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Annals of Clinical & Laboratory Science 37:152-157 (2007)
© 2007 Association of Clinical Scientists

Simple Immunohistochemical Staining Method Using Large Sized Gold Colloid Conjugated Secondary Antibody

Eun Jung Lee1, Yonggoo Kim1, Jihyang Lim1, Myungshin Kim1, Chang Suk Kang2, Jae Hoon Lee2, Rak Sun Mok2, Young Nam Park2, Ha-Young Choi1 and Kyungja Han1
1 Department of Laboratory Medicine, Hospital Pathology, Catholic University Medical College, Seoul; 2 Research and Development Center, YD Diagnostics Corp., Yongin Kyunggi-Do, Korea

Address correspondence to Kyungja Han, M.D., Department of Laboratory Medicine, Catholic University Medical College, St. Mary’s Hospital, Youngdeungpo-gu, Youidodong 62, Seoul, Korea [South] 150-713; tel 82 2 3779 1297; fax 82 2 783 6648; e-mail hankja{at}catholic.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To test the feasibility of semiquantitative immunohistochemical staining (IHC) with large sized gold conjugated secondary antibody (gold-2°Ab), we used beads covered with a known amount of primary antibody and a secondary antibody conjugated with gold colloid particles (20 and 40 nm in diameter), and we compared the results to those obtained by enzyme IHC. Beads coated with 6 graded amounts of mouse IgG molecules showed 6 levels of color intensity. The graded color intensities could readily be distinguished. The color developed as soon as we added gold-2°Ab, and the intensities were stable for 1 wk. Enzyme IHC using identical beads showed dregs of pigment after incubation in DAB for 5 min. The large sized gold-2°Ab showed strong signals on cell surfaces; application of the large sized gold-2°Ab to paraffin-embedded tissue sections was also feasible. The color was bright red and was easier to differentiate from hemosiderin pigment than the color developed by enzyme IHC. In conclusion, gold IHC with large sized gold-2°Ab is superior to enzyme IHC for quantification of antigens via IHC. Gold IHC is especially recommended for tissues with many macrophages, such as bone marrow and spleen.

Keywords: gold colloid particles, immunohistochemistry, gold conjugated secondary antibody


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colloidal gold has been used as a marker particle for the past 2 decades because of its simplicity of preparation, its high resolution, and its suitability for quantitative immunostaining [1–6]. Application of colloidal gold to ultrastructural studies has been widely performed [3,7–12]. However, the labeling is too weak to detect the signals by light microscopy unless an enhancement step, like silver enhancement, is used [13,14]. The diameter of gold colloid particles used as immunogold probes has ranged from 1 to 15 nm. In principle, the smaller gold particles produce higher labeling intensity of the target antigen because of reduced steric hindrance [7,15]. However if steric hindrance does not make a significant difference, then larger gold particles could produce stronger color intensity than smaller particles under the light microscope. Steric hindrance causes significant problems when the antigen is bound with primary antibody, but it may not always occur when the primary antibody is bound with secondary antibody.

The importance of quantitative IHC evaluation of specific antigens (eg, Her-2 protein, hormone receptor status of breast cancer, and p53 protein) has recently been emphasized [16–21]. However, the inter-observer reproducibility and the between-test reproducibility are both low for enzyme IHC [16,17,22,23]. There are inherent difficulties for such common analytes as estrogen and progesterone receptors [23–27]. Even when a commercial enzyme IHC kit is standardized with a control cell line, scoring of cases for Her2 by pathologists shows clinically significant variations and low precision [22]. Enzyme IHC and gold IHC with silver enhancement both have high sensitivity, but because of the strong amplification, the results are difficult to estimate quantitatively.

To test the feasibility of performing semi-quantitative IHC with a large sized gold conjugated secondary antibody, we used beads that were covered with a known amount of primary antibody, and we used a secondary antibody conjugated with large sized gold particles (20 and 40 nm diameter). We compared the results with those obtained by conventional enzyme IHC.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gold conjugation of the secondary antibodies.  Polycloncal goat anti-mouse IgG (Arista, USA) (polyclonal secondary antibody) and rat IgG anti-mouse IgG2a (Beckman Coulter, USA) (IgG secondary antibody) were used. Gold conjugation was performed by the R & D Center, YD Diagnostics (Korea). The diameter of the colloidal gold particles was 40 nm with conjugation to polyclonal antibody and 20 nm with conjugation to IgG secondary antibody. Carbonate buffer (0.5 M, pH 9.5) was used for the conjugation of polyclonal antibody and carbonate buffer (0.5 M, pH 7.4) was used for conjugation of the IgG secondary antibody; both conjugations were done for 20 min and the volume ratio of antibody to gold colloid was 1 to 10. The conjugated gold colloid was suspended in phosphate buffer (10 mM, pH 7.4) with 1% bovine serum albumin; the final concentration of the antibody was 10 µg/ml.

