Detection of Antigen-antibody Reaction Using a Fluorescent Probe and Its Application to Homogeneous Competitive-type Immunoassay for Insulin

The interaction of bovine insulin with anti-human insulin antibody (mAb) was examined using a fluorescent probe. The fluorescence intensity of fluoresceinthiocarbamyl (FTC)-insulin was increased by adding mAb, and the increase was saturated at 53% at a molar ratio of FTC-insulin to mAb of 2.0. Based on the change in fluorescence intensity, a standard curve of the homogeneous competitive-type immunoassay was constructed, and the detection range of insulin was found to be 50–400 nM.     

Increased tumor cell reactivity and complement-dependent cytotoxicity with mixtures of monoclonal antibodies against different gangliosides

Melanomas and other cancers of neuroectodermal origin express multiple cell-surface gangliosides in patterns that vary significantly even within the same tumor type. Monoclonal antibodies (mAb) against four of these gangliosides (GM2, GD2, 9-O-acetyl-GD3 and GD3) were tested alone and in combination on 14 tumor cell lines (7 melanomas, 3 neuroblastomas, 3 sarcomas and 1 astrocytoma) using flow cytometry and complement-dependent cytotoxicity (CDC) assays. Increased tumor cell recognition and CDC resulting from the combination of three or four mAb were found in 14/14 tested cell lines, and this was most striking when each mAb was used at suboptimal concentration. At these concentrations, the average mean fluorescence intensity of the 14 cell lines with individual mAb was between 3.0 and 6.8 and increased to 10.8 and 18.8 with the three- and four-mAb mixtures. The average percentage CDC-specific release with individual mAb was 2.0%–8.3%, and 12.3% and 16.6% with the three- and four-mAb combinations. The number of cell lines showing significant mean fluorescence intensity and CDC increased from 2–8/14 with single mAb to 13–14/14 with the mixtures of three or four mAb. Our experimental results support the rationale for active immunization with a polyvalent ganglioside vaccine or passive therapy with a combination of mAb to different gangliosides in patients with tumors of neuroectodermal origin. In addition, our studies have demonstrated that 9-O-acetyl-GD3 is a surprisingly effective target for immune attack, although it is a minor constituent of these cells.     

Alanine scanning mutants of rat proinsulin I show functional diversity of anti-insulin monoclonal antibodies

In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.     

胰岛素对βTC-3 细胞胰岛素受体表达的作用

目的:观察外源性胰岛素对小鼠胰岛β细胞瘤细胞株βTC-3细胞胰岛素受体表达的影响。方法:采用免疫荧光细胞化学技术结合激光扫描共聚焦显微镜观察高浓度胰岛素(100 IU/ml)刺激不同时间(0 min、30 min、60 min、120 min、240 min),培养的βTC-3细胞胰岛素受体的表达,用Image pro plus软件对胰岛素受体的荧光强度进行了半定量分析。结果:与0 min比较,胰岛素孵育30 min、60 min、120 min、240 min时胰岛素受体荧光强度均明显下降(P<0.05)。结论:高浓度胰岛素孵育βTC3细胞后,可明显下调胰岛素受体的表达,这可能是高胰岛素血症导致胰岛素抵抗产生的机制之一。     

Detection of an equilibrium intermediate in the folding of a monomeric insulin analog.

To determine the conformational properties of the C-terminal region of the insulin B-chain relative to the helical core of the molecule, we have investigated the fluorescence properties of an insulin analog in which amino acids B28 and B29 have been substituted with a tryptophan and proline residue respectively, ([WB28,PB29]insulin). The biological properties and far-UV circular dichroism (CD) spectrum of the molecule indicate that the conformation is similar to that of native human insulin. Guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation of the analog as monitored by CD intensity at 224 nm indicates a single cooperative transition with a midpoint of 4.9 M GdnHCl. In contrast, when the equilibrium denaturation is observed by steady-state fluorescence emission intensity at 350 nm, two distinct transitions are observed. The first transition accounts for 60% of the observed signal and has a midpoint of 1.5 M GdnHCl. The second transition roughly parallels that observed by CD measurements with an approximate midpoint of 4.5 M GdnHCl. The near-UV CD spectrum, size-exclusion, and ultracentrifugation properties of [WB28,PB29]insulin indicate that this analog does not self-associate in a concentration-dependent manner as does human insulin. Thus, the observed fluorescence changes must be due to specific conformational transitions which occur upon unfolding of the insulin monomer with the product of the first transition representing a stable folding intermediate of this molecule.     

