An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

The Biochemical Properties of Antibodies and Their Fragments.

Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.

Profiling antibodies to class II HLA in transplant patient sera.

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3µg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to-patient antibody concentrations did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

Purification of human immunoglobulins A, G and M from Cohn fraction II/III by small peptide affinity chromatography.

This work describes attempts to purify human IgG, IgA and IgM from Cohn fraction II/III using HWRGWV affinity peptide resin. The effects of peptide density and different elution additives on recovery of the three antibodies were investigated. At low peptide density, salting-in salts such as magnesium chloride and calcium chloride facilitated antibody elution. Ethylene glycol, urea and arginine also facilitated elution because of their ability to decrease hydrophobic interactions, hydrogen bonding and electrostatic interactions. However, at high peptide density, no recovery improvements were observed because of increased non-specific hydrophobic interactions. The final elution conditions for each antibody were chosen based on the resulting yields and purities when a 10:2:1mg/mL mixture of human IgG, IgA and IgM was used as starting material. Different pretreatment methods were employed in order to improve the purity of antibodies from Cohn fraction II/III. After pretreatment with caprylic acid precipitation or combination of caprylic acid and polyethylene glycol precipitation, purities over 95% and yields of about 60% were obtained for hIgG, which are comparable to current chromatographic purification methods involving two chromatography steps when hIgG is isolated from plasma fractions. A hIgA-enriched fraction with 42% hIgA and 56% hIgG, as well as a hIgM enriched fraction with 46% hIgM, 28% hIgA and 24% hIgG, were obtained as the by-products.

Experimental infection of chickens with Histomonas meleagridis confirms the presence of antibodies in different parts of the intestine.

Specific pathogen-free chickens were infected with a clonal culture of the protozoan parasite, Histomonas meleagridis. Severe lesions were found within the caeca of all birds euthanized at 7 and 14 days post-infection (d.p.i.). Following necropsy of birds, intestinal samples were taken to establish ex vivo tissue cultures to determine the IgG, IgA and IgM antibody levels in the supernatants before and after incubation with a recently established ELISA. Presence of antibodies was also determined in the sera and first optical density values for IgG above the cut-off were detected at 14 d.p.i. IgA levels remained low in the serum with a small peak 4 weeks p.i., a phenomenon also found for IgM. The intestinal tissue samples showed very strong immunological reactions in the parasitized caeca with an initial peak of IgM, high levels of IgG and a continuous increase of IgA. In the duodena and jejuna, IgA values reached similar high levels as those obtained in the caeca, whereas IgG and IgM increased only slightly.

Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum.

All four subclasses of human serum IgG contain a single N-glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core-fucosylated and partially truncated oligosaccharides, that may carry a bisecting N-acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N-glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N-glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96-well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano-LC-MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N-glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass-specific analysis of IgG glycosylation from clinical samples.

Discovery of a new class of immunoglobulin heavy chain from fugu.

In teleosts, the genomic organization of the immunoglobulin (Ig) heavy (H)-chain locus was thought to follow a typical translocon-type multigene structure; however, recent studies have indicated a variation in the structure and this might be teleost specific. Isotypes of the Ig H-chain, namely IgM, IgD, IgZ and IgT, have been identified. In this study, we report the discovery of a new class of IgH from fugu. This isotype was first identified from the genomic sequence of the fugu IgH locus. This novel IgH gene is composed of two constant (C) domains, a hinge region, and two exons encoding membrane regions. Surprisingly, the new IgH gene is present between the variable (V)H and Cmu regions of the locus. The C domains of the new isotype do not show any significant similarity to mammalian or fish IgH genes. The cloned cDNA from the new isotype has typical Ig H-chain characteristics and is expressed as both secretory and membrane form. Transcript analyses suggest that the new IgH from fugu might only use the joining (J)H segments present in front of the new CH domains and that the usage of DH and JH segments is specific to the isotype expressed. The expression pattern of the gene has been confirmed by in situ hybridization and PCR studies.

Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau.

Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised.

Salivary albumin, total protein, IgA, IgG and IgM concentrations and occurrence of some periodontopathogens in HIV-infected patients: a 2-year follow-up study.

HIV infection reduces oral defensive mechanisms and may affect mucosal integrity. Differences in salivary protein concentrations and periodontopathogenic bacteria were studied in 56 HIV-infected patients with respect to their disease phase. Thirty-three patients were followed up for 2 years. Fifty-three healthy subjects of corresponding age and sex were studied as controls. At baseline, salivary albumin, total protein, IgA, and IgM levels were significantly higher (P<0.05-0.0001) in all phases of HIV infection, except the asymptomatic (ASX) phase, when compared with the control group. IgG levels were significantly increased in all phases except the ASX phase (P<0.05). After 2 years, salivary total protein, IgG, and IgM levels were still higher (P<0.05-0.005) in all HIV phases when compared with the control group (P<0.05-0.005). The albumin level was significantly higher in the ASX phase (P<0.005) and in the AIDS-related complex phase (ARC) (P<0.05), while the increase in IgA level was significant only in the ARC phase (P<0.005). Periodontopathogenic bacteria analyzed by PCR were detected both in the patients and the non-infected, but a statistically significant difference in the carriage percentage between the follow-up lymphadenopathy syndrome phase (LAS) and the control group was found only in Porphyromonas gingivalis (P<0.05) and Bacteroides forsythus (P< 0.0001). Thus, HIV infection appeared to cause a significant increase in the studied salivary proteins, suggesting leakage of serum components into the mouth.

