INFLUENCE OF ANTIBODY DILUENT pH ON IMMUNOHISTOCHEMISTRY DURING PRIMARY ANTIBODY INCUBATION Niina Toikkanen1, Tommi Ijäs1, Johanna Lappeteläinen1, Charlotta Putkonen1, and Teppo Haapaniemi1

1) BioSiteHisto Ltd, Tampere, Finland

Introduction Methods Results

Conclusion

• The pH of commercially available antibody diluents is typically adjusted to close to physiological pH, but some specialized antibody diluents are available with a different pH range e.g., pH 6-8.

• A significant influence of the pH and saline concentration of the antibody diluent during primary antibody incubation in immunohistochemistry (IHC) has been reported in several publications [1].

• In this study, 15 Optibody antibodies were diluted into antibody diluents where the pH was adjusted to pH 6 or pH 8. An antibody diluent with a non-adjusted pH of 7.2 was used as a reference.

• No significant differences in staining pattern or signal to noise (S/N) ratio were observed using pH adjusted antibody diluents with the antibodies used in this study.

Antibodies• Antibodies were selected according to the cellular location

of their respective antigenic targets. In total, 15 monoclonal antibodies targeting 5 nuclear, 5 cytoplasmic and 5 membranous proteins were used (Table 2).

• Of the 15 antibodies used, 5 were of rabbit origin and 10 of mouse origin.

• The pH of the antibody diluent (BCB-20006, Nordic BioSite) was adjusted to pH 6 or pH 8 using HCl or NaOH, respectively.

Immunohistochemistry• Multi-tissue sections with core sizes of 6 mm in diameter were

used as tissue sample material. Adjacent sections with 4 µm section thickness were sectioned. Two sections per slide were stained in order to minimize the influence of variations in section thickness during interpretation. The tissue material used contained negative, low, and high antigen-containing tissues.

• Staining was performed using LabVision autostainer platform and a polymer-based 2-step HRP detection method with DAB visualization.

• The primary antibody incubation period was 30 minutes all cases, and a volume of 300 µl was used per slide. Dilution of each primary antibody was 1:200.

• Interpretations were made according to the scoring system presented in Table 1. Slides were digitalized using 3D-Histech Pannoramic MIDI scanner.

• No significant influence of the antibody diluent pH during primary antibody incubation.

• No intensity or S/N differences were observed using antibodies against nuclear markers (Table 2).

• Slight differences in S/N ratio between pH 6 and pH 8 were observed using CD3, CD4, CD79a, Desmin, CK20 and CKpan Optibodies™ (Figure 1).

• No significant influence of the antibody diluent pH was observed.

• Low variations in staining intensity were observed with antibodies against epitopes located in the plasma membrane and cytoplasm. No variation in staining intensity was observed with antibodies against nuclear markers (Table 2).

• Commercial antibody diluents with variations in pH might impact the staining reaction and the antibody-epitope interactions during primary antibody incubation to a greater extent than in-house modified antibody diluents.

References:1) Boenisch, Thomas. (1999). Diluent Buffer Ions and pH: Their Influence on the Performance of Monoclonal Antibodies in Immunohistochemistry. Applied Immunohistochemistry & Molecular Morphology. 7(4): 300. doi:10.1097/00129039-199912000-00009.

Score Description of score-2 Significant negative impact on staining intensity and

specificity-1 Weak negative impact on staining intensity and

specificity0 No difference in staining intensity and specificity1 Weak positive impact on staining intensity and

specificity2 Significant positive impact on staining intensity and

specificity

Scoring of the stained slide Intensity and specificity of the staining

Marker Clone Location of antigen Host pH 6 pH 7.2 pH 8

P53 BS12 Nuclear Mouse 0 0 0

Ki67 BS4 Nuclear Mouse 0 0 0

P63 BSR6 Nuclear Rabbit 0 0 0

SOX10 BS7 Nuclear Mouse 0 0 0

BCL6 BS19 Nuclear Mouse 0 0 0

CD4 BSR4 Membranous Rabbit -1 0 1

CD79a BSR20 Membranous Rabbit 1 0 -1

CD3 BSR10 Membranous Rabbit 0 0 -1

E-Cadherin BS38 Membranous Mouse 0 0 0

Ep-CAM BS14 Membranous Mouse 0 0 0

SMA BS66 Cytoplasm Mouse -1 0 0

Desmin BS21 Cytoplasm Mouse 0 0 0

CKpan BS5 Cytoplasm Mouse -1 0 0

CK20 BS101 Cytoplasm Mouse 0 0 0

CK20 BSR11 Cytoplasm Rabbit 1 0 0

Figure 1. Influence of antibody diluent pH during primary antibody incubation. Sufficient staining patterns were observed with all antibodies when using antibody diluents with pH 6 and pH 8. No differences in antibody specificity were observed when using different pH antibody diluents. However, slight variations in staining intensity were observed using pH 6 and pH 8 antibody diluents. Significant differences in staining intensity were not observed in this study.

Figure 2. Comparison of the staining pattern of Optibodies™ using different antibody diluent pH’s (pH6, pH7.2, and pH8). Multi-tissue sections were stained according to optimized protocols, and two serial sections in the same slide were stained in order to minimize the influence of variation of the tissue section thickness. Non-modified antibody diluent (pH 7.2) was used as a reference. Specificity and intensity were interpreted from every tissue core of the multi-block sections from digitalized slides. All of these stainings were sufficient without significant difference in S/N ratio, even when low antigen containing-tissues were stained.

pH 6 pH 8 pH 6 pH 8

pH 6 pH 7.2 pH 8

CD3, Tonsil

CD79a, DLBCL

CD4, Tonsil

Desmin, Appendix

CKpan, Appendix

CK20 (BSR11), appendix

SOX10, Melanoma

Ki67, Tonsil

Desmin, Leiomyoma

SMA, Liver

CKpan, Liver

CD4, Tonsil

CD3, Tonsil

CD79a, Tonsil