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CD99: Development and Evaluation of a Novel Monoclonal Antibody for use in Manual and Automated Immunohistochemistry

CD99 is a transmembrane glycoprotein which is expressed in a wide range of tissues. Although its function is not fully understood it has been implicated in a variety of cellular processes including the homotypic aggregation of the T cells, apoptosis of immature thymocytes, up-regulation of T cell receptor and MHC molecules and leukocyte diapedesis.

We have developed a novel and robust monoclonal antibody to an epitope within 101 amino acids of the N-terminal region of the human CD99 molecule. This antibody is effective in both manual and automated immunohistochemistry on formalin-fixed, paraffin-embedded tissues.

Clone PCB1 was evaluated on 329 normal and tumor tissues. Strong membranous expression was noted particularly in thymocytes, pancreatic islets and cases of Ewing’s sarcoma, peripheral neuroectodermal tumors and malignant/lymphoblastic lymphomas. Clone PCB1 will be particularly useful for the differentiation of the Ewing’s sarcoma family of tumors from other types of small round cell tumors.


Ewing’s sarcoma and peripheral neuroectodermal tumors (pNET) are members of a histologically similar group of tumors known as small round cell tumors of childhood and adolescence (SRCT).1 The tumors are tan gray, often with necrotic and hemorrhagic zones. The histological pattern of SRCT ranges from uniform small round cells with round nuclei, fine chromatin and scant cytoplasm with indistinct borders to larger irregular cells with irregular nuclear contours and pseudorosettes, nesting pattern, and even spindle cells. The differential diagnosis includes tumors of neural (neuroblastomas), mesenchymal (rhabdomyosarcomas, Ewing’s sarcoma, pNET, desmoplastic small round cell tumor), neuroendocrine carcinomas, acute lymphoblastic leukemia and myeloid leukemia.1, 2 These tumors differ considerably in respect to their site of origin, etiology and prognosis. Ewing’s sarcoma/pNET is the second most common bone and soft tissue sarcoma in children. It occurs more frequently in bone than extra-skeletal.

There is a male predominance. Ewing’s sarcoma and pNET are diagnosed predominantly in the second decade of life.1, 3 These tumors behave aggressively and have a tendency to metastasize rapidly.1, 4 The Ewing’s sarcoma family of tumors are associated with a reciprocal chromosomal translocation between the long arms of chromosomes 11 and 22 in approximately 85% of cases t(11;22) (q24;q12) resulting in the formation of the EWS-FLI-1 fusion gene.1, 3, 5 This aberrantly expressed fusion protein is believed to contribute to the development of Ewing’s sarcoma family tumors by acting as a transcription factor which alters the expression of target genes.5 This translocation is not thought to be found in lymphomas, neuroblastomas or rhabdomyosarcomas.2

CD99 is a transmembrane glycoprotein, encoded by the MIC2 gene6 and characteristically expressed by Ewing’s sarcoma and pNET. It is also detectable in acute lymphoblastic leukaemia/lymphoma, acute myelogenous leukaemia, myeloid sarcoma, synovial sarcoma and mesenchymal chondrosarcoma. It is found in the pseudo-autosomal region of the X and Y chromosomes and is thought to escape X-chromosome inactivation. Recently the MIC2 gene has been shown to encode two distinct proteins, which are produced by alternative splicing of the CD99 transcript, and may be identified in western blots as bands of 30 kDa and 32 kDa.7, 8 The two splice variants of CD99 have been shown to be differentially expressed in a variety of human cells tested.9–11 They have also been shown to play opposing roles during the intercellular adhesion molecule 1 (ICAM1) mediated B cell adhesion process, with the minor truncated variant inhibiting homotypic adhesion of B cells and the major form inducing homotypic adhesion of B cells.9–11 Although its function is not fully understood CD99 has also been implicated in a variety of other cellular processes including apoptosis of immature thymocytes, up-regulation of T cell receptor and MHC molecules6, 10, 12 and leukocyte diapedesis.13

