Immunoreactivity Of mAb Research Articles

Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin.

Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

A new antigenic marker specifically labels a subpopulation of the class II Kenyon cells in the brain of the European honeybee Apis mellifera.

The mushroom bodies are the higher-order integration center in the insect brain and are involved in higher brain functions such as learning and memory. In the social hymenopteran insects such as honeybees, the mushroom bodies are the prominent brain structures. The mushroom bodies are composed of lobed neuropils formed by thousands of parallel-projecting axons of intrinsic neurons, and the lobes are divided into parallel subdivisions. In the present paper, we report a new antigenic marker to label a single layer in the vertical lobes of the European honeybee Apis mellifera. In the brain of A. mellifera, a monoclonal antibody (mAb) 15C3, which was originally developed against an insect ecdysone receptor (EcR) protein, immunolabels a single layer of the vertical lobes that correspond to the most dorsal layer of the γ-lobe. The 15C3 mAb recognizes a single ~200 kDa protein expressed in the adult honeybee brain. In addition, the 15C3 mAb immunoreactivity was also observed in the lobes of the developing pupal mushroom bodies. Since γ-lobe is well known to their extensive reorganization that occurs during metamorphosis in Drosophila, the novel antigenic marker for the honeybee γ-lobe allows us to investigate morphological changes of the mushroom bodies during metamorphosis.

Prognostic Role of 14F7 Mab Immunoreactivity against N-Glycolyl GM3 Ganglioside in Colon Cancer

Purpose. To assess the prognostic role of 14F7 Mab immunoreactivity, against N-Glycolyl GM3 ganglioside, in patients with colon cancer (CC) and to evaluate the relationship between its expression and clinicopathological features. Methods. Paraffin-embedded specimens were retrospectively collected from 50 patients with CC operated between 2004 and 2008. 14F7 Mab staining was determined by immunohistochemistry technique and its relation with survival and clinicopathologic features was evaluated. Results. The reactivity of 14F7 Mab was detected in all cases. Most cases had high level of immunostaining (70%) that showed statistical correlation with TNM stage (P = 0.025). In univariate survival analysis, level of 14F7 Mab immunoreactivity (P = 0.0078), TNM Stage (P = 0.0007) and lymphovascular invasion (0.027) were significant prognostic factors for overall survival. Among these variables, level of 14F7 Mab immunoreactivity (HR = 0.268; 95% CI 0.078–0.920; P = 0.036) and TNM stage (HR = 0.249; 95% CI 0.066–0.932; P = 0.039) were independent prognostic factors on multivariate analysis. Conclusions. This study is the first approach on the prognostic significance of 14F7 Mab immunoreactivity in patients with colon adenocarcinoma and this assessment might be used in the prognostic estimate of CC, although further studies will be required to validate these findings.

N-glycosylation of CD97 within the EGF domains is crucial for epitope accessibility in normal and malignant cells as well as CD55 ligand binding.

CD97 is an EGF-TM7 receptor found on various carcinomas where expression levels correlate with dedifferentiation and tumor stage, smooth muscle cells and leukocytes. CD97 acts as an adhesion molecule by binding to its cellular ligand, CD55. In this study, we demonstrate that 2 immunodominant CD97 epitopes are not equally present in the various cell types. Differences were apparent in gastrointestinal tumors and smooth muscle cells where monoclonal antibodies (mAbs) to the first epidermal growth factor (EGF) domain (CD97(EGF)) showed a more restricted staining pattern than mAbs to the stalk region (CD97(stalk)). This discrepancy was not detectable in cultured gastrointestinal tumor cell lines. In fact, the selection of the CD97 mAb influences the result of clinical studies. Thus, we clarified the reason(s) for these differences in CD97 mAb staining on various cell types. We provide evidence that epitope accessibility for CD97(EGF) mAbs depends on N-glycosylation. Immunoprecipitation of CD97 from the Colo 205 tumor cell line revealed the established 78 and 83 kDa products, while a 52 and 57 kDa band were obtained from smooth muscle cells. N-glycosidase F reduced the size of CD97 in Colo 205 cells to 52-57 kDa. Culturing these cells with tunicamycin resulted in the same decrease in size and impaired CD97(EGF) mAb binding. As shown by site-directed mutagenesis, deletion of the N-glycosylation sites located within the EGF domains efficiently disturbed CD97(EGF) mAb immunoreactivity and, importantly, binding of CD55. In conclusion, CD97(EGF) epitope accessibility for mAbs and ligand binding is influenced by cell type-specific N-glycosylation.

