Labeled Antibody Method Research Articles

A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

Protocol for Creating Antibodies with Complex Fluorescence Spectra.

Fluorescent antibodies are a workhorse of biomedical science, but fluorescence multiplexing has been notoriously difficult due to spectral overlap between fluorophores. We recently established proof-of-principal for fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC), which uses combinations of existing fluorophores to create unique spectral signatures for increased multiplexing. However, a method for labeling antibodies with MuSIC probes has not yet been developed. Here, we present a method for labeling antibodies with MuSIC probes. We conjugate a DBCO-Peg5-NHS ester linker to antibodies and a single-stranded DNA "docking strand" to the linker and, finally, hybridize two MuSIC-compatible, fluorescently labeled oligos to the docking strand. We validate the labeling protocol with spin-column purification and absorbance measurements. We demonstrate the approach using (i) Cy3, (ii) Tex615, and (iii) a Cy3-Tex615 combination as three different MuSIC probes attached to three separate batches of antibodies. We created single-, double-, and triple-positive beads that are analogous to single cells by incubating MuSIC probe-labeled antibodies with protein A beads. Spectral flow cytometry experiments demonstrate that each MuSIC probe can be uniquely distinguished, and the fraction of beads in a mixture with different staining patterns are accurately inferred. The approach is general and might be more broadly applied to cell-type profiling or tissue heterogeneity studies in clinical, biomedical, and drug discovery research.

Iodination of Antibodies with Immobilized Iodogen.

Iodination, a chemical or enzymatic incorporation of 125I to specific amino acid side chains, is a commonly used method for labeling antibodies with radioisotopes. Commercially available products make iodination of antibodies a simple and quick process. One example, used here and available at Pierce, is the "Iodination bead," or N-chloro-benzenesulfonamide immobilized on nonporous, polystyrene beads.

One-step site-specific antibody fragment auto-conjugation using SNAP-tag technology.

Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site-specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme's active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV-SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv-SNAP-tag-based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame.

Heat-Induced Antigen Retrieval in Immunohistochemistry: Mechanisms and Applications.

Heat-induced antigen retrieval (HIAR) is an essential technique for current immunohistochemistry. We describe the mechanisms of HIAR and its protocols for formalin-fixed, paraffin-embedded specimens and frozen sections, which are the most popular materials for immunohistochemistry using light microscopy. In addition, we describe the antigen retrieval method for highly masked epitopes and double immunostaining using the horseradish peroxidase (HRP)-labeled antibody method and immunofluorescence method in this chapter.

A new protein crosslinking method for labeling and modifying antibodies (TECH2P.758)

Abstract The modification and labeling of a protein with a tag macromolecule is an important task in biological research. There are a few methods available for crosslinking two macromolecules. The direct crosslinking of two macromolecules suffer extremely low yield and difficult purification. A common method is to use a small crosslinker (such as SMCC) in numerous bioconjugations, but SMCC-modified protein is extremely unstable and often self-reactive during storage since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. We have recently developed an effective crosslinking method to selectively link two macromolecules. The new crosslinking method uses two unique crosslinkers that readily react upon mixing. Specifically, a pair of crosslinkers is used, including Buccutite FOL and Buccutite MTA. A protein (e.g., antibody) is first labeled with Buccutite FOL, and the tag macromolecule is labeled with Buccutite MTA. The two resulted Buccutite-modified conjugates can be readily purified by a desalting column or dialysis. Buccutite FOL-labeled protein and Buccutite MTA-tag macromolecule are mixed under extremely mild neutral conditions to give the desired protein-tag macromolecule conjugate. The Buccutite bioconjugation system is run at neutral pH, and completed within 1 hour. The conjugation can be run at low concentration, thus eliminating the pretreatment step for some diluted proteins. No catalyst is required.

The Gold Medal of the Austrian Dermatologic Society

By honoring me with this gracious award, you also honored my father, Reinhard Beutner, who introduced me to observation-based biomedical research and to the University at Buffalo that has made our work possible for the past 50 years. At the University at Buffalo, Ernest Witebsky showed me the concepts of serologic specificity of the antibody reaction. He suggested that we apply these concepts to labeled antibody methods, immunofluorescence (IF). These relate structure and function and now serve as vital tools for diverse studies. Witebsky also taught us about the methods used by Karl Landsteiner for studies of red blood cell autoantibodies and other views on autoimmunity. These served as a model for studies on autoimmunity to skin cells. As an immunologist, these studies of autoimmune skin diseases would not have been possible for me without considerable help from the dermatologic community. First, the Chief of Dermatology at the University at Buffalo in

Labeling of Antibodies by in Situ Modification of Thiol Groups Generated from Selenol-Catalyzed Reduction of Native Disulfide Bonds

A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available—selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.

