ARTICLE |
Correspondence to: Manoj Raje, Inst. of Microbial Technology, Sector 39A, Chandigarh 160036, India. E-mail: manoj@imtech.res.in
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Summary |
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We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM. (J Histochem Cytochem 50:863873, 2002)
Key Words: antigen detection, antigen quantitation, transmission electron, microscopy, nonspecific binding, artificial immunospecimen, ELISA, blocking, signal-to-noise ratio, immunogold labeling
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Introduction |
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IMMUNOGOLD LABELING ELECTRON MICROSCOPY is a powerful technique for the localization and quantification of antigens in cells and tissues. A crucial factor for the successful application of this method is the ability to decide on the use of appropriate reagents that minimize nonspecific binding (NSB) to the resin sections while retaining a high level of signal. In practice, some level of background labeling usually occurs in all cases (
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Materials and Methods |
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VHb Antigen and Anti-VHb Antibody
E. coli DH5 cells harboring the plasmid PUC8:15 expressing VHb protein (
ELISA of VHb Antigen
Fifty µl of lysate containing 2.5 µg of protein (
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Dot-blotting of Antigen
Five microliters of cell lysate containing 20 µg of total protein was spotted onto pieces of nitrocellulose paper (Advanced Microdevices; Ambala, India) and allowed to dry for a few minutes under an infrared lamp. The spots were placed into individual wells of a 12-well cell culture plate and blocked for 2 hr by immersion in 2 ml of various blocking agents accompanied by continuous rocking on a shaker. Then the spots were washed three times with PBST and incubated in 1 ml of rabbit anti-VHb, diluted 1:500 in PBST, for 1 hr at RT. After three washes with PBST, goat anti-rabbit IgG HRP conjugate (Sigma) diluted 1:16,000 in PBST was added as second antibody and allowed to react further for 1 hr at RT, with the wells covered with aluminum foil. After washing as before, the reaction product was developed using 3,3' 5,5'-tetramethylbenzidine for localization of HRP (Bangalore Genie) for 15 min. The reaction was stopped by washing the spots with distilled water. In two parallel sets of control experiments; (a) the antigen containing cell lysate was replaced with antigen-negative cell lysate and (b) rabbit anti-VHb antibody was replaced with NRS.
Western Blotting Analysis
Western blotting of cell lysate was carried out according to the procedure of
Processing of Cells for TEM
E. coli cells were fixed for 30 min in 1% glutaraldehyde (EM Sciences; Ft Washington, PA) + 4% paraformaldehyde (Polysciences; Warrington, PA) constituted in PBS, dehydrated in alcohol, and processed for embedding in LR White resin (EM Sciences) at 60C for 24 hr. Ultrathin sections were cut, flattened with xylene vapor, collected on nickel grids, processed for immunogold labeling, stained with 2% aqueous uranyl acetate, and observed in a JEOL 1200 EX II TEM.
Immunogold Labeling Method
Sections on grids were first blocked for 30 min by floating on drops of PBS containing different blocking agents along with 0.01% Tween-20, followed by washing on drops of PBST (five changes, 5 min each). This was followed by incubation overnight (1618 hr) at 4C with rabbit anti-VHb antibody diluted 1:500. All antibody dilutions were made in respective blocking buffers that had been diluted tenfold with PBS. Then the grids were washed and bound antibodies were localized by incubating the sections for 1 hr on goat anti-rabbit gold conjugate (10 nm; Sigma) diluted 1:20. Finally, grids were washed on drops of PBST and water before being stained with 2% aqueous uranyl acetate. All incubations, except for the primary antibody step, were carried out at RT.
Controls Used
The specificity of immunogold labeling was judged by (a) comparing the probe density on the cells with that on the adjacent plastic resin, (b) comparing the probe density on the cells from which the first antibody had been omitted, (c) comparing the probe density on sections of antigen-negative cells that had been labeled in a similar manner as test sections and, finally, (d) by substituting the rabbit anti-VHb antiserum with NRS.