Quantum Dots as alternatives to organic fluorophores for Cryptosporidium detection using conventional flow cytometry and specific monoclonal antibodies: lessons learned.

BACKGROUND: Significant developments in biological applications are occurring through the incorporation of Quantum Dots (QDs) as biological labels. The demonstration of QDs unique optical properties may have important implications for the study of environmental samples, where microorganisms of interest need to be isolated away from the background debris. METHODS: Flow cytometric analysis was used to determine the fluorescence intensity of oocysts after mAb staining by QDs or organic fluorophore conjugates. In addition, the level of non-specific binding to detrital particles within a control water concentrate was estimated using the optimal staining concentration determined for each mAb analyzed. RESULTS: Under 488 nm excitation, oocysts stained with QD-conjugates exhibited significantly lower fluorescence intensity than organic conjugates. Moreover, the level of non-specific binding by QD-conjugates to detrital particles present in the water concentrate was significantly higher that of the organic conjugates. CONCLUSIONS: While QDs are noted for their superior spectral characteristics, they have been shown here to be unsuitable for conventional flow cytometric detection of Cryptosporidium. Therefore, we conclude that in their current form, QD's are severely limited for fluorescent detection of pathogens in environmental applications.     

胰岛素对βTC-3细胞胰岛素受体表达的作用

目的：观察外源性胰岛素对小鼠胰岛β细胞瘤细胞株βTC-3细胞胰岛素受体表达的影响。方法：采用免疫荧光细胞化学技术结合激光扫描共聚焦显微镜观察高浓度胰岛素（100 IU/ml）刺激不同时间（0 min、30 min、60 min、120 min、240 min）,培养的βTC-3细胞胰岛素受体的表达,用Image pro plus软件对胰岛素受体的荧光强度进行了半定量分析。结果：与0 min比较,胰岛素孵育30 min、60 min、120 min、240 min时胰岛素受体荧光强度均明显下降（P〈0.05）。结论：高浓度胰岛素孵育βTC3细胞后,可明显下调胰岛素受体的表达,这可能是高胰岛素血症导致胰岛素抵抗产生的机制之一。     

S152 (CD27). A modulating disulfide-linked T cell activation antigen

A large subset of normal resting peripheral blood T cells express a protein at low density defined by the murine mAb S152. Reactive T cells are present in both the CD4+ and CD8+ subpopulations. This determinant, however, is not expressed on growth factor-independent T cell lines. After activation of mononuclear cells by either Con A or PHA, greater than 80% of the cells stained at a mean fluorescence intensity that was more than six times that seen on resting cells. When PWM- or mixed lymphocyte reaction-activated cultures were studied, 7 to 22% of S152+ cells stained at high intensity whereas most cells stained at the baseline low intensity. The increased fraction of S152+ cells staining at high intensity after activation paralleled both the increased percentage of anti-Tac+ and 5E9+ cells and cellular proliferation measured by thymidine incorporation. Modulation of the S152 Ag was induced when either Con A- or PHA-activated mononuclear cells were placed into secondary cultures containing S152 mAb. Expression of the S152 Ag began to decrease after 2 h and reached a minimum after 6 h. Resting T cells, however, did not appear to modulate when cultured with S152 mAb. Immunoprecipitation and gel electrophoresis analysis revealed the S152 molecule to have a mobility of 120 kDa before reduction. After reduction the molecule was shown to be composed of two 55-kDa molecules with an isoelectric point of 5 to 6 indicating that the S152 Ag is a disulfide-linked homodimer. These studies confirm that the S152 mAb reacts with the newly defined CD27 molecule. The presence of the S152 Ag on resting and activated cells, its parallel increase with the Tac and 5E9 Ag, and its ability to modulate on activated cells suggest that this molecule may play a functional role during T cell activation.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors

The possibility of obtaining, from any antibody, a fluorescent conjugate which responds to the binding of the antigen by a variation of fluorescence, would be of great interest in the micro- and nano-analytical sciences. This possibility was explored with antibody mAb4E11, which is directed against the dengue virus and for which no structural data is available. Three rules of design were developed to identify residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. (i) The target residue belonged to the hypervariable loops of the antibody. (ii) It was adjacent, along the amino acid sequence of the antibody, to a residue which was functionally important for the interaction with the antigen. (iii) It was not important in itself for the interaction with the antigen. Eight conjugates between a single chain variable fragment of mAb4E11 and an environment-sensitive fluorophore were constructed. Three of them showed an increase in their fluorescence intensity by 1.5-2.8-fold on antigen binding, without loss of affinity. This increase allowed the titration of the antigen in serum above a threshold concentration of 10nM. Experiments of quenching with potassium iodide suggested that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the binding of the antigen, and that therefore its mechanism is general.     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis.

