Generation of functional antibodies for mammalian membrane protein crystallography

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High-efficiency generation of monoclonal antibody for Vitreoscilla hemoglobin protein

Bacterial hemoglobin from Vitreoscilla (VHb) is recognized as a good fusion protein for the soluble expression of foreign protein. In this study, we generated a monoclonal antibody (MAb) against VHb for its detection. For the rapid screening of MAb, a protein chip technology based on the Alexa-488 (A488) dye labeling method was introduced. In order to fabricate the chip, the VHb protein was chemically coupled to the chip surface and then the culture supernatants of 84 hybridoma cell lines were spotted onto the VHb chip. The bound MAbs were measured by A488- modified anti-mouse IgG. A single spot (MAb A10) exhibited significantly high signal intensity. The immunoblot analysis evidenced that the MAb A10 can detect VHb-fused proteins with high specificity.     

Biological screening of monoclonal antibodies

A method was developed to screen hybridomas secreting immunoglobulin to cell surface receptors by observing the ability of antibodies to inhibit cell attachment and survival. The model used to develop the screening procedure involved mouse hybridomas secreting monoclonal IgG to human epidermal growth factor (EGF) receptors. Conditioned medium from these hybridomas inhibited the attachment and subsequent growth of human foreskin fibroblasts unless excess EGF was added to the cultures. This procedure allows for the selection of hybridomas producing increased levels of immunoglobulin.     

High throughput screening setup of a scale-down device for membrane chromatography-aggregate removal of monoclonal antibodies

In biopharmaceutical process development, resin-based high throughput screening (HTS) is well known for overcoming experimental limitations by permitting automated parallel processing at miniaturized scale, which results in fast data generation and reduced feed consumption. For membrane adsorber (MA), HTS solutions have so far only been available to a partial extent. Three case studies were performed with the aim of aligning HTS applications for MAs with those established for column chromatography: Process parameter range determination, mechanistic modeling (MM), and scalability. In order to exploit the MA typically features, such as high mass transfer and easy scalability, for scalable high throughput process development, a scale-down device (SDD) for MA was developed. Its applicability is confirmed for a monoclonal antibody aggregate removal step. The first case study explores the experimental application of the SDD developed. It uses bind and elute mode and variations of pH and salt concentration to obtain process operation windows for ion-exchange MAs Sartobind® S and Q. In the second case study, we successfully developed a mechanistic model based on parameters obtained from the SDD–HTS setup. The results proved to validate the use of the SDD developed for parameter estimation and thus model-based process development. The third case study shows the transferability and scalability of data from the SDD–HTS setup using both a direct scale factor and MM. Both approaches show good applicability with a deviation below 20% in the prediction of 10% dynamic breakthrough capacity and reliable scale-up from 0.42 to 800 ml.     

Overcoming the challenges of membrane protein crystallography

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.     

Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of salmonella

S. KERR. H.J. BALL, D.P. MACKIE, D.A. POLLOCK AND D.A. FINLAY. 1992. Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars and several related enterobacteria. Those detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media by conventional culture. Of these 58 were detected by the ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. The results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures at an early stage.     

Lipidic phase membrane protein serial femtosecond crystallography

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.     

Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of Salmonella.

Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars and several related enterobacteria. Those detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media by conventional culture. Of these 58 were detected by the ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. The results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures at an early stage.     

Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP auxin-binding protein - DEAE diethylaminoethyl - Ig immunoglobulin - kDa kilodalton - NAA naphthalene-1-acetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

The potential protective effect of monoclonal antibodies to gonococcal outer membrane protein IA

Hybrid cell lines were derived which produced monoclonal antibodies directed against gonococcal outer membrane protein IA. One antibody, SM101, recognized 24 P.IA-expressing strains out of 25 tested--the rest exhibited relatively less cross-reactivity. Competitive radioimmunoassays revealed that each antibody could effectively inhibit binding of the others, suggesting close proximity of the epitopes recognized. The antibodies were used in assays in vitro to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, the antibodies afforded some protection to epithelial cells challenged with gonococci. They were very effective at bactericidal killing in the presence of complement and, in addition, were opsonic for homologous and heterologous strains. The cross-reacting antibody, SM101, was one of the most effective in both assays. The results show that the conserved epitope on P.IA recognized by antibody SM101 is potentially an effective target on the gonococcal surface for immunoprophylaxis.     

Basement membrane diversity detected by monoclonal antibodies

Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno) chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin.     

Basement membrane diversity detected by monoclonal antibodies

Abstract. Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent-cent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno)chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin     

An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies

The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains.     

Detection by monoclonal antibodies of an early membrane protein induced by Epstein-Barr virus.

Two monoclonal antibodies, E8B3 and E8D2, were raised against Epstein-Barr virus (EBV)-producing cells and were shown to immunoprecipitate a protein with an approximate molecular weight of 105,000 (p105). The protein was detectable only in EBV-containing cells which were supporting the virus lytic cycle, and its synthesis increased after cells were induced with phorbol esters. The molecule was radiolabeled and immunoprecipitated from virus-producing cells that had been extrinsically labeled with 125I, and the antibodies E8B3 and E8D2 reacted in immunofluorescence assays with infected cells; the molecule was also associated with virion particles. Synthesis of p105 was not blocked by phosphonoacetic acid and could be induced in Raji cells by superinfection with virus derived from P3HR1 cells. These data support the conclusion that p105 is an EBV-specific early membrane protein.     

Computational tool for the early screening of monoclonal antibodies for their viscosities

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures). The SCM tool has been extensively validated at 3 different organizations, and has proved successful in correctly identifying highly viscous antibodies. As a quantitative tool, SCM is amenable to high-throughput automated analysis, and can be effectively implemented during the antibody screening or engineering phase for the selection of low-viscosity antibodies.     

Preparation of "histocomposites" for direct immunohistochemical screening of monoclonal antibodies

Screening and selection of hybrids producing relevant antibodies in monoclonal technology usually rely on rapid and sensitive adsorption assays of the ELISA type. To identify clones producing antibodies with unexpected specificities direct immunohistological screening may be applied, but this is both tedious and expensive. Histocomposites made from a number of tissue types permit testing of supernatants at the required early stage after fusion. The multiple antigenic specificities displayed in such test specimens ensure detection of a broad range of antibodies. A simple method for production of the histocomposites is described.     

A monoclonal antibody for G protein-coupled receptor crystallography

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.     

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.     

Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs

Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.     

Development of a simplified method for subclass isotyping and screening monoclonal antibodies

A simplified dot-blot procedure for screening and subclass isotyping of monoclonal antibodies is described in which only 0.5-50 ng of antigen and 1 microliter of antibody are needed to perform the test. The results on the nitrocellulose membrane can be stored indefinitely for future reference. This method is less expensive, uses smaller quantities of antigen and antibody, and is faster than presently used enzyme-linked immunosorbent assay techniques or other dot-blot methods. Monoclonal antibodies against Mycoplasma gallisepticum were screened and isotyped using this technique.