A Comparison Between The Streptavidin-Biotin-Alkaline Phosphatase Method And The Immunogold Technique Using Ultra Small Gold Particles And Silver Enhancement

R. Willemsen, PhD Department of Clinical Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.

Introduction

Fragile X syndrome is the most common basis of inherited mental retardation and is caused by a mutation in the FMR1 gene which involves an increase in length of a stretch of CGG repeats just upstream of the coding region. In the normal population the number of CGG repeats varies between 6-50 repeats. CGG repeat numbers between 50 and 200 (premutation) are observed in unaffected carrier males and females. In patients with fragile X syndrome this repeat shows an expansion of more than 200 repeats (full mutation), associated in all males with mental retardation. Due to this large number of repeats, transcription suppression through methylation of the promotor region of the FMR1 gene occurs, resulting in absence of the encoded protein FMRP. This lack of FMRP protein is responsible for the fragile X phenotype. Characterization of the gene defect of fragile X syndrome led to improved molecular diagnosis of the syndrome via Southern blot analysis and a PCR test. Recently, an immunocytochemical test at the light microscopic level on blood smears is described to identify fragile X patients. This immunocytochemical test is based on the presence of FMRP protein in normal lymphocytes and the absence of FMRP protein in lymphocytes from fragile X patients. The FMRP protein was visualized in blood smears by a three step immunoincubation procedure using a streptavidin-biotin-alkaline phosphatase complex. This presentation deals with the comparison of the alkaline phosphatase labelling technique and the immunogold method using ultra small gold particles and silver enhancement with Aurion R- Gent.

Methods

Fixation and Permeabilization

Blood smears were made from one drop of blood immediately after bleeding. Slides were airdried and either stored at room temperature (max. 3 weeks) or directly fixed in 0.1 M phosphate buffer (Sörrensen, pH 7.3), containing 3% paraformaldehyde for 10 minutes at room temperature. Cells were permeabilized by treatment with 100 % methanol for 20 minutes at room temperature. Subsequently, slides were washed in PBS (10 mM Phosphate buffer, 150 mM NaCl) +0.1% BSA-c for 10 minutes.

Immunoincubation

Immunoincubation was performed using the Shandon disposable coverplate incubation system. Slides were washed 2X10 minutes with PBS, 0.1% BSA-c.

Alkaline Phosphatase Labelling (3 Step Immunoincubation)

1. Incubation with mouse monoclonal antibodies 1A1 against FMRP protein (1:1200 with PBS, 0.1% BSAc) for 16 hours at 4°C followed by 3X5 minutes washing with PBS, 0.1% BSA-c.
2. Incubation with goat anti-mouse immunoglobulins conjugated with biotin for 60 minutes (1:200 with PBS, 0.1% BSA-c) at room temperature followed by 3X5 minutes washing with PBS, 0.1% BSA-c.
3. Incubation with streptavidin-biotinylated alkaline phosphatase complex for 45 minutes at room temperature (1:100 with PBS, 0.1% BSA-c) followed by 3X10 minutes washing with PBS, 0.1% BSA-c.
4. Wash in 0.1 M TRIS for 5 minutes.
5. New Fuchsin substrate-chromogen system for 25 minutes at room temperature. Levamisole is added in the substrate solution to block endogenous alkaline phosphatase activity.
6. Wash in 0.1 M TRIS for 5 minutes.
7. New Fuchsin substrate-chromogen system for 25 minutes at room temperature.
8. Wash in aqua dest for 5 minutes.
9. Slides are counter stained with Gill's haematoxylin for 2 seconds and mounted with aquamount.

