensor exhibited a linear range from 1.0 × 10(-9) to 1.0 × 10(-5) mg mL(-1) with a detection limit of 1.0 × 10(-9) mg mL(-1) for HIV-1 p24 antigen. The proposed ECL immunosensor was used to analyze HIV-1 p24 antigen in human serum, and satisfactory recoveries were obtained, indicating that the proposed method is promising for practical applications in the clinical diagnosis of HIV infection.

[Effect of pH of Adsorption Buffers on the Number and Antigen-Binding Activity of Monoclonal Antibodies Immobilized on the Surface of Polystyrene Microplates].

The change in the concentration and antigen-binding activity of 28 monoclonal antibodies was studied after their adsorption on the surface of polystyrene microplates in buffers with different pH values (1.0, 2.8, 7.5, 9.6, and 11.9). We used 16 clones to the HIV p24 protein and 12 clones to the surface antigen of Hepatitis B Virus. The binding efficiency of adsorbed antibodies to the labeled antigen was evaluated by the slope of the linear region of the binding curve to the concentration axis. It was shown that the antigen-binding activity of six antibodies (21.5%) statistically significantly increased after adsorption at pH 2.8 and 11.9 as compared to pH 7.5 and 9.5. The maximum amount of antibodies was found to be adsorbed on the solid surface at pH 7.5. The analysis of the binding of 125I-HBs-antigen to adsorbed antibodies made it possible to evaluate the concentration of active antibodies on the polystyrene surface. It was shown that the increase in the antigen-binding activity was due to an increase in the proportion of antibodies with retained activity after adsorption at pH 2.8 and 11.9. Under these conditions, about 20% of the antibodies retained their antigen-binding activity, and 6% did so after immobilization at pH 7.5.