Which fluorophore is brightest? A comparison of the staining obtained using fluorescein,tetramethylrhodamine, lissamine rhodamine,texas red,and cyanine 3.18

Summary There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.     

Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.

There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.     

GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

Intracellular antibodies and cancer: New technologies offer therapeutic opportunities

Since the realisation that the antigen‐binding regions of antibodies, the variable (V) regions, can be uncoupled from the rest of the molecule to create fragments that recognise and abrogate particular protein functions in cells, the use of antibody fragments inside cells has become an important tool in bioscience. Diverse libraries of antibody fragments plus in vivo screening can be used to isolate single chain variable fragments comprising VH and VL segments or single V‐region domains. Some of these are interfering antibody fragments that compete with protein‐protein interactions, providing lead molecules for drug interactions that until now have been considered difficult or undruggable. It may be possible to deliver or express antibody fragments in target cells as macrodrugs per se. In future incarnations of intracellular antibodies, however, the structural information of the interaction interface of target and antibody fragment should facilitate development of binding site mimics as small drug‐like molecules. This is a new dawn for intracellular antibody fragments both as macrodrugs and as precursors of drugs to treat human diseases and should finally lead to the removal of the epithet of the ‘undruggable’ protein‐protein interactions.     

GABARAPL1 antibodies: Target one protein,get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA_A receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: generic, parallel, and scalable protein purification

We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin-binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins).     

Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.     

Hydrophobic residues 382-386 of antithrombin III, Ala-Ala-Ala-Ser-Thr, serve as the epitope for an antibody which facilitates hydrolysis of the inhibitor by thrombin.

In the presence of a monoclonal antibody raised against the human thrombin-antithrombin III complex, the reaction between thrombin and antithrombin III proceeded to form preferentially a two-chain form of the inhibitor rather than to follow the major pathway of stable acyl complex formation. We thus propose the term "switching antibody" for an antibody that switches the enzyme-inhibitor reaction (Asakura, S., Matsuda, M., Yoshida, N., Terukina, S., and Kihara, H. (1989) J. Biol. Chem. 264, 13736-13739). By analyzing a CNBr fragment of the thrombin-antithrombin III complex that reacts with the antibody we localized the epitope for the antibody to a strongly hydrophobic residue 382-386 peptide segment, Ala-Ala-Ala-Ser-Thr, of the inhibitor, which is also contiguous with a hydrophobic amino acid Ala at its carboxyl terminus. This particular region should be cryptic in nascent antithrombin III, but could have been exposed to provide the reactive site for the antibody at an early stage of the reaction. Thereby a conformational change may have been induced at or near the reactive site of the complex, facilitating hydrolysis of the inhibitor by the enzyme. Interestingly, this hydrophobic region is highly conserved among members of the serpin family.     

The use of aIF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins

Recent data have indicated that specific keratin molecules can provide useful markers for studying different types and stages of epithelial differentiation. To utilize these protein markers, however, it is important to establish the keratin nature of the molecules and identify unambiguously the individual keratin species. In this paper, we show that this can be done relatively easily by one- and two-dimensional gel electrophoresis combined with immunoblotting using three monoclonal antibodies (aIF, AE1, and AE3). The aIF antibody has previously been shown to crossreact with all classes of intermediate-filament proteins. Using one- and two-dimensional immunoblotting, we establish that this antibody recognizes all known epithelial keratins of human and rabbit, although the reaction is relatively strong for the larger, basic keratins and is relatively weak for some of the smaller, acidic keratins. In contrast, AE1 and AE3 monoclonal antibodies have previously been shown to be highly specific for the acidic and basic subfamilies of the keratins, respectively. The combined use of the broadly reacting aIF antibody and the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies should facilitate the immunological definition, identification, and classification of mammalian epithelial keratins.     

Improved immunocytochemical staining through the use of Fab fragments of primary antibody, Fab-specific second antibody, and Fab-horseradish peroxidase

A modification of the unlabeled antibody method of immunocytochemistry is described here that offers increased immunoreagent penetration and greatly reduced background staining. The method involves the following alterations to the conventional technique; the use of Fab fragments of primary antibody, rather than whole immunoglobulin G (IgG) or serum; the use of a second, or link, anti-rabbit IgG serum that is Fab fragment-specific, rather than directed against the whole rabbit IgG molecule; the use of the Fab--horseradish peroxidase complex described by JR Slemmon, PM Salvaterra, and K Saito (J Histochem Cytochem 28:10, 1980), rather than peroxidase--antiperoxidase (PAP). Steps 2 and 3 alone brought about a significant reduction in background staining, but did not increase the depth of immunostaining, as compared to the PAP technique. When all three steps were combined, however, background staining was further reduced, and there was a five- to tenfold increase in the depth of immunostaining. These readily made changes should be useful in preembedding immunocytochemistry whenever enhanced reagent penetration is required.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.     

Antibody against a novel, myriocin (ISP-I)-based immunosuppressant, FTY720

An antibody was prepared by immunizing rabbits with an ovalbumin conjugate of 2-amino-2-(2-(4-(4-mercaptobutyl)phenyl)ethyl)propane-1,3-diol HCl (AMPD-4), which contains the essential structure of the novel immunosuppressant FTY720. As the antibody reacted to not only AMPD-4, but also FTY720, it should be useful for immunoassay of FTY720 in body fluids, tissues and cells.     