CD99 is expressed on most human tissues including cortical thymocytes,1, 12 pancreatic islet cells,1, 14 Leydig and Sertoli cells,1, 15 virtually all hematopoietic cell types (except granulocytes), peripheral blood lymphocytes,6 ovarian granulosa cells,1, 7 endothelial cells,1, 13 and basal/parabasal squamous epithelial cells.16 Although CD99 is characteristically expressed in Ewing’s sarcoma, it is particularly useful in differentiating Ewing’s and other small round cell tumor, particularly neuroblastoma. Expression of CD99 has been demonstrated in a wide range of tumors including lymphomas, rhabdomyosarcomas, ovarian tumors, ependymomas,10, 16 pancreatic endocrine tumors, sex cord stromal tumors and breast carcinomas.1,14 The aim of this study was to develop a novel monoclonal antibody to CD99 that would give superior staining results, in terms of specificity and intensity, when compared to the existing NovocastraTM clone HO36-1.1 in both manual and automated immunohistochemistry.

Materials and Methods

Production of Soluble Human CD99 Recombinant Protein
Total RNA was extracted from washed HPB-ALL cells using an RNeasy Mini Kit (Qiagen, West Sussex, UK). For reverse transcription, 1mg of total RNA was primed with a specific primer and cDNA synthesis was carried out using the Reverse Transcription System Kit (Promega, USA) in accordance with the manufacturer’s protocol.

Amplification of a 303-base pair N-terminal region of CD99 (EMBL-EBI accession number X16996) was carried out by PCR using a second CD99 specific primer and half of the reverse transcription mix as a template in a 50 µL PCR reaction mix (2 mM MgCl2, 0.2 mM dNTPs, 0.3 mM of primer, 2.5 U Taq-DNA polymerase and 1x RT transcription buffer reaction mix (Promega, USA). A total of 30 cycles of PCR (94°C/30 sec, 60°C/30 sec and 72°C/30 sec) was performed.

A PCR product of the appropriate size was confirmed by agarose gel electrophoresis and then cloned into the pGEM-Teasy vector (Promega, USA). Clones were identified by digestion with the appropriate restriction enzymes and their identity confirmed by DNA sequencing (The Central Molecular Biology Facility, Newcastle University, UK). The cloned CD99 gene fragment was sub-cloned into the expression vector pET41b (Novagen, USA) which incorporates a GST tag on to the N-terminal end of the recombinant and also adds oligo-histidine tags to aid purification by immobilised metal ion chromatography. The resulting construct was transformed into Escherichia coli BL21 DE3 (pLysS) and protein expression was induced in cultures with the addition of isopropyl thiogalactoside (IPTG, 1 mmol/L).

The CD99 recombinant fusion protein was purified by column chromatography using a Ni2+-IDA metal chelate resin (Generon).

Purified protein was assessed by SDS-PAGE (data not shown). When assessing hybridoma clones it was essential to use a GST screening antigen to screen out clones secreting antibody raised against the GST protein. A GST screening protein was produced using molecular recombinant technology.

Monoclonal Antibody
ProductionHybridomas were generated as previously described.17 Hybridoma supernatants were screened by enzyme linked immunosorbent assay (ELISA). This involved screening the supernatants against the CD99 recombinant protein and GST to identify specific reactivity. Positive hybridoma supernatants were also screened by immunohistochemistry. Hybridomas producing CD99 antibodies were selected and clones established by limiting dilution.

Immunohistochemical validation was performed on a range of normal and tumor tissues using the selected mouse monoclonal CD99 antibody, clone PCB1, in conjunction with the Novolinkª Polymer Detection System RE7140-K (250 tests). Paraffin sections, 4mm thick, were cut on to Leica Microsystems charged coated slides (S21.2113.A). To facilitate adhesion, these slides were then dried overnight at 37°C and finally baked for 1 hour at 56°C. Sections were deparaffinised in xylene and rehydrated through graded alcohols.