Structural Variability of CD44v Molecules and Reliability of Immunodetection of CD44 Isoforms Using mAbs Specific for CD44 Variant Exon Products

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.

Method for radioiodination of proteins using N-succinimidyl 3-hydroxy-4-iodobenzoate.

A conjugation method has been developed for the radioiodination of proteins which should be adaptable to kit formulation. m-Hydroxybenzoic acid was converted to 3-hydroxy-4-[131I]iodobenzoic acid in 65% radiochemical yield using Chloramine-T as the oxidant. This intermediate was then converted to N-succinimidyl 3-hydroxy-4-[131I]iodobenzoate ([131I]mSHIB) in 75% yield by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide in a reaction time of only 10 min. Monoclonal antibody (mAb) 81C6 was labeled in 40-60% yield by reaction with [131I]mSHIB. Performing purifications of radioiodinated compounds using cartridges instead of HPLC did not alter conjugation efficiency, mAb immunoreactivity, or tissue distribution. Thyroid uptake of labeled mAb was low but up to 2.4 times higher than that seen when the mAb was labeled with N-succinimidyl 3-[125I]-iodobenzoate. These results suggest that [131I]mSHIB may be a useful reagent for the radioiodination of proteins, particularly in contexts when less complicated purification methods would be advantageous.

The Anti-Human Prostasome MAB 78 binds to an Antigen Distinct from PSA and PAP

Purpose The characteristics of an antigen corresponding to a monoclonal antibody (mAb 78) against human prostasomes were compared with prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP). The correspondence of Ag 78 to two other prostate-derived antigens, prostasin and peptide pGlu-Phe-Pro-NH 2, were also considered. Materials and Methods The immunoreactivity of mAb 78 and the cross-reactivity of mAb 78 with PSA and PAP were studied with immunohistochemical and enhanced chemiluminescence (ECL) Western blotting methods. Results The mAb 78 did not bind to PSA blots, and anti-PSA antibody did not label prostasome blots. Neither did PSA and PAP impede the binding of mAb 78 onto prostasome blots nor to paraffin sections of prostate epithelium. After Western blots, mAb 78 bound diffusely to a band with a molecular weight of 35 kDa, but did not bind to PSA (33 kDa) or PAP (monomer 50 kDa, intact molecule 100 kDa) bands. From purified 35 kDa bands, three fractions were sequenced, which showed no similarities to PSA and PAP. Conclusions The Ag 78 is different from PSA and PAP, and probably also from prostasin and peptide pGlu-Phe-Pro-NH 2. The mAb 78 can be used as a new marker for human prostasomes.

Purification by affinity chromatography with anti-idiotypic monoclonal antibodies of immunoreactive monoclonal antibodies following labeling with 188Re.

Because of its high energy beta emissions and imageable gamma emissions, 188Re represents an attractive isotope to radiolabel monoclonal antibodies (mAb) recognizing human tumor-associated antigens for radioimaging and radioimmunotherapy in patients with malignant diseases. The application of 188Re is, however, hindered by the denaturation of a sizable proportion of antibody molecules during the labeling process. To overcome this problem, we have combined radiolabeling of mAb with 188Re with purification of immunoreactive 188Re-labeled mAb by affinity chromatography over columns of anti-idiotypic (anti-id) mAb. Utilizing the anti-high-molecular-weight melanoma-associated antigen (HMW-MAA) mAb 763.74 as a model system, we found that the immunoreactivity of mAb 763.74 labeled with 188Re to a specific activity of 1 mCi/mg increased from about 50% to at least 80% following passage over columns of immobilized anti-id mAb. Moreover, between 90-100% of immunoreactive mAb contained in radiolabeled preparations could be recovered from anti-id mAb columns. These results indicate that the procedure we have described may facilitate the application of 188Re for immunoscintigraphy and immunotherapy of malignant diseases.