Immunogold Labelling (2 Step Immunoincubation)

1. Incubation with mouse monoclonal antibodies 1A1 against FMRP protein (1:4000 with PBS, 0.1% BSAc) for 16 hours at 4°C followed by 3X5 minutes washing with PBS, 0.1% BSA-c.
2. Incubation with goat anti mouse immunoglobulins conjugated with ultra small gold particles for 60 minutes at room temperature (1:80 with PBS, 0.1% BSA-c) followed by 3X10 minutes washing with PBS, 0.1% BSA-c.
3. Wash in PBS for 10 minutes.
4. Postfixation with PBS, containing 2% glutaraldehyde for 10 minutes.
5. Wash 3X10 minutes in PBS.
6. Wash 3X10 minutes in aqua dest.
7. Silver enhancement with Aurion R-Gent for 20 minutes at room temperature.
8. Wash 2X5 minutes in aqua dest.
9. Silver enhancement with Aurion R-Gent for 20 minutes at room temperature.
10. Wash 2X5 minutes in aqua dest.
11. Slides were counter stained in Nuclear Fast Red for 45 seconds, dehydrated in ethanol/xylene and mounted with Pertex.

Immunogold Labelling (3 Step Immunoincubation)

1. Incubation with mouse monoclonal antibodies 1A1 against FMRP protein (1:4000 with PBS, 0.1% BSAc) for 16 hours at 4°C followed by 3X5 minutes washing with PBS, 0.1% BSA-c.
2. Incubation with goat anti-mouse immunoglobulins conjugated with biotin (1:200 with PBS, 0.1% BSA-c) for 60 minutes at room temperature followed by 3X5 minutes washing with PBS, 0.1% BSA-c.
3. Incubation with goat anti-biotin conjugated with ultra small gold particles for 60 minutes at room temperature (1:30 with PBS, 0.1% BSA-c) followed by 3X10 minutes washing with PBS, 0.1% BSA-c.
4. Wash in PBS for 10 minutes.
5. Postfixation with PBS, containing 2% glutaraldehyde for 10 minutes.
6. Wash 3X10 minutes in PBS.
7. Wash 3X10 minutes in aqua dest.
8. Silver enhancement with Aurion R-Gent for 20 minutes at room temperature.
9. Wash 2X5 minutes in aqua dest.
10. Slides were counter stained in Nuclear Fast Red for 45 seconds, dehydrated in ethanol/xylene and mounted with Pertex.

Results and Conclusion

All three methods show expression of FMRP protein in lymphocytes from a healthy individual, whereas no expression is seen in lymphocytes of a fragile X patient.

In lymphocytes, the concentration of FMRP protein is very low, hence utilising alkaline phosphatase a three step immunoincubation is necessary to obtain sufficient reaction product. The alkaline phosphatase protocol has proven to be a reliable test for screening large numbers of male patients for the fragile X syndrome. However, a major disadvantage of this method might be the presence of endogenous alkaline phosphatase activity in blood cells.

The detection methods with the gold conjugates as described here illustrates that the results obtained using this method are highly comparable to those obtained with the alkaline phosphatase method. The three step immunogold labelling procedure yielded strong labelling of normal lymphocytes with 20 minutes silver enhancement. Even the two step immunogold labelling procedure in combination with two silver enhancement incubations results in sufficient reaction product. The discrimination between the nuclear staining and the coloured reaction product is more easy with the immunogold labelling method than with the alkaline phosphatase method. Both methods show no labelling of lymphocytes from patients with the fragile X syndrome, indicating the high specificity of both labelling procedures. In conclusion : the immunogold labelling method using ultra small gold particles followed by silver enhancement is a good alternative for the alkaline phosphatase method, especially in cases whereby tissues or cells are used with high endogenous alkaline phosphatase activity. In addition, the immunogold method is more sensitive and the reaction product is easier to discriminate when a nuclear counterstaining (Haematoxylin) is necessary.

Literature

Willemsen R, Mohkamsing S, de Vries B, Devys D, van den Ouweland A, Mandel JL, Galjaard H and Oostra B: Rapid antibody test for fragile X syndrome. Lancet 345: 1147, 1995.

Willemsen R, Smits A, Mohkamsing S, van Beerendonk H, de Haan A, de Vries B, van den Ouweland A, Sistermans E, Galjaard H and Oostra BA: Rapid antibody test for diagnosing fragile X syndrome: a validation of the technique. Hum Genet 99: 308, 1997.