Immunoassay, DNA Analysis, and Other Ligand Binding Assay Techniques: From Electropherograms to Multiplexed, Ultrasensitive Microarrays on a Chip

Ligand or assays have made a major impact on biomedical research and clinical diagnosis since their development in the late 1950s. Immunoassay techniques (relying on specific antibodies to bind the target analyte) represent the best-known example, but analogous DNA and RNA analysis methods (using oligonucleotides to recognize defined polynucleotide sequences) are rapidly gaining in importance and are likely to exert profound effects on human society. The evolution of these methods may be divided into three phases: (i) the initial development and widespread use of sensitive competitive assays relying on radioisotopically labeled analyte to monitor the binding reaction; (ii) the introduction in the 1980s of ultrasensitive, noncompetitive, labeled antibody methods relying on high-specific-activity nonisotopic labels, leading to the emergence of the automatic analyzers that now dominate the field, and (iii) the present development of microarraymethods based on antibody or oligonucleotide microspots (each recognizing an individual analyte) arrayed on a solid support and relying on observation (typically by confocal microscopy) of fluorescent signals emitted from each spot. Miniaturized microarray methods permitting ultrasensitive measurement of hundreds of different analytes in a minute sample are likely to revolutionize medicine and related fields within the next decade.

A New Attractive Approach of Three- Dimensional(3D) Reconstruction of the Relationships Between Hormone Secreating Cells and its Microvessel Network Patterns in Rat Pituitary Gland by Confocal Laser Scanning Miroscopy(CLSM) With Combined Fluorescent and Non-Fluorescent Probes.

Abstract There have been considerable interests in the relationship between hormone secreting endocrine cells and microvessel network patterns in human pituitary gland. However, its 3D microcirculatory networks have not been studied extensively. Therefore, this study was designed to visualize hormone secreting endcrine cells and microvessels in 3D by CLSM and to reveal relationship between hormone secretion and microvessel networks in the rat pituitary gland. CLSM has been an exciting new apparatus because of its more powerful resolution than conventional light microscopy and the high-performance on 3D optical sectioning. The images taken with CLSM can be digitized and made susceptible to image analysis manipulation and computer-assisted 3D reconstructions. The female normal adult Wistar rats were used in this study. To demonstrate microvessels, fluorescent probe( 30 mg/ml)-conjugated gelatin solution was injected to left ventricle of the heart After complete perfusion, the pituitary glands were resected and immediately fixed in cold graded PFA(0.5- 8 %) containing 15 % picric acid solutions (4 °C. GH, ACTH cells or S100 protein positive fcfliculostellate (FS) cells were demonstrated by indirect TRTTC-labeled or HRP labeled antibody methods.

A modified immunohistochemical procedure for the detection of estrogen receptor in mouse tissues.

A horseradish peroxidase (HRP) labeled antibody method was developed for use with a monoclonal antibody to detect estrogen receptor (ER) in mouse tissue. Combined use of HRP labeled F(ab')2 fragment absorbed with mouse liver protein to minimize background staining and imidazol-DAB reaction gave the most reliable and sensitive immunostaining. The method was applied to uterine, vaginal, pituitary and liver tissues in ovariectomized adult mice. In uterus and vagina, ER was recognized in nuclei of epithelial cells, stromal cells and smooth muscle cells of the muscle layer and blood vessels. Liver tissue showed positive nuclear immunostaining in parenchymal cells; however, no reaction was present in endothelial cells, Kupffer cells, bile ductal cells, and smooth muscle cells of blood vessels. ER was localized in the nuclei of anterior pituitary cells while weak reaction was also recognized in cells of the intermediate lobe. No staining was detected in the posterior pituitary. Results demonstrate that both occupied and unoccupied ER are localized in the cell nucleus from several target tissues. Weak immunostaining in samples could not be enhanced by multiple procedures. It is suggested that nuclear ER is partially hidden by nuclear components such as nucleic acid and chromatin proteins.

AN IMMUNO‐ELECTRON MICROSCOPIC STUDY ON INTRA and EXTRACELLULAR LOCALIZATION OF ALPHA‐FETOPROTEIN IN HUMAN HEPATOCELLULAR CARCINOMA

Intra and extracellular localization of alpha-fetoprotein (AFP) has been studied by an indirect peroxidase labeled antibody method using 12 cases of human hepatocellular carcinoma (HCC). With light microscopic observation, positive immuno-staining for AFP was observed in 6 out of 12 cases and demonstrated as granular or diffuse deposits in the cytoplasm of neoplastic hepatocytes. In electron microscopic studies, 8 cases showed the positive immuno-staining for AFP in the neoplastic hepatocytes. Intracellular antigen was well circumscribed within certain cell organelles with the positive immuno-staining for AFP being observed in perinuclear space, cisternae of rough endoplasmic reticulum (RER), Golgi complexes, and secretory vesicles. In addition, the positive immuno-staining for AFP was observed in bile canaliculus-like space in most cases with increased levels serum AFP and in some cases which showed normal levels of serum AFP. Furthermore, the positive immuno-staining for AFP was also observed in intercellular, Disse's-like and sinusoid-like spaces, and micropinocytotic and lysosome-like vesicles in the endothelial cells in a few cases which showed excessively high value of serum AFP.

