Hybridoma 1992.11:803-813. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/09/15. For personal use only.
HYBRIDOMA Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers
Monoclonal Antibodies to Human Thyroglobulin: Evaluation of Immunoreactivity A.A. D.
NARKAR,1
SWAROOP,2
SHAH,1 J. YADAV,2
D.H. and R.
MULHERKAR3
1Radiation Medicine Center, B.A.R.C., c/o Tata Memorial Center Annexe, Parel, Bombay 400 012, India 2Tata Memorial Hospital, Parel, Bombay 400 012, India 3Cancer Research Institute, Parel, Bombay 400 012, India -
-
-
ABSTRACT We have earlier reported production and characterization of monoclonal antibodies (MAbs) to human thyroglobulin (h-tg).In the present study HI MAb was evaluated for its immunoreactivity towards different forms of tg and various human thyroid tumours. The specificity of HI MAb was validated by the absence of cross reaction with tri-iodothyronine (T ) Thyroxine (T ) and human gamma globulins. Sodium-dodicyl-sulphate polyacrylamide gel electrophoresed (SDS-PAGE) immunoblot of h-tg on the nitrocellulose membrane revealed multiple immunoreactive bands on reaction with polyclonal antibody (PAb) in comparison with total lack of reactivity with HI MAb. The absence of immunoreactivity of HI MAb was demonstrated with SDs treated, Dithiothreitol (DT) treated and heat denatured tg using dot immunobinding technique. However, the HI MAb was able to react with tg treated with unfolding agents such as urea and guanidine hydrochloride. All the treated forms of tg were equally recognized by PAb. The immunoreactivity of the oxidized/reduced tg towards HI MAb was markedly reduced (60.0\) as compared to that obtained with native tg. It appears that HI MAb is directed towards conformational epitope involving sulphydryl bonds.
Immunohistochemically, a comparable immunoreactivity between PAb and MAb was thyroid tissues, follicular thyroid tissues, Hurthle cell and poorly differentiated thyroid tumor tissues using immunoperoxidase staining. The sections from papillary carcinoma tissue (thyroid as well as metastatic lymph node) exhibited intense immunoreactivity with PAb. Thyroglobulin present on these sections was not recognized by HI MAb. observed with normal tissues carcinoma
Nonetheless, HI MAb was able to detect tg in follicular differentiation wherever present. The absence of immunoreactivity of HI MAb in papillary carcinoma strongly suggests that this neoplasm produces tg which is antigenically different from the protein present in the normal tissue. The reactivity of HI MAb with metastatic lymph node of an unknown primary origin suggests its usefulness in the identification of prevalent metastasis of differentiated thyroid carcinoma other than
papillary type.
803
Hybridoma 1992.11:803-813. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/09/15. For personal use only.
INTRODUCTION
Thyroglobulin (tg) has been a crucial biomolecule in the study of thyroid diseases because of its vital role in the thyroid hormone(s) synthesis and This molecule has been extensively investigated biochemically in normal storage. and pathological conditions. Immunologically, tg had been considered as a secluded antigen and its exposure was believed to provoke production of auto-antibodies. It has now been shown to be a normal physiological component of the serum and is present in the detectable quantities in about 80% of the normal healthy subjects (1). Serum tg level is raised in most of the thyroid disorders and hence is of limited value in the differential diagnosis of tumours. Nonetheless, it is a reliable tumour marker in the management of proved patients of differentiated thyroid carcinoma. Since the availability of monoclonal antibodies (MAbs) as a powerful tool for elucidation of structure and function of proteins, several laboratories have
produced MAbs to tg to use as a probe for structural analysis (2-6), to indentify epitopes involved in autoimmune processes (7,8) and for localisation of tg in thyroid tumours (9-11) We have earlier reported the production and characterization of MAbs to human its in in-vitro and usefulness immunoassay as well as tg (h-tg)
immunohistochemistry (5). In the present immunoreactivity of HI MAb towards modified tg
paper
and
we
further report the human thyroid
different
tumours.
MATERIALS & METHODS
Production of MAbs to h-tq: Human
and
tg
MAbs
to
h-tg
were
prepared
as
described earlier (5). Briefly,
fused with spleen cells of BALB/c mouse immunized with h-tg. The supernatants were screened with indirect ELISA for the presence of anti-h-tg antibodies. Of the seven stable clones secreting specific antibodies, HI MAb was used in the present to IgG subclass with studyf HI Mab Îelonged was an equillibrium constant ka of 2.4 X 10 1/m. The HI MAb partially purified on DEAE-cellulose using NaCI gradient (12). This partially purified preparation was used for evaluation of immunoreactivity in the present study.
SP2/0
mouse
myeloma cells
Specificity of HI globulins (h-Iqs).