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 micrograms/ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 microgram/ml (P less than 0.01) but the staining intensity at 0 microgram/ml was not different from that at 3 micrograms/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo- mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

Conformation and proteolysis of glucagon and insulin in surfactant and lipid solutions

The effects of micelles of nonionic, zwitterionic, anionic and cationic surfactants and lipids on the conformation of glucagon and insulin have been investigated by circular dichroism and intrinsic protein fluorescence. The influence of these amphipathic compounds on the hydrolysis, monitored by HPLC, of glucagon and insulin by trypsin and chymotrypsin has also been studied. The alpha-helix content of glucagon was increased to a similar extent by all the micelles, irrespective of their charge and of whether they were synthetic surfactants or phospholipids. The amphipathic compounds always induced a blue-shift in the wavelength of maximum emission of fluorescence of glucagon of about 9 nm, whereas the fluorescence intensity was increased in some cases and decreased in others. The circular dichroism of insulin was also modified in some cases. Some amphipathic compounds protected glucagon against proteolysis by trypsin and chymotrypsin very markedly, whereas others did not protect at all or only slightly protected the hormone. Two hypotheses have been formulated to explain the different results. Hydrolysis of insulin was generally not influenced by surfactants and lipids.     

Reflective FT-NIR and SERS studies of HER-II breast cancer biomarker using plasmonic-active nanostructured thin film immobilized oriented antibody

We describe the fabrication of plasmonic-active nanostructured thin film substrate as a label-free surface-enhanced Raman scattering (SERS)-based biosensor immobilized covalently with monoclonal HER-II antibody (mAb) to detect overexpressed HER-II as a biomarker in breast cancer serum (BCS). Oriented conjugation of mAb via hydrazone linkage to provide higher mAb accessibility was characterized by UV-vis and reflective Fourier transform near-infrared (FT-NIR) spectroscopic techniques. The interaction of BCS with mAb was studied by FT-NIR and nonresonant SERS at 637 nm. The results showed detection of glycoprotein content at different laser powers including a rise in amino acid and glycan content with varying results at higher power. With nonresonant SERS we observed nonlinear behavior of peak intensity. Analysis of variance was implemented to determine the effect of laser power which was found not to be a contributing factor. However, at the nanoscale, factors including the heating effect and aggregation of molecules can contribute to the nonlinearity of peak intensity.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Intracellular detection of Bcl-2 and p53 proteins by flow cytometry: comparison of monoclonal antibodies and sample preparation protocols

Several techniques have been proposed for flow cytometric evaluation of intracellular antigens. This approach is particularly important for detection at the single cell level of proteins which correlate to tumour progression. Bcl-2 and p53 are two of the most relevant proteins. In the present study we have compared five different cell fixation-permeabilisation protocols and nine fluorochrome-conjugated (FITC or PE) monoclonal antibodies (mAb): four mAb directed against Bcl-2 and five against p53. For detection of Bcl-2 we have analysed three Bcl-2 positive cell lines (K562, Daudi and MCF-7), and peripheral blood samples obtained from nine healthy subjects. To distinguish internal positive (lymphocytes) and negative control cells (granulocytes), it was necessary to perform simultaneous detection of surface and intracellular antigens. For detection of p53 three cell lines, two p53 positive (Raji and CEM) and one p53 negative (HL-60), were analysed. Using these cells we have performed a combined analysis of the efficiency of monoclonal antibodies and sample preparation techniques. In conclusion, clones 124-FITC and Bcl-2/100-PE (Bcl-2), and clones BP53,12-FITC and G59-12-PE (p53) provided the highest specific fluorescence intensity of the respective markers independent of cell preparation protocols. Importantly, our results show that mAb background may depend on the specific fixation/permeabilisation kit and that mAb titration using negative and positive control cells is essential to determine the specificity and the sensitivity of the mAb used.     