Heat induced epitope retrieval was performed using Epitope Retrieval Solution pH6.0 (RE7113) in a Prestige pressure cooker for 8 minutes at full pressure. Endogenous peroxidase activity was blocked by incubation for 5 min in peroxide block solution. Slides were washed in 50 mM Tris buffered saline (TBS, pH 7.6) for 5 minutes, incubated with protein block for 5 minutes and washed in TBS for a further 5 minutes. Sections were then incubated for 30 minutes at 25¡C with CD99 primary antibody diluted 1:100 in IHC diluent (RE7133). Following two sequential 5 minute wash steps in TBS, sections were incubated in rabbit anti-mouse Post Primary for 30 minutes at 25°C. Two sequential 5 minute wash steps in TBS were again performed and sections incubated for 30 minutes at 25°C with Novolink Polymer. Two further sequential 5 minute wash steps in TBS were performed and bound peroxidase visualised using DAB chromogen. The DAB working solution was prepared by adding 50 µL of DAB chromogen per mL of DAB Substrate Buffer. Slides were washed in water and then sections were counterstained with hematoxylin. Finally sections were dehydrated, cleared and mounted in DPX.

All scoring was carried out by senior scientists experienced in the assessment of immunohistochemical staining and tissue histology. In normal tissues, membrane staining of tissue elements was noted and the staining intensity recorded as either, negative, weak, moderate or strong. In tumor tissues, any membrane staining of tumor elements was classified as “positive” regardless of the staining intensity.

Automated Immunohistochemistry
Clone PCB1 was shown to be effective on the Leica automated BondTM system using BondTM Epitope Retrieval Solution 1 (AR9961) and BondTM Polymer Refine Detection (DS9800). Protocol F applied epitope retrieval for 20 minutes, incubation with clone PCB1 for 15 minutes, incubation with Post Primary for 8 minutes, Polymer for 8 minutes, DAB for 10 minutes and hematoxylin for 5 minutes. Staining was unaffected by the position of the peroxidise block step in the protocol.

External Evaluation
Clone PCB1 was evaluated by Dr Korinna J_hrens at the Charité – Universitätsmedizin Berlin, Germany.

Figure 1.

Immunohistochemical staining for CD99 using clone PCB1 on A) pancreas and
B) tonsil (original magnification x200). Manual staining of paraffin section.

Figure 2.

Immunohistochemical staining for CD99 using clone PCB1 on A) Ewing’s sarcoma (original magnification x200)
and B) pNET (original magnification x200). Bondª automated system staining of paraffin sections.


Normal Tissues
Table 1 shows the staining for CD99 in normal tissues. Clone PCB1 detected the CD99 protein in a variety of tissues including lymphocytes, vessel endothelium and basal epithelia of tonsil and pancreatic islets (Figure 1). (Total number of cases stained = 80).

Abnormal Tissues
Clone PCB1 detected the CD99 protein in 31/40 soft tissue tumors and sarcomas (Table 2), where staining was particularly noted in pNET and Ewing’s sarcoma (Figure 2). Clone PCB1 also stained 19/51 hematological malignancies (Table 3), 14/68 lung tumors (Table 4) and 28/34 prostate tumours (Gleeson grade 6-9) (Table 5). (Total number of cases stained = 247).

Table 1. Immunostaining for CD99 in Normal Tissues


Staining Results




Strong membrane staining of epithelium

Bone marrow

Moderate membrane staining of a subset of lymphocytes

Bowel, large

Strong membrane staining of epithelium

Bowel, small

Strong membrane staining of epithelium

Brain, cerebellum

Strong staining of white matter


Strong membrane staining of occasional cells in glandular and ductal epithelium


Strong membrane staining of basal epithelium


Moderate membrane staining of epithelium


Strong membrane staining of glomerular capillaries


Strong membrane staining of hepatocytes


Strong membrane staining of bronchial epithelium

Muscle, skeletal





Moderate membrane staining in stromal cells




Strong membrane and cytoplasmic staining of islets of Langerhans and occasional acinar epithelial cells


Moderate membrane and cytoplasmic staining of pituicytes


Strong membrane staining of acinar epithelium

Salivary Glands

Moderate membrane staining of glandular and ductal epithelium


Moderate membrane staining of splenocytes in white pulp


Strong membrane staining of basal epithelium


Strong membrane staining of epithelium and gastric glands


Strong membrane and cytoplasmic staining of tubules


Strong membrane staining of thymocytes


Strong membrane staining of occasional follicular epithelial cells


Strong membrane staining of basal epithelium

Umbilical cord

Strong membrane staining of mesothelium

Uterus (myometrium)

Weak/moderate membrane staining of myometrial stromal cells

In positive tissues peripheral lymphocytes and vessel endothelium were positive (where present) with clone PCB1, all other tissue elements were negative.