Immunochemical quantification of myoglobin heat denaturation: Comparative studies with monoclonal and polyclonal antibodies

Solutions of bovine myoglobin in 0.1 M‐phosphate buffer, pH 6.0, were heated over the temperature range 55–85°C for 60 min. Their immunological reactivity was tested after 0.2 μm filtration using polyclonal antibodies in the single radial immunodiffusion assay (SRID) and six monoclonal antibodies (MAbs) in a competitive and a sandwich ELISA. Reproducible results were obtained in the SRID assay (coefficient of variation (CV) = 4.0%), with a mid‐point denaturation of 79.0 ± 0.4°C. Similar results were obtained with two MAbs (mid‐point denaturation at 79.4 ± 0.5°C and 78.3 ± 0.8°C for competitive and sandwich ELISAs respectively), but the CV% values were higher (8 and 13% respectively). With the remaining four MAbs, quantification was unreliable as immunoreactivity increased during heating with large inter‐assay variations. A careful control of MAb immunoreactivity in situations covering all possible denaturation states of the test protein is an absolute prerequisite to their use in ELISAs for the immunoquan...

Evidence obtained with monoclonal antibodies that O antigen is the major antigen responsible for the cross-reactivities between serotypes 4 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae.

Monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae serotype 4 (reference strain M62 and field isolate F6) were produced and characterized. Three hybridoma clones were raised against strain M62, and 13 were raised against strain F6. The predominant antibody class was immunoglobulin M (IgM), although IgG2a and IgG2b were also obtained. Three of the MAbs produced to field isolate F6 (5C5, 1E10, and 5H7) did not recognize the reference strain of serotype 4, another (6F7) was reactive with both reference strains of serotypes 4 and 7, and the remaining 12 MAbs reacted only with the reference strain of the homologous serotype. All epitopes recognized by MAbs, except for one (6F7), were sensitive to periodic acid oxidation, and all of them were resistant to boiling in the presence of sodium dodecyl sulfate and reducing conditions, as evidenced by immunodot. Enhanced chemioluminiscence-immunoblot assays revealed that 10 MAbs (3E12, 5B8, 7C3, 6F7, 7F5, 7E6, 5G4, 4F1, 7E10, and 4B8) recognized a ladder-like banding pattern, which is in accordance with the O side chain antigen of lipopolysaccharide (LPS), while the remaining 6 MAbs (5C5, 5H7, 1E10, 6D11, 6B4, and 5E4) blotted with high-molecular-weight regions composed of a single banding pattern. The suitability of MAbs for serotyping of 78 field isolates was also examined. A high correlation was found between the results previously established by indirect hemagglutination with polyclonal sera and those obtained by enzyme-linked immunosorbent assay with MAbs. According to the different immunoreactivity of MAbs, three groups were established: group I (MAbs 3E12, 5B8, 7C3, 6F7, and 7F5), group II (MAbs 7E6, 5G4, 4F1, 7E10, and 4B8), and group III (MAbs 5C5, 5H7, and 1E10). MAbs 6D11, 6B4, and 5E4 could not be included in any of the described above. At least six different immunodominant epitopes on the O antigen of the A. pleuropneumoniae serotype 4 LPS were identified. Finally, the implications of the effect of the O antigen of LPS in cross-reactions between serotypes 4 and 7 are clearly evidenced.

Immunohistochemical localization of neuronal nicotinic receptor subtypes at the pre- and postjunctional sites in mouse diaphragm muscle

The existence of neuronal nicotinic acetylcholine receptor (nAChR) subunits was investigated in the cryostat sections of mouse diaphragm muscles using the indirect immunofluorescence technique. The specific immunolabelings with monoclonal antibodies (mAbs) to β2 and to α8 subunits of neuronal nAChR were observed at the endplate determined by labeling with a fluorescent dye (BODIPY)-conjugated α-bungarotoxin. The immunoreactivity of mAb to the α3 subunit of neuronal nAChR was detected on the motor nerve fibers including the nerve terminals. These results provide evidence that the subtypes of postsynaptic nAChR, recognized by the anti-β2 and/or anti-α8 mAbs, and the presynaptic nAChR recognized by the anti-α3 mAb, are present at the neuromuscular junction, in addition to the classical muscle nAChR.