MAb
were
:
Cross
reactivity
to
h-tg,
T,-Ti
an
HYBRIDOMA Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers
Monoclonal Antibodies to Human Thyroglobulin: Evaluation of Immunoreactivity A.A. D.
NARKAR,1
SWAROOP,2
SHAH,1 J. YADAV,2
D.H. and R.
MULHERKAR3
1Radiation Medicine Center, B.A.R.C., c/o Tata Memorial Center Annexe, Parel, Bombay 400 012, India 2Tata Memorial Hospital, Parel, Bombay 400 012, India 3Cancer Research Institute, Parel, Bombay 400 012, India -
-
-
ABSTRACT We have earlier reported production and characterization of monoclonal antibodies (MAbs) to human thyroglobulin (h-tg).In the present study HI MAb was evaluated for its immunoreactivity towards different forms of tg and various human thyroid tumours. The specificity of HI MAb was validated by the absence of cross reaction with tri-iodothyronine (T ) Thyroxine (T ) and human gamma globulins. Sodium-dodicyl-sulphate polyacrylamide gel electrophoresed (SDS-PAGE) immunoblot of h-tg on the nitrocellulose membrane revealed multiple immunoreactive bands on reaction with polyclonal antibody (PAb) in comparison with total lack of reactivity with HI MAb. The absence of immunoreactivity of HI MAb was demonstrated with SDs treated, Dithiothreitol (DT) treated and heat denatured tg using dot immunobinding technique. However, the HI MAb was able to react with tg treated with unfolding agents such as urea and guanidine hydrochloride. All the treated forms of tg were equally recognized by PAb. The immunoreactivity of the oxidized/reduced tg towards HI MAb was markedly reduced (60.0\) as compared to that obtained with native tg. It appears that HI MAb is directed towards conformational epitope involving sulphydryl bonds.
Immunohistochemically, a comparable immunoreactivity between PAb and MAb was thyroid tissues, follicular thyroid tissues, Hurthle cell and poorly differentiated thyroid tumor tissues using immunoperoxidase staining. The sections from papillary carcinoma tissue (thyroid as well as metastatic lymph node) exhibited intense immunoreactivity with PAb. Thyroglobulin present on these sections was not recognized by HI MAb. observed with normal tissues carcinoma
Nonetheless, HI MAb was able to detect tg in follicular differentiation wherever present. The absence of immunoreactivity of HI MAb in papillary carcinoma strongly suggests that this neoplasm produces tg which is antigenically different from the protein present in the normal tissue. The reactivity of HI MAb with metastatic lymph node of an unknown primary origin suggests its usefulness in the identification of prevalent metastasis of differentiated thyroid carcinoma other than
papillary type.
803
Hybridoma 1992.11:803-813. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/09/15. For personal use only.
INTRODUCTION
Thyroglobulin (tg) has been a crucial biomolecule in the study of thyroid diseases because of its vital role in the thyroid hormone(s) synthesis and This molecule has been extensively investigated biochemically in normal storage. and pathological conditions. Immunologically, tg had been considered as a secluded antigen and its exposure was believed to provoke production of auto-antibodies. It has now been shown to be a normal physiological component of the serum and is present in the detectable quantities in about 80% of the normal healthy subjects (1). Serum tg level is raised in most of the thyroid disorders and hence is of limited value in the differential diagnosis of tumours. Nonetheless, it is a reliable tumour marker in the management of proved patients of differentiated thyroid carcinoma. Since the availability of monoclonal antibodies (MAbs) as a powerful tool for elucidation of structure and function of proteins, several laboratories have
produced MAbs to tg to use as a probe for structural analysis (2-6), to indentify epitopes involved in autoimmune processes (7,8) and for localisation of tg in thyroid tumours (9-11) We have earlier reported the production and characterization of MAbs to human its in in-vitro and usefulness immunoassay as well as tg (h-tg)
immunohistochemistry (5). In the present immunoreactivity of HI MAb towards modified tg
paper
and
we
further report the human thyroid
different
tumours.
MATERIALS & METHODS
Production of MAbs to h-tq: Human
and
tg
MAbs
to
h-tg
were
prepared
as
described earlier (5). Briefly,
fused with spleen cells of BALB/c mouse immunized with h-tg. The supernatants were screened with indirect ELISA for the presence of anti-h-tg antibodies. Of the seven stable clones secreting specific antibodies, HI MAb was used in the present to IgG subclass with studyf HI Mab Îelonged was an equillibrium constant ka of 2.4 X 10 1/m. The HI MAb partially purified on DEAE-cellulose using NaCI gradient (12). This partially purified preparation was used for evaluation of immunoreactivity in the present study.
SP2/0
mouse
myeloma cells
Specificity of HI globulins (h-Iqs).
MAb
were
:
Cross
reactivity
to
h-tg,
T,-Ti
an