123I radiolabeling of monoclonal antibodies for in vivo procedures.

When labeled to monoclonal antibodies (MAbs) or their fragments, 123I can be used for imaging or for predicting the treatment potential and radiation dosimetry of 131I labeled to the same molecular species. Because 123I (p,5n) from the Crocker Nuclear Laboratory is in dilute solution, when compared with commercial 125I of labeling grade, we have evaluated labeling parameters using Chloramine-T as the oxidant and derived an optimum set of labeling conditions that provide a 60-80% radiochemical yield of highly immunoreactive antibody. When Lym-1, an IgG-2a murine antibody against human lymphoma, was used, yields of labeled immunoglobulin were decreased by protein or Chloramine-T concentrations less than 0.4 microgram/microliter and 0.8 microgram/microliter, respectively; denaturation of the immunoglobulin occurred when the Chloramine-T concentration was greater than 1.0 microgram/microliter. Optimum labeling occurred at pH 7-8 with deleterious effects when the pH was below 5 or above 10. An optimum method for labeling antibodies with multimillicurie amounts of 123I (less than one iodine atom per 100 antibody molecules) is described. Some of the observations derived from this study are also applicable to the preparation of treatment doses of 131I-labeled antibodies, wherein the amount of antibody can be a restrictive factor.

4 Diagnosis of hyperthyroidism: The newer biochemical tests

Investigation of suspected hyperthyroidism is conventionally based on measurement of total or free T4 as the initial test, followed in equivocal cases by total or free T3, and the TRH test. Recent developments in techniques for measuring free hormones and TSH promise to change this approach. Free T4 and free T3 can now be rapidly and simply quantitated in whole serum using labelled analogue radioimmunoassays. Specific and highly sensitive assays using labelled monoclonal antibodies are now available for serum TSH that permit the suppressed levels found in most cases of hyperthyroidism to be distinguished from euthyroid levels. These newer assays are available at a cost per test that is often similar to that of the more established tests. Available evidence indicates that measurement of basal serum TSH by a sensitive labelled antibody method can serve as a first line test, at least in uncomplicated cases of suspected hyperthyroidism. In patients with a suppressed TSH, a serum free T4, and in equivocal cases free T3, will distinguish the clinical and subclinical forms of hyperthyroidism. Such an approach would obviate the need for the TRH test. It must be emphasized, however, that experience with this new approach is limited. Caution is advised in the interpretation of low TSH and free hormone levels when there are associated complicating features, such as severe non-thyroidal illness, or pregnancy. These developments mark a trend in thyroid function testing away from measurement of circulating total hormone levels. The newer tests provide an assessment of end organ (thyrotroph) response, and an assessment of biologically active (free) hormone to which the tissues are exposed. These complementary approaches have the potential to identify relatively minor degrees of thyroid dysfunction.

Immunocytochemical and enzymecytochemical studies on the intracellular transport mechanism of secretory immunoglobulin A and lactoferrin in human salivary glands.

The intracellular transport mechanism of secretory immunoglobulin A (sIgA) has been immunocytochemically defined in human submandibular glands. To examine the properties of the intracytoplasmic vesicles which contain IgA, the enzyme labeled antibody method for SC and IgA and Novikoff's method for acid phosphatase (ACPase) activity were employed on the same sections. The intracytoplasmic vesicles containing IgA and SC in the serous acinar cells were free of ACPase activity and were thus distinguishable from lysosomes. Neither IgA nor SC was localized in the secretory granules. Lactoferrin was localized in the secretory granules and glandular lumen of the serous acinar cells, but not in the cytoplasmic vesicles, which were also free of ACPase activity. These findings suggests that the transport of sIgA was performed by intracytoplasmic vesicles and that lactoferrin is discharged from secretory granules into the lumen and finally makes a "rendezvous" with sIgA in the lumen of acinar cells.

Combined application of monoclonal antibody to human la antigens and avidinblotin-peroxidase staining method in immunoelectron microscopy

Immunological detection of cellular and tissue antigens have been based on the widely used original enzyme labeled antibody method or the unlabeled antibody, peroxidase-anti-peroxidase (PAP) method. Recently, an improved immunoenzymatic technique for light microscopy using the avidin-biotinperoxidase complex (ABC) system was developed for cell and tissue staining, and found to be superior to the PAP technique in terms of sensitivity and specificity. The application of the ABC method in immunoelectron microscopy (IEM), however, has not been extensively evaluated. This study demonstrates that the ABC method is suitable for localizing specific cellular antigen at the ultrastructural level with monoclonal antibody (MAb), the production of which by somatic cell hybridization is now a well established procedure.