Quantitation of Probe Density
The resultant probe density, using test and control serum, was quantified from random micrographs of cell sections as described previously (
Analysis
The total probe density on cells and clear plastic was calculated for the test and control of each sample processed. Densities were expressed as the number of gold particles/µm2. Comparisons between samples were done by two-tailed t-test.
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Results |
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Evaluation of Blocking Agents by Dot-blotting Assay
The efficiency of various blocking agents in suppressing nonspecific background labeling was first tested by dot-blotting assay. When anti-VHb antiserum was used to probe antigen +ve lysate, spotted onto nitrocellulose membrane, a uniform level of test as well as background signal was obtained in almost all cases (Fig 1). When antigen -ve cell lysate (-ve immunospecimen)-spotted membranes were used, no appreciable signal was observed in any of the samples. Similar negative signal was observed when NRS was used in place of anti-VHb antiserum (data not shown).
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Evaluation of Blocking Agents Using Western Blotting
Results of Western blotting of antigen +ve cell lysate are presented in Fig 2A2J. A prominent band representing the antigen was seen with all blocking reagents, along with some additional faint bands, especially when blocking agents other than casein were used. When similar experiments were carried out using antigen -ve cell lysate or where anti-VHb antiserum was replaced with NRS, no significant binding was observed (data not shown).
Evaluation of Blocking Efficacy Using ELISA
ELISA was used to evaluate the blocking efficiency of various proteins in suppressing nonspecific labeling. Results of +ve signal and background binding are presented in Table 2. When anti-VHb antiserum was used for probing wells coated with Ag+ve E. coli cell lysate (positive immunospecimen) almost uniform results (OD 0.30.4) were obtained. In experiments using Ag -ve E. coli cell lysate (negative immunospecimen), signals lower than the corresponding Ag+ve cell lysate coated wells were obtained in all cases (p<0.01). In this case, wells blocked with CAE demonstrated the lowest OD (0.05) followed by SM (0.08). Both of these values were significantly less than (p<0.05) the corresponding OD values of wells blocked with other blocking agents in which the absorbance ranged from 0.1 to 0.16. On carrying out ELISA of only clear plastic (uncoated) wells that had been subjected to blocking, once again CAE and SM-blocked wells gave lowest signal (OD = 0.005 and 0.006, respectively). These values are significantly lower (p<0.05) than those obtained with all other blocking agents. The rest of the blocking agents evaluated gave OD values ranging from 0.01 to 0.06.
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For antigen +ve E. coli cell lysate coated wells probed with NRS, the lowest signal was again observed in wells blocked with CAE (OD = 0.04). This was significantly lower than those obtained in other cases. CAE was followed by SM, which had a slightly higher signal (OD = 0.06).
Probe Density
The numbers of gold particles/µm2 of total cell or clear plastic area are shown in Table 3. The representative areas are shown in Fig 3 Fig 4 Fig 5 Fig 6. On cell sections of antigen +ve E. coli cells labeled with anti-VHb antibody (Fig 3A, Fig 3C, Fig 4A, Fig 4C, Fig 5A, Fig 5C, Fig 6A, and Fig 6C), the probe density was always higher than the level of nonspecific binding in adjacent plastic. It was also significantly higher (p<0.001) than the probe density on cell sections of antigen -ve E. coli cells (Fig 3B, Fig 3D, Fig 4B, Fig 4D, Fig 5B, Fig 5D, Fig 6B, and Fig 6D) or sections of antigen +ve E. coli cells probed with; normal rabbit serum (in place of the first antibody) or only with the gold conjugate (not shown). Probe density results confirmed the ELISA finding that CAE and SM gave lowest levels of background labeling. As observed in ELISA, use of CAE as a blocking agent gave the lowest levels (p<0.001) of nonspecific binding on antigen -ve E. coli cells (0.28 ± 0.09) followed by SM (1.95 ± 0.28). Other blocking agents resulted in higher levels of nonspecific binding. Use of CAE for blocking also resulted in the lowest density of nonspecific labeling on clear plastic resin (0.80) and also when NRS was used in place of first antibody to label antigen +ve E. coli cell sections.