Intrinsic fluorescence, difference spectrophotometry and theoretical studies on tertiary structure of calf thymus histone H1

Tyr-72 is included in the hydrophobic cleft which is formed in the histone H1 globular head. Tyr-72 is screened against polar aqueous environment and its intramolecular mobility is sharply retarded. This microenvironment causes a red shift (lambda max = 279 nm) and a sharpening of the longer wavelength shoulder of absorption spectra, a high fluoresence anisotropy value (A = 0,11), high quantum yield of fluoresence (approximately 0.2) and a decrease of the Stern-Volmer Constant during quenching of histone H1 fluorescence by acrylamide. It has been found that the change in the intensity of histone fluorescence at lambda excit = 265 nm, but not at lambda excit = 280 nm, is due to the changes in the quantum yield of fluorescence. The increase of fluorescence intensity at lambda excit = 280 nm depends on the changes in the quantum yield and molar extinction coefficient of histone H1 tyrosyl chromophore. The change in the ratio of fluorescence intensity exited at 280 nm (F280) to the fluorescence intensity excited at 265 nm (F265) corresponds to the change of delta epsilon 286 in difference absorption spectra. The introduction of the parameter Cf = F280/F265 allows one to go over to studying excitation spectrum shifts instead of histone absorption spectrum shifts, which is much more convenient methodologically since in this case it is possible to carry out research using lower protein concentrations and turbid solutions. The results make it possible to designate Tyr-72 of histone H1 as a special class of fluorescent tyrosyls whose properties differ from those of tyrosyls of other tryptophane-free proteins: RNAase, insulin, core histones--H2A, H2B, H3, H4 and some others.     

Utilization of fluorescence tracer in hyperinsulinemic-euglycemic clamp test in mice

The hyperinsulinemic–euglycemic clamp test is considered to be a gold standard for the evaluation of insulin sensitivity. Here, a new version of the clamp test that used the fluorescence tracer 2-NBDG was tested. C57BL/6J mice were induced insulin resistant (IR) with a high-calorie diet. Rosiglitazone was administrated to IR mice and diabetic db/db mice. Insulin resistance was estimated with the oral glucose tolerance test (OGTT), the insulin tolerance test (ITT), the serum insulin level and the homeostasis model assessment of insulin resistance (HOMA-IR), and then confirmed by the hyperinsulinemic–euglycemic clamp test with 2-NBDG. The 2-NBDG content was measured by the fluorescence intensity. The characteristics of insulin resistance were shown remarkably with the increased values of serum insulin and HOMA-IR in IR mice, and with the results from OGTT and ITT in both IR and db/db mice. In the hyperinsulinemic–euglycemic clamp test, the glucose infusion rate and amount of 2-NBDG taken up into the liver, adipose, and skeletal muscle were decreased significantly in IR mice and db/db mice, respectively. The clearing rates of 2-NBDG from the circulation were much slower in both mouse models. All markers were reversed significantly by rosiglitazone treatment. The results indicate that with the fluorescence tracer 2-NBDG, the hyperinsulinemic–euglycemic clamp test can be used to estimate insulin sensitivity in vivo.     

Influence of calcium ions in the flow cytometric analysis of human CD8-positive cells.

BACKGROUND: The CD8 co-receptor is an important marker used to identify various lymphocyte subsets. A significant decrease in CD8alpha staining intensity was observed in the presence of divalent cation chelators. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were treated with calcium chelators, stained with different anti-human CD8 mAbs, and analyzed by flow cytometry. RESULTS: Calcium chelators caused a dose-dependent decrease in fluorescence intensity, using specific anti-human CD8alpha mAbs. This phenomenon was not due to CD8 internalization and could be reversed by the addition of calcium ions. In contrast, calcium depletion increased staining intensity with one anti-CD8beta mAb. CONCLUSIONS: Divalent cation chelators are used as cell anti-clumping agents in MACS or FACS applications. Researchers should be aware that such treatment could lead to the almost complete loss of fluorescence with selected anti-human CD8alpha mAbs. Since CD8 staining is used in conjunction with tetramer staining to identify antigen-specific cytotoxic human T cells, the effect of calcium depletion should be taken into account in experimental design.     

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.     

The application of anti‐ESAT‐6 monoclonal antibody fluorescent probe in ex vivo near‐infrared fluorescence imaging in mice with pulmonary tuberculosis

Here, we aimed to assess the feasibility of anti‐ESAT‐6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT‐6 expression in tuberculosis tissue of mice using near‐infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti‐ESAT‐6 mAb or IgG. Mice in the experimental group were injected with fluorescence‐labeled mAb probe, and mice in the control group were injected with fluorescence‐labeled non‐specific IgG antibody. Twenty‐four hours later, the lung tissue of mice was examined using ex vivo near‐infrared fluorescence imaging. In addition, the contrast‐to‐noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near‐infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti‐ESAT‐6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.