THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOHISTOCHEMISTRY PREPARATION AND PROPERTIES OF SOLUBLE ANTIGEN-ANTIBODY COMPLEX (HORSERADISH PEROXIDASE-ANTIHORSERADISH PEROXIDASE) AND ITS USE IN IDENTIFICATION OF SPIROCHETES

LUDWIG A. STERNBERGER ¹, PAUL H. HARDY JR. ¹, JOHN J. CUCULIS ¹, and HOWARD G. MEYER ¹

¹ Basic Sciences Department, Medical Research Laboratory, Edgewood Arsenal, Maryland 21010, and Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å. s_20,w, 11.98 x 10^–13; d_20,w, 2.48 x 10^–7; molecular weight by sedimentation velocity, 429,000, and equilibrium, 413,000. Sensitivity and specificity of immunohistochemical staining of spirochetes was about 100- to 1000-fold that of immunofluorescence. The unexpected ratio of PO to anti-PO is presumed to be due to stabilization by the pentagonal shape in which three corners are suspected to be PO and two antibody fragment Fc.

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				R. Munda, Y. Berlatzky, M. Jonung, R. F. Murphy, K. Brackett, S. N. Joffe, and J. W. Alexander Studies on Segmental Pancreatic Autotransplants in Dogs Arch Surg, November 1, 1983; 118(11): 1310 - 1315. [Abstract] [PDF]

				C. Margo, A. Hidayat, J. Kopelman, and L. E. Zimmerman Retinocytoma: A Benign Variant of Retinoblastoma Arch Ophthalmol, October 1, 1983; 101(10): 1519 - 1531. [Abstract] [PDF]

				G. Rowden, E. M. Connelly, and R. K. Winkelmann Cutaneous Histiocytosis X: The Presence of S-100 Protein and Its Use in Diagnosis Arch Dermatol, July 1, 1983; 119(7): 553 - 559. [Abstract] [PDF]

				H. Gharib, H. M. Frey, E. R. Laws Jr, R. V. Randall, and B. W. Scheithauer Coexistent Primary Empty Sella Syndrome and Hyperprolactinemia: Report of 11 Cases Arch Intern Med, July 1, 1983; 143(7): 1383 - 1386. [Abstract] [PDF]

				H. B. BREWER Jr., L. A. ZECH, R. E. GREGG, D. SCHWARTZ, and E. J. SCHAEFER Type III Hyperlipoproteinemia: Diagnosis, Molecular Defects, Pathology, and Treatment Ann Intern Med, May 1, 1983; 98(5_Part_1): 623 - 640. [Abstract] [PDF]

				S.M. Goldin, S.Y. Chan, D.M. Papazian, E.J. Hess, and H. Rahamimoff Purification and Characterization of ATP-dependent Calcium Pumps from Synaptosomes Cold Spring Harb Symp Quant Biol, January 1, 1983; 48(0): 287 - 295. [Abstract] [PDF]

				J. Rossier, D. Liston, G. Patey, M. Chaminade, A.S. Foutz, A. Cupo, P. Giraud, M.P. Roisin, J.P. Henry, P. Verbanck, et al. The Enkephalinergic Neuron: Implications of a Polyenkephalin Precursor Cold Spring Harb Symp Quant Biol, January 1, 1983; 48(0): 393 - 404. [Abstract] [PDF]

				S. Watson, N. Seidah, and M Chretien The carboxy terminus of the precursor to vasopressin and neurophysin: immunocytochemistry in rat brain Science, August 27, 1982; 217(4562): 853 - 855. [Abstract] [PDF]

				S. Watson, H Akil, W Fischli, A Goldstein, E Zimmerman, G Nilaver, and T. van wimersma Griedanus Dynorphin and vasopressin: common localization in magnocellular neurons Science, April 2, 1982; 216(4541): 85 - 87. [Abstract] [PDF]

				A. H. Koeppen and R. J. Cassidy Oligodendroglioma of the Medulla Oblongata in a Neonate Arch Neurol, August 1, 1981; 38(8): 520 - 523. [Abstract] [PDF]

				C. F. Winkler, G. K. Goodman, R. A. Eiferman, and L. T. Yam Orbital Metastasis From Prostatic Carcinoma: Identification by an Immunoperoxidase Technique Arch Ophthalmol, August 1, 1981; 99(8): 1406 - 1408. [Abstract] [PDF]

				N. Halmi, M. Peterson, G. Colurso, A. Liotta, and D. Krieger Pituitary intermediate lobe in dog: two cell types and high bioactive adrenocorticotropin content Science, January 2, 1981; 211(4477): 72 - 74. [Abstract] [PDF]

				J. Bolaffi, S Reichlin, D. Goodman, and J. Forrest Jr Somatostatin: occurrence in urinary bladder epithelium and renal tubules of the toad, Bufo marinus Science, November 7, 1980; 210(4470): 644 - 646. [Abstract] [PDF]

				H Kimura, P. McGeer, F Peng, and E. McGeer Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochemistry Science, May 30, 1980; 208(4447): 1057 - 1059. [Abstract] [PDF]

				T. R. Callihan and E. S. Jaffe Identification of Crystalline Deposits Arch Ophthalmol, February 1, 1980; 98(2): 386 - 386. [Abstract] [PDF]

				R. F. Gagel, M. E. Costanza, R. A. DeLellis, R. A. Norton, S. R. Bloom, H. H. Miller, A. Ucci, and L. Nathanson Streptozocin-Treated Verner-Morrison Syndrome: Plasma Vasoactive Intestinal Peptide and Tumor Responses Arch Intern Med, December 1, 1976; 136(12): 1429 - 1435. [Abstract] [PDF]

				N.-C. A. Chang, S.-I. Hung, K.-Y. Hwa, I. Kato, J.-E. Chen, C.-H. Liu, and A. C. Chang A Macrophage Protein, Ym1, Transiently Expressed during Inflammation Is a Novel Mammalian Lectin J. Biol. Chem., May 11, 2001; 276(20): 17497 - 17506. [Abstract] [Full Text] [PDF]

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