Immunoelectron microscopic localization of thyroglobulin in the human thyroid gland.

The microidentification of the organelles containing thyroglobulin (TG) in the follicular cells of human thyroid glands were studied by the immunoelectron microscopic method. Fresh human thyroid glands were used for the purification of TG. TG was purified by differential salt fractionation and DEAE-cellulose chromatography. Anti-human TG rabbit IgG antibody was obtained by immunization of this purified TG. F(ab')2 fragments of anti-TG rabbit IgG antibody were prepared by pepsin digestion. The specificity of the antibody was tested on an Ouchterlony immunodiffusion plate. Conjugation of the purified F(ab')2 fragments of anti-TG rabbit IgG antibody to horse radish peroxidase was performed in order to use the direct peroxidase labelled antibody method. Under immunohistochemical light microscopy, the luminal colloid and the follicular cells appeared heavily stained. Under immunoelectron microscopy, positive reactions were observed in the rough endoplasmic reticulum, the Golgi apparatus, and the apical cell border. To date, no clear direct evidence of the presence of TG in the rough endoplasmic reticulum and the Golgi apparatus of human thyroid cells has ever been reported in immunoelectron microscopic studies. This study indicates that the use of F(ab')2 fragments of anti-TG rabbit IgG antibody in immunoelectron microscopic study is useful for the identification of TG in organelles of human thyroid follicular cells.

Intramitochondrial localization of glutathione-peroxidase (GSH-PO) in the rat adrenocortical cells.

Immunocytochemical localization of glutathione-peroxidase (GSH-PO) in the rat adrenocortical cells was observed not only in cytosol (CYTOSOL GSH-PO) but in mitochondria (MITOCHONDRIAL GSH-PO) by the direct peroxidase labeled antibody method using anti-GSH-PO IgG Fab' fragment. In the untreated rat adrenocortical cells, CYTOSOL GSH-PO was observed mainly in the outer part of the zona fasciculata and zona reticularis. MITOCHONDRIAL GSH-PO was observed mainly in the inner part of zona fasciculata. Biochemical GSH-PO activity in the cytosol and mitochondrial fractions obtained from whole adrenal gland was comparable to the immunocytochemical results. In the hypophysectomized rat adrenocortical cells, markedly atrophic inner part of zona fasciculata showed only CYTOSOL GSH-PO, and biochemical activity of the cytosol GSH-PO was markedly increased. It has been reported by us that this atrophic zone contains accumulated cholesterol esters. By ACTH administration to the hypophysectomized rats, markedly, hypertrophic outer part of zona fasciculata showed increased immunocytochemical localization of MITOCHONDRIAL GSH-PO. And biochemically, mitochondrial GSH-PO activity in this group was higher than that of the hypophysectomized group.These results suggest that the CYTOSOL GSH-PO has its role in the reduction of cholesterol ester hydroperoxides and that the MITOCHONDRIAL GSH-PO in the reduction of sterol hydroperoxides produced during steroidogenesis.

Immunocytochemical localization of T antigen in cells of BK virus-induced hamster brain tumor.

The localization of both the large T and small t tumor (T) antigens in cultured cells (Vn 12 cells) of hamster brain tumors induced with BK virus (BKV), a new human papovavirus, was studied by an enzyme labelled antibody method at both the light and electron microscopic levels. Under the light microscope, BKV T antigen was observed in the nucleus, except for the nucleoli, of cells in interphase, and under the electron microscope it was observed in the nucleus except for the nucleoli and nuclear membrane. BKV T antigen appears to be closely associated with nuclear chromatin as previously reported for simian virus 40 tumor antigen (SV40 T antigen). The intracellular localization of BKV T antigen was the same as that of SV40 T antigen. In metaphase, BKV T antigen seems to be distributed diffusely throughout the cytoplasm except for the chromosomes. In telophase, BKV T antigen transfers from the cytoplasm to the nucleus. The migration of BKV T antigen during the cell cycle is thought to be related to the function of T antigen.

Dermatitis herpetiformis with linear IgA deposition: ultrastructural localization of in vivo bound IgA

Ultrastructural localization of bound IgA in the skin of a patient suffering from dermatitis herpetiformis with linear IgA deposition at the dermo-epidermal junction was studied using the horseradish peroxidase labelled antibody method. The reaction products were observed as electron-dense materials, not only immediately below the cytomembrane of the basal cells but also in the uppermost dermis just below the basal lamina. The major portion of the lamina lucida was free of the reaction products. These findings suggest a diversity in the mode of IgA deposition and the possibility of the presence of different antigens in the basement membrane zone, to which IgA class antibodies may be bound respectively.