Immunohistochemical staining with gold conjugated secondary antibody (Gold IHC). 

  1. Detection of different numbers of mouse IgG molecules on the beads with using gold IHC. The beads were coated with 6 different numbers of mouse IgG molecules: 0, 3,200, 14,000, 69,000, 212,000, and 649,000 per bead (Dako Cytomation, Denmark). Forty µl of bead suspension was mixed with 40 µl of 20 nm gold-conjugated IgG secondary antibody and 40 nm gold-conjugated polyclonal antibody. After periods of 30 sec, 1, 5, or 20 min, and 3, 5, or 7 days at room temperature, 10 µl of the mixture was dropped on a slide and examined by light microscopy without washing.
  2. Detection of CD5 on peripheral blood lymphocytes with using gold IHC. A 3 ml peripheral blood sample was drawn from a healthy volunteer (K. Han) and anticoagulated with EDTA. Three tubes were set out. In each tube, 20 µl of the blood sample was mixed with 10 µl of anti-CD5-PE (Becton-Dickinson, USA) and the mixture was left for 15 min at room temperature. Then 2 ml of lysing solution (Becton-Dickinson) was added and the mixture was kept for 10 min at room temperature. After washing with phosphate buffered saline (PBS, pH 7.4), 1 tube was mixed with 1 ml of PBS; this was then analyzed using flow cytometry (FACS-CaliBur, Becton-Dickinson). The second tube was mixed with 20 µl of 40 nm gold-conjugated polyclonal secondary antibody and the third tube was mixed with 20 µl of 20 nm gold-conjugated IgG secondary antibody. After periods of 5, 10, 30 min, 1 hr, or 3 days at room temperature, slides were prepared by using 1 drop of the mixture; the slides were examined by light microscopy without washing.
  3. Detection of CD14 in bone marrow biopsy by using gold IHC. Paraffin-embedded bone marrow biopsy blocks were sectioned at 4 µm, deparaffinized, and rehydrated. After the sections had been heated in an autoclave in citrate buffer (pH 6.0) for 10 min for antigen retrieval and then washed, the slides were incubated with the blocking antibody for 5 min at room temperature. The slides were incubated for 60 min at room temperature with monoclonal antibody against CD14 (Labvision, USA) that was diluted 1:20. The slides were washed and then incubated with 20 nm gold-conjugated IgG secondary antibody for 5 min. The slides were washed, counterstained with Mayer’s hematoxylin, and mounted with glycerin mounting medium.

Enzyme immunohistochemical staining (enzyme IHC). 

  1. Detection of different numbers of mouse IgG molecules on the beads with using enzyme IHC. The same type of beads used for gold IHC was used for this procedure (Dako Cytomation). Forty µl of bead suspension was mixed with 30 µl of secondary antibody coupled with peroxidase linked polymer (Labvision, USA) for 30 min at room temperature. After being washed with distilled water, the resuspended beads were dropped onto slides and several drops of diaminobenzidine (DAB) solution were added. After periods of 30 sec, 1, 5, or 20 minutes at room temperature, the slides were examined by light microscopy without being washed.
  2. Detection of CD14 in bone marrow biopsy by using enzyme IHC. The paraffin-embedded bone marrow biopsy blocks were sectioned at 4 µm, deparaffinized, and rehydrated. After they had been heated in an autoclave in citrate buffer (pH 6.0) for 10 min for antigen retrieval, the slides were immersed in 0.3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. The slides were washed and then incubated with the blocking antibody for 5 min at room temperature. The slides were then incubated for 60 min at room temperature with monoclonal antibody against CD 14 (Labvision) that was diluted 1:20. After they had been washed, the slides were covered with primary antibody enhancer for 20 min. The slides were covered with 30 µl of secondary antibody coupled with peroxidase-linked polymer (Labvision) for 30 min at room temperature. After the slides were washed with distilled water, several drops of DAB solution were added. After 30 sec, the slides were observed under the microscope for the development of color. After 2 min, the slides were washed using distilled water, counter-stained with Mayer’s hematoxylin. and then mounted with glycerin mounting medium.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IHC using gold conjugated secondary antibody. 