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Discussion |
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The current study demonstrates the advantage of ELISA as a convenient method for selection of optimal blocking reagents to carry out immunogold labeling electron microscopy. Blocking to suppress nonspecific labeling is a crucial aspect for the sensitive and specific localization and quantitation of antigen in cells and tissue (
The dot-blotting method totally failed in accurately predicting the different levels of nonspecific binding, either on clear plastic or on cell components that would occur when samples were evaluated by TEM. The use of this method would lead to the erroneous conclusion that all of the blocking reagents, with the exception of BQBSA, would give similar low levels of NSB because only BQBSA-blocked samples showed some significant background labeling on the nitrocellulose matrix. As in the case of dot-blotting, the use of Western blotting also did not provide any clear indication of the relative merits of various background-suppressing reagents. Moreover, these two methods were also not sensitive enough to detect any NSB of primary antibody to cell components other than the antigen (by use of antigen-negative cell lysates) and were also unable to detect the background labeling that occurs due to control serum (NRS). Inability to differentiate background labeling due to nonspecific binding reflects the limitation in sensitivity of the Western blotting method (even densitometric scans of the blots failed to differentiate among various blocking agents; data not shown). In addition, this lack of ability to differentiate among different blocking agents in terms of NSB may also be due to the biophysical nature of the interaction involving nitrocellulose support, the blocking agent, and the antibody molecules.
In contrast to the above two approaches, ELISA provided a clear-cut distinction in background level of signals when different blocking reagents were used. This method accurately predicted the significantly lower (p<0.001) levels of NSB observed in all cases when immunogold labeling TEM was carried out utilizing casein (and, to a lesser extent, skim milk) for blocking. The close similarity between the effects of CAE and SM could be due to the fact that the major component of skim milk is casein. In accordance with ELISA results for nonspecific signal arising from the use of control serum, immunogold labeling showed the lowest level of binding for CAE, 0.0 ± 0.0 gold particles/µm2. In the case of 2% FSG and 2% AuBSA, although the level of NSB by primary antibody to antigen negative cells was significantly higher than CAE and SM, the background labeling by gold conjugate alone, to antigen-positive cells as well as to clear plastic, was much lower than all the other blocking agents. In these two cases the positive signal, due to primary antibody binding to antigen-positive cells was also significantly less (p<0.001) than the labeling on cells when any of the other blocking agents were utilized. This may be caused by obscuring of exposed antigenic sites in cut cell sections at these higher concentrations of FSG and AuBSA, which may be due to the differences in the physical and chemical nature of these two proteins. Casein and BSA are globular proteins, and the major components of horse and goat sera, albumin and globulin, are also globular in nature. On the other hand, gelatin (a preparation from collagen) is a fibrous protein with a preponderance of both charged and hydrophobic amino acids. AuBSA is a partially linearized (to facilitate exposure of hydrophobic groups) form of BSA developed by Aurion and has an increased net negative charge due to acetylation of amino groups. The manufacturers recommend the use of these proteins only at 0.1 to 0.2% rather than at concentrations as high as 2% (
Earlier we have demonstrated the utility of ELISA-based methods for rapid selection of sample preparation conditions for quantitative detection of antigen in cell sections by immunogold labeling TEM (
In the present work we have presented data using a soluble intracellular antigen. However, in principle the ELISA technique can also be applicable to other proteins including membrane bound antigens (
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Acknowledgments |
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We wish to thank Drs A. Mondal and G.C. Varshney for critical reading and correction of manuscript. The skillful technical assistance of Mr Anil Theophilus is gratefully acknowledged. This is IMTech Communication No. 030/2001.
Received for publication October 2, 2001; accepted January 16, 2002.
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