Immunohistological characterization of mucin epitopes by pre-treatment of gastro-intestinal sections with periodic acid

Periodate pretreatment of paraffin sections of ethanol-fixed gastrointestinal mucosae was used to characterize the carbohydrate or peptidic nature of mucin epitopes by immunoperoxidase. Immunoreactivity of monoclonal antibodies (MAbs) against histo-blood group related carbohydrate epitopes such as A, Lea, Lec, Sialosyl Lea, H type 2, I, T, Tn and sialosyl Tn dramatically decreased or even disappeared after periodate pretreatment of deparaffinized sections. In contrast, the immunoreactivity of MAbs against peptide mucin epitopes such as the gastric M1 mucin epitopes was almost unaffected by this treatment. Moreover, periodate treatment revealed cryptic peptide M1 mucin epitopes and the peptide MUSE11 epitope associated with the 20 amino acid tandem repeat (PDTRPAPGSTAPPAHGVTSA). An increase of cross-reactions of anti-human M1 MAbs with gastric epithelium of different vertebrate species was detected with periodate treatment. Our results suggest that this method can be useful for preliminary characterization of the biochemical nature of mucin epitopes (peptidic or saccharidic) and for demasking peptidic tumour markers which are hidden by saccharide molecules in normal tissues.

Stability of monoclonal antibody-defined epitopes

Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and defines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42° C and 65° C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability.

Epitope mapping of the HIV-1 gag region with monoclonal antibodies

A new type of immunochemical mapping of the human immunodeficiency virus type 1 (HIV-1) gag region was performed. By use of native HIV-1 viral lysates or the gag recombinant p24-15 antigen, a new set of monoclonal antibodies (Mabs) to the gag region proteins was generated. Synthetic HIV-1 peptides covering the entire gag region were used to specifically localize the continuous epitopes by direct binding to the Mabs and by blocking the Mab immunoreactivity. The identified immunogenic epitopes were localized between the gag amino acids (aa) 108–127, 203–217, 208–222, 248–282, 273–302, 288–307, 308–322, 331–354 and 408–422. These continuous epitopes formed seven immunogenic regions. One strongly p17-reactive Mab appeared to react with a discontinuous epitope, the components of which were 110 aa distant in the linear sequence: aa 23–27 and 128–132. The synthetic peptides appeared to be more congruent with the Mab-reactive sites in solution than when coated to a solid phase.

Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenografts in nude mice.

Doxorubicin (DXR) was covalently conjugated to a monoclonal antibody (mAb), 9.2.27 (IgG2a), which recognizes a chondroitin sulfate proteoglycan expressed preferentially on the surface of human melanoma cells. Immunoconjugates with a molar ratio of DXR to mAb ranging from 2:1 to 10:1 were obtained by coupling the drug via an acid-sensitive linker, cis-aconitic anhydride. The immunoreactivity of mAb 9.2.27 was well retained after conjugation. DXR-mAb 9.2.27 conjugates were found to be 2 orders of magnitude more potent in killing tumor cells in vitro (IC50 = 0.1 microM) than free drug targeted to drug receptor(s). Most significantly, DXR-mAb 9.2.27 immunoconjugates specifically suppressed the growth of established tumors in vivo and prolonged the life-span of tumor-bearing nude mice. This suppression of melanoma growth achieved by the immunoconjugate was both tumor and antibody specific. A biodistribution study indicated that DXR-mAb 9.2.27 conjugates delivered at least 4 times more DXR (3.7% total injected dose per g of tumor) as compared to free DXR alone (0.8% total injected dose per g of tumor) in tumor-bearing nude mice 48 hr postinjection. The tumor-suppressive effects of DXR-mAb 9.2.27 conjugates are even more remarkable since free DXR did not suppress tumor growth in vivo and also because this drug per se is known to be quite ineffective for the treatment of human melanoma.

Aneuploidy and expression of gastric-associated mucus antigens M1 and CEA in colorectal adenomas.

A series of 55 flow cytometric characterized colorectal adenomas was analyzed with four different monoclonal antibodies (Mabs) for the occurrence of M1 antigens (associated with gastric fucomucins) and one Mab for carcinoembryonic antigen (CEA). The antigens were detected with an indirect immunoperoxidase technic on paraffin sections after pretreatment with pronase for M1 antigens and without pretreatment for CEA. The staining pattern revealed different correlations with the various parameters, i.e., size, histologic type, atypia, and ploidy of the adenomas. Especially the cytoplasmic staining of anti-M1, Mab (1-13 M1) correlated well with aneuploidy (R = 0.43; P less than or equal to 0.001) and with the DNA index (R = 0.34; P less than or equal to 0.01). Staining of the anti-CEA Mab only correlated with the size of the adenomas (R = 0.29; P less than or equal to 0.03). It is concluded that the immunoreactivity of Mab (1-13 M1), which significantly correlated with aneuploidy, may be associated with malignant transformation in the adenoma-carcinoma sequence.