  1. Detection of different numbers of mouse IgG molecules on beads using gold-conjugated antibody. Beads coated with 6 graded numbers of mouse IgG molecules showed 6 levels of color intensities (Fig. 1Go). The color was very clear and it was easy to differentiate the intensities. The color developed within 30 sec after addition of the gold-conjugated secondary antibody and the intensities remained stable for 1 wk (Fig. 1Go). The color intensities using 40 nm gold colloid polyclonal secondary antibody and 20 nm gold colloid anti-IgG were similar.
  2. Detection of CD5 on peripheral blood lymphocytes using gold-conjugated secondary antibody. Based on flow cytometry, 29.5% of the leukocytes showed CD5 expression. Based on IHC using anti-CD5 and gold conjugated secondary antibody, about 25% of the cells showed red staining on the surface, like a rim (Fig. 3Go). The fine dot-like red signals began to develop after 30 min incubation with 20 nm gold conjugated IgG secondary antibody, and these positive cells were easy to differentiate from negative cells. For some cells, the color was more concentrated and denser than that in other cellular areas (Fig. 3Go). The red signals began to develop after 30 min incubation with 40 nm gold conjugated polyclonal secondary antibody, and these signals were similar to those with 20 nm gold conjugated IgG secondary antibody (Fig. 3Go). After 3 days of incubation with gold conjugated secondary antibody, the signals with 20 nm gold conjugated IgG secondary antibody were clear and similar to those after 30 min incubation; however, the signals with 40 nm polyclonal secondary antibody were larger and darker than after 30 min incubation (Fig. 3Go).
  3. Detection of CD14 in bone marrow biopsy using gold-conjugated secondary antibody. Some histiocytes and monocytes in the bone marrow biopsy showed a positive reaction to CD14 with a clear background (Fig. 4Go). The color was red and stable during both counterstaining and the glycerin mounting process. The red signals were easy to differentiate from the yellow-brown hemosiderin pigment (Fig. 4Go).


Figure 1
Figure 1
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  		Fig. 1. Detection of graded numbers of mouse IgG molecules on beads using 20 nm gold colloid particle conjugated secondary antibody after incubation for 2 min (left, x400) and 5 days (right, x200). The color is clear, the graded color intensities are easy to differentiate, and the intensities are stable for 5 days.

 

Figure 3
Figure 3
Figure 3
Figure 3
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  		Fig. 3. Detection of CD5 molecules on leukocytes using anti-CD5-PE and gold colloid conjugated secondary antibody. The fine dot-like red signals are found on the cell surfaces and are easy to differentiate from negative cells. In some cells, the color is concentrated and dense (upper left). The red signals after 30 min incubation with 40 nm gold conjugated polyclonal secondary antibody (upper left) are similar to those with 20 nm gold conjugated IgG secondary antibody (upper right). After 3 days of incubation, the signals with 20 nm gold conjugated IgG secondary antibody (lower left) are similar to those after 30 min incubation. However, after 3 days of incubation, signals with 40 nm polyclonal secondary antibody are larger and darker (lower right) than after 30 min incubation (x1,000).

 

Figure 4
Figure 4
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  		Fig. 4. Detection of CD14 on bone marrow biopsy by enzyme IHC (left) and by using 20 nm gold colloid conjugated secondary antibody (right). Some histiocytes and monocytes in the bone marrow biopsy show positive reaction to CD14 with both methods. However, the color with the gold IHC method is red and easier to distinguish from yellow-brown hemosiderin pigment (arrows).

 
Enzyme IHC. 
  1. Detection of different numbers of mouse IgG molecules on beads using enzyme IHC. The same type of beads used for the gold IHC showed several different intensities of brown color (Fig. 1Go). The color was clear and strong, but it was difficult to differentiate the graded intensities. The color developed one min after adding the sustrate (DAB) and the intensities rapidly deepened according to the exposure time (Fig. 2Go).
  2. Detection of CD14 in bone marrow biopsy using enzyme IHC. Positive signals did not appear until 1 min after adding DAB, and the color was strong after 2 min. Some histiocytes and monocytes in the bone marrow biopsy showed a positive reaction (brown color) to CD14 (Fig. 4Go). The color was similar to that of hemosiderin.