Purification of horse immunoglobulin isotypes based on differential elution properties of isotypes from protein A and protein G columns.

Elution properties of horse immunoglobulin isotypes from protein A and protein G columns were examined. IgGa and IgGb isotypes were bound to protein A and protein G columns and were eluted by adjusting the pH of the elution buffer from 8.0 to 2.0. IgGc bound to protein G column but not to protein A column while IgG(T) bound to both columns. IgM and IgA apparently appeared not to bind to either column. New methods for purification of serum isotypes were developed using protein A and protein G columns as well as formerly established methods. Using these methods, it was possible to obtain purified isotypes for establishment of immunological assays for practical clinical use.

Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments.

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1-IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy.

Neurocysticercosis: detection of IgG, IgA and IgE antibodies in cerebrospinal fluid, serum and saliva samples by ELISA with Taenia solium and Taenia crassiceps antigens

We assayed samples of cerebrospinal fluid (CSF), serum and saliva from patients with neurocysticercoses, control group and individuals with other parasitoses, by ELISA with Taenia crassiceps vesicular fluid antigen (Tcra) and Taenia solium total antigen (Tso) for the detection of antibodies. The sensitivity for IgG-Tcra was 100 per cent for CSF and serum, and 32.0 per cent for saliva; and for IgG-Tso 100 per cent for CSF, 80.0 per cent for serum and 24 per cent for saliva. Specificity was 100 per cent for CSF and 80.0 per cent for serum with both antigens, and 100 per cent for saliva with Tcra and 87.5 per cent with Tso. The sensitivity and specificity for IgA-Tcra was, respectively, 40.0 per cent and 100 per cent for CSF, 36.0 per cent and 97.1 per cent for serum, and 4.0 per cent and 90.0 per cent for saliva. IgE detection showed 24.0 per cent sensitivity and 97.1 per cent specificity for serum, with no detection in CSF samples. The search for antibodies revealed the presence of IgG > IgA > IgE in CSF, serum and saliva samples, with IgG being present in all phases of the disease, while IgA/IgE were more frequent in the inactive form.

Evaluation of a high IgE-responder mouse model of allergy to bovine beta-lactoglobulin (BLG): development of sandwich immunoassays for total and allergen-specific IgE, IgG1 and IgG2a in BLG-sensitized mice.

An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.

Cutting edge: mouse IgG1 antibodies comprise two functionally distinct types that are differentially regulated by IL-4 and IL-12.

IL-4-dependent and -independent IgG1 Abs differ in their ability to induce mast cell degranulation as measured by passive cutaneous anaphylaxis (PCA). Mice immunized with OVA or PIII (fraction of Ascaris suum) produced high titers of IgG1 as shown by ELISA and PCA. In contrast, another A. suum fraction, PI, elicited IgG1 Abs with no PCA activity. IgG1 with anaphylactic activity required IL-4, as IgG1 responses to OVA and PIII in IL-4-/- mice gave no PCA. PI-specific IgG1 was IL-4-independent, because no difference was found between the responses of IL-4-/- and IL-4+/+ mice. Significant PCA reactions were elicited, however, with PI-specific IgG1 from IL-12-/- or anti-IFN-gamma Ab-treated mice, although less Ab was measured by ELISA. These results indicate that one type of IgG1 has anaphylactic activity and its synthesis is IL-4-dependent, being inhibited by IL-12 or IFN-gamma; the other lacks this activity and its synthesis is stimulated by IL-12 or IFN-gamma.

Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins--evaluation of two anti-His-tag monoclonal antibodies.

A tag comprising four to six histidines genetically fused to the protein of interest (His-tag) has been widely used to purify proteins by immobilized metal-ion affinity chromatography (IMAC). Here we report the utilization of the same tag twice in series, first for IMAC and subsequently for immunoaffinity purification. Both steps are based on completely different physical principles and can therefore remove different contaminants. Two anti-His-tag antibodies (3D5 and PentaHis) were characterized for their binding and elution properties using the BIAcore surface plasmon resonance biosensor. The dissociation constant of the PentaHis antibody was determined to be 1 x 10(-8) M and for the 3D5 antibody 3.4 x 10(-7) M at pH 7.4. Imidazole in the sample did interfere with binding, whereas chelating agents such as EDTA and high salt did not. The antibody 3D5 was coupled to a column matrix and used for a coupled two-step purification, in which the IMAC column is eluted with EDTA and the eluent is loaded directly on the immunoaffinity column. This method may constitute a very general procedure to purify proteins to near homogeneity without the need to tailor conditions individually, and it may thus be very attractive for high-throughput screening programs and for developing general protocols for clinical grade material.

Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for staphylococcal protein A and streptococcal protein G.

Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purification of the subisotypes of equine IgG. Purification of IgGa and IgGb was achieved by the separation of a 'fall-through' peak from ion-exchange chromatography consisting of IgGa and IgGb into two fractions (peaks C and D) by FPLC protein A and protein G affinity chromatography. Peak C consisted of IgGb and peak D consisted of IgGa exhibiting slightly faster cathodal migration than peak C in IEP analysis. Affinity chromatography using protein A and G columns also indicated that there may be two different components of IgG(T); one with a low affinity for protein G and the other having a greater affinity for protein G.

A second immunoglobulin light chain isotype in the rainbow trout.

A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.