Specificity,polyspecificity, and heterospecificity of antibody‐antigen recognition

The concept of antibody specificity is analyzed and shown to reside in the ability of an antibody to discriminate between two antigens. Initially, antibody specificity was attributed to sequence differences in complementarity determining regions (CDRs), but as increasing numbers of crystallographic antibody‐antigen complexes were elucidated, specificity was analyzed in terms of six antigen‐binding regions (ABRs) that only roughly correspond to CDRs. It was found that each ABR differs significantly in its amino acid composition and tends to bind different types of amino acids at the surface of proteins. In spite of these differences, the combined preference of the six ABRs does not allow epitopes to be distinguished from the rest of the protein surface. These findings explain the poor success of past and newly proposed methods for predicting protein epitopes. Antibody polyspecificity refers to the ability of one antibody to bind a large variety of epitopes in different antigens, and this property explains how the immune system develops an antibody repertoire that is able to recognize every antigen the system is likely to encounter. Antibody heterospecificity arises when an antibody reacts better with another antigen than with the one used to raise the antibody. As a result, an antibody may sometimes appear to have been elicited by an antigen with which it is unable to react. The implications of antibody polyspecificity and heterospecificity in vaccine development are pointed out. Copyright © 2014 John Wiley & Sons, Ltd.     

Properties of a monoclonal antibody for the detection of abasic sites, a common DNA lesion.

The abasic site is one of the most frequent changes occurring in DNA and has been shown to be lethal and mutagenic. An abasic site in DNA can be tagged by reaction with O-4-nitrobenzylhydroxylamine (NBHA), resulting in the formation of an oxime linkage between the abasic site and the NBHA moiety. In order to measure NBHA-tagged abasic sites, a monoclonal antibody was elicited against a 5'-phosphodeoxyribosyl O-4-nitrobenzyl hydroxylamine-BSA conjugate. The antibody was specific for the NBHA residue as demonstrated by hapten inhibition, with IC50 values for 5'-phosphodeoxyribosyl-NBHA, deoxyribosyl-NBHA, ribosyl-NBHA and NBHA of 0.3 microM, 5 microM, 5 microM and 7 microM, respectively. Other haptens examined, including benzylhydroxylamine, 5'-phosphodeoxyribosyl-, deoxyribosyl-, and ribosyl-benzylhydroxylamine, showed no inhibition even at 1 mM. The antibody showed high specificity for NBHA-modified AP sites in DNA and exhibited no cross reactivity with normal DNA bases, otherwise-modified DNA bases or unmodified AP sites. Using a direct ELISA assay, the antibody detected 1 AP site (after NBHA-modification) per 10,000 base-pairs or approximately 10 femtomoles of AP sites in DNA. DNA lesions were detectable in 60Co gamma-irradiated DNA at a dose as low as 10 rad (0.1 Gy) and the production of antibody detectable sites was proportional to the gamma-ray dose. Since NBHA reacts with lesions containing an aldehyde group, the simplicity and sensitivity of the antibody assay should provide a useful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.     

Murjne Anti-Idiotypic Monoclonal Antibodies to Syngeneic Antihuman High Molecular Weight-Melanoma Associated Antigen Monoclonal Antibodies: Development,Characterization, and Clinical Applications

Following a discussion of the rationale underlying the selection of human melanoma to test the usefulness of anti-idiotypic monoclonal antibodies in the therapy of solid tumors, the development of the anti-idiotypic monoclonal antibody MoAb) MF11–30 is described. This antibody recognizes a private idiotope within the antigen-combining site of the immunizing antihuman high molecular weight melanoma-associated antigen MoAb 225.28. The results of a phase I clinical trial with the MoAb MF11–30 in patients with advanced melanoma are described. The lack of toxic effects and the minor responses in six patients suggest that these studies should be extended to a larger number of patients with an emphasis on the analysis of the mechanisms underlying the clinical response.     

Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

Drug dosing is commonly based on the dogma that, increasing the dose maximizes the therapeutic response until a dose level that is prohibitively toxic is reached. This doctrine also applies to antibody therapy, as several protocols have explored dose escalation. We have analyzed the effect of different amounts of a homophilic Herceptin targeting a human lung tumor cell line, and discovered that the normal dose-potency relationship does not apply. To study this paradoxical effect of antibody concentration on potency, we examined the molecular species of the homophilic Herceptin under different concentrations using size exclusion chromatography and gel electrophoresis. We also varied experimental conditions in FACS tumor targeting, such as concentration of antibody, membrane immobilization, temperature, and antibody homo/dimer immobilization. We observed that high concentrations of homophilic Herceptin reduce targeting, and also noted the tumor growth arrest in the xenograft mice after the tumor reached a critical size. The therapeutic window appears to be defined by tumor size and antibody concentration. Since the concentration of this homophilic antibody defines the optimum targeting window, our data suggest the therapeutic dose of antibody should be matched with the tumor burden.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

Engineering,Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.     

Amplified detection, using a monoclonal antibody, of an aphid-specific epitope exposed during digestion in the gut of a predator.

Monoclonal antibodies are invaluable tools for identifying and quantifying prey remains in the fore-guts of predators. However, they must be target-specific, detect an epitope that is well replicated within the prey (to enhance assay sensitivity) and, critically, recognise a site that can resist digestion. A monoclonal antibody is reported that proved to be aphid-specific and capable of detecting, and accurately identifying, as little as 16.5 ng of aphid protein within a heterologous mixture of invertebrate material. The antibody was selected by screening hybridoma lines for antibodies that bound with semi-digested aphid proteins. The antibody detected an epitope that was found, against expectation, to significantly increase in concentration with time (by approx. 50% over 6 h) in the gut of the carabid predator Pterostichus melanarius. The resultant extended antigen detection period and half-life, and the high specificity of this antibody, showed it to be an enhanced tool for studying interactions between aphids and their predators in the field. It was concluded that the antibody was initially generated to a surface epitope on a high molecular weight native protein (> 200 kD). This epitope, however, was then either replicated on internal sites progressively revealed by digestion, or new epitopes became available as the conformation of the protein changed during digestion.