Figure 2
Figure 2
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  		Fig. 2. Detection of graded numbers of mouse IgG molecules on beads using enzyme IHC after incubation in diaminobenzidine (DAB) solution for 2 min (left) and 5 min (right). The color is clear and strong, but it is not easy to differentiate the graded intensities. The intensities deepen after 5 min incubation and dregs of pigment are seen on the surface of the beads (x400).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The conventional enzyme IHC procedure consists of many steps, including incubation with an endogenous enzyme blocking reagent and a primary antibody, washing, incubation with a biotinylated secondary antibody, washing, incubation with peroxidase-conjugated streptavidin, washing, visualization by immersing slides in DAB, washing, and counterstaining. In the gold IHC procedure, several of these steps (ie, incubation with an endogenous enzyme blocking reagent, washing, incubation with peroxidase-conjugated streptavidin, washing, and visualization) are omitted. Thereby, the staining time is shortened by about 1 hr and variability of the staining intensity is reduced. Although the avidin-biotin complex method can amplify the signal intensity, it may be a disadvantage for evaluating antigen quantitatively. Secondary antibody coupled with enzyme-linked polymer has recently been developed and the signal was also amplified. Although colloidal gold particles are unstable in the presence of electrolytes, various macromolecules will bind to colloidal gold particles and cause them to become more hydrophilic and stable in the presence of electrolytes [28]. Protein A-gold complex has been proposed as a second step reagent for IHC [29]. However, gold conjugated secondary antibody has not been used in IHC, probably because of weak signals.

In this study, gold conjugated secondary antibody using large sized gold colloid particles, 20 nm and 40 nm in diameter, showed beautiful red signals. This result indicates that if the size of the gold colloid particle is large enough, the gold conjugated secondary antibody is suitable for performing IHC. Another point is steric hindrance of the large sized gold particles [7,15]. This study showed that the secondary antibody labeled with large particles can bind to the primary antibody without a problem of steric hindrance, unlike the binding of primary antibody to antigen. The beads coated with 6 graded numbers of mouse IgG molecules showed 6 levels of color intensities using 20 nm and 40 nm gold colloid particles conjugated to secondary antibody. This indicates that IHC that uses large size colloidal gold particles conjugated to secondary antibody without enhancement can give quantitative information about the presence of antigen in the tissue. The color is very clear and develops within 30 sec after addition of the gold-conjugated secondary antibody, The color intensity is stable up to 1 week.

In this study, the enzyme IHC revealed dregs of pigment even after 5 min incubation in DAB, and the intensity of color was much stronger than after 2 min incubation. The incubation time in the substrate solution is a critical factor that determines the color intensity in enzyme IHC. This result suggests that gold IHC is better than enzyme IHC for quantification of antigens by IHC. These large sized gold conjugated secondary antibodies showed strong signals on the cell surfaces. The color was clear red and it was sensitive enough to show a count similar to flow cytometry. Although incubation with 40 nm gold conjugated polyclonal antibody for 3 days made the signal darker and larger than after 30 min incubation, the color and appearance of the signals were very stable. Steric hindrance seemed not to cause any significant problem because lymphocytes showed well localized, strong signals on their surface. If the large size of the particles caused steric hindrance, the color should be weak or invisible and an intensely concentrated spot should not appear.

Application of the large sized gold colloid conjugated secondary antibodies to paraffin embedded tissue sections is also possible. The color is bright red and easy to differentiate from hemosiderin pigment. On the other hand, the brown color developed by incubation in DAB is similar to hemosiderin. The color of gold labeled antibody is stable for about 6 months. The stability has not yet been checked for a longer time. The gold labeled antibody is easier to precipitate than the enzyme labeled antibody. Counterstaining with hematoxylin is possible, if glycerin mounting medium is used.

In summary, gold IHC with large sized gold colloid particles (20 to 40 nm in diameter) is superior to enzyme IHC for quantification of antigens. Furthermore, the gold IHC method is simpler and more rapid than enzyme IHC. The color is bright red and is easier to differentiate from hemosiderin pigment than the brown color formed in enzyme IHC. For these reasons, gold IHC is recommended, especially for tissues with many macrophages such as bone marrow and spleen.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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