Table 2. Immunostaining for CD99 in Soft Tissue Tumors and Sarcomas


IHC Positivity





Synovial sarcoma


Sarcoma epithelioides


Ewing’s sarcoma


Desmoplastic small round cell tumor


Clear cell sarcoma


Malignant rhabdoid tumor


Alveolar soft part sarcoma


Table 3. Immunostaining for CD99 in Hematological MalignanciesTable 3. Immunostaining for CD99 in Hematological Malignancies


IHC Positivity

Diffuse large B cell lymphoma


Follicular lymphoma


Hodgkin’s lymphoma


Anaplastic large cell lymphoma


Peripheral T cell lymphoma




T lymphoblastic lymphoma


Marginal zone lymphoma


Mantle cell lymphoma


Non-Hodgkin’s lymphoma (unclassified)


Burkitt’s lymphoma


NK/T cell lymphoma


Unspecified lymphoma


Table 4. Immunostaining for CD99 in Lung Tumors


IHC Positivity



Squamous cell carcinoma


Small cell carcinoma


Large cell carcinoma


Atypical carcinoma


Papillary adenocarcinoma


Bronchioalveolar carcinoma


Giant cell carcinoma


Non-small cell carcinoma


Table 5. Immunostaining for CD99 in Miscellaneous Tumors


IHC positivity

Prostate – Gleeson grade 6-9


Pancreas – duct adenocarcinoma


Pancreas – islet cell carcinoma


Squamous cell carcinomas (various locations)


Liver – hepatocellular carcinoma


Liver – cholangiocarcinoma


Ovary – transitional cell carcinoma


Ovary – mucinous cystadenocarcinoma


Ovary – clear cell carcinoma


Colorectal – adenocarcinoma


Stomach – adenocarcinoma


Thyroid – papillary carcinoma


Thyroid – follicular carcinoma


Kidney – clear cell RCC


Brain – glioblastoma


Brain – glioma


Breast – ductal carcinoma


Novocastra NCL-CD99 (clone HO36-1.1)

Immunohistochemical staining using our existing NovocastraTM product, NCL-CD99 (clone HO36-1.1), highlighted non-specific cross-reactive staining in several tissues.

Western blotting also highlighted that in addition to a weak band at the correct molecular weight (32 kDa), clone HO36-1.1 also produced an additional strong band at a higher molecular weight (IHC and blotting data not shown). Western blotting confirmed that clone PCB1 produced a single band at 32 kDa (data not shown).


We have developed a novel monoclonal antibody to CD99 for use in formalin-fixed, paraffin-embedded tissue immunohistochemistry. NCL-L-CD99-187, clone PCB1, was shown to be effective at a dilution of 1:100 using Heat Induced Epitope Retrieval solution pH 6 and the Novolink Polymer Detection System. Staining was unaffected by the position of the peroxidase block step in the protocol or the use of TBS or PBS-based diluents and wash buffers. The antibody is also effective on the automated Bond system using Bond Epitope Retrieval Solution 1 (AR9961) and Bond Polymer Refine Detection (DS9800).

Clone PCB1 is an important immunological tool for the differentiation of Ewing’s sarcoma and other peripheral neuroectodermal tumors from other types of small round cell tumor.


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  9. Lee H, Kim B, Hahn J, et al. Functional involvement of src and focal adhesion kinase in a CD99 splice variant-induced motility of human breast cancer cells. Experimental and Molecular Medicine. 2002; 34 (3): 177–183.
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  17. Rees ML, Marshall I, McIntosh GG, et al. Wild-type estrogen receptor beta expression in normal and neoplastic paraffin-embedded tissues. Hybridoma and Hybridomics. 2004; 23 (1): 11–18.

Unless otherwise stated all references quoted refer to data on CD99 derived from established scientific publications involving various antibodies and techniques and do not refer to IHC staining expression directly attributable to NCL-L-CD99-187.