A. C. Jöbsis

Demonstration of the prostatic origin of metastases. An immunohistochemical method for formalin‐fixed embedded tissue

An indirect immunohistochemical technique is described for identification of the prostatic origin of metastases in formalin fixed, paraffin or paraplast embedded material. A rabbit antiserum against the prostate specific acid phosphatase isoenzyme was developed. The method is applicable with or without previous decalcification. In 30 cases of prostatic carcinoma there was only one negative result, and in 20 cases of metastases from prostatic carcinoma positive results were obtained in every instance. All carcinomas (primary focus or metastasis) of non prostatic origin (55) stained negatively with the developed antiserum. The application and possible limitations of the method are discussed.

The immunohistochemical detection of prostatic acid phosphatase: its possibilities and limitations in tumour histochemistry.

SummaryThe immunological specificity of the Amsterdam rabbit antiserum against human prostatic and phosphatase was studied on paraffin sections of 200 prostatic carcinomas and 330 control tissues using an indirect peroxidase technique. Peripheral blood leucocyte smears were also investigated with a fluorescent technique. In a limited number of cases, the mixed aggregation immunocytochemical method was also applied as post-primary incubation procedure. The diaminobenzidine (DAB) final reaction product of the peroxidase technique, carried out under standard conditions, was quantified in some cases using the Leyden Television Analysis System (LEYTAS) with a built-in standard.A positive reaction was obtained in 96.5% of the prostatic carcinomas. Only 2.1% of the non-prostatic tumour cases (23 types) showed a positive reaction, namely six out of 10 insulomas and one out of 10 carcinoid tumours. The β-cells of the normal islet of Langerhans and the leucocytes in the smears showed a positive reaction. The sensitivity of the peroxidase method, judged subjectively, is not only dependent on the circumstances of fixation, embedding and incubation but also on the degree of tumour differentiation. None of the three prostatic carcinomas studied reached the level of DAB staining intensity shown by the hyperplastic prostatic epithelium.

Human placental steroid sulphatase--purification and monospecific antibody production in rabbits

Human steroid sulphatase was purified 43-fold from placental microsomes using a four step procedure: solubilization with Miranol H2M, Bio-Gel A 1.5m chromatography, column chromatofocusing and Sephadex G-75 chromatography. The purified enzyme that appeared electrophoretically homogeneous was used to immunize rabbits. Protein blotting demonstrated that the resulting antiserum mainly reacted with a polypeptide of 63 000 dalton, which is about the size of placental steroid sulphatase. The antiserum was freed from minor impurities by absorbing it to Sepharose 4B with immobilized antigens prepared from a steroid sulphatase deficient placenta.

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

SummaryThe reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.

Biochemical Investigation to the Reliability of the Histochemical Demonstration of Microsomal Arylsulphatase Activity in Cryostat Sections

SummaryPolyacrylamide gel-electrophoresis was performed with an extract from cultivated skin fibroblasts. Arylsulphatase activity is measured and visualised using the biochemical substrate dehydroepiandrosterone sulphate and the histochemical substrate 6-bromo-2-naphthyl sulphate respectively. The histochemical substrate was hydrolysed at Rf=0.49 and 0.58 while the biochemical substrate was hydrolysed only at 0.49. We conclude that two different microsomal arylsulphatases exist: a sulphatase able to hydrolyse steroid sulphatases (Rf=0.49) and one unable to hydrolyse steroid sulphatases (Rf=0.58). In consequence it is recommended to carry out an electrophoresis experiment after the histochemical investigation, in order to discriminate between these two types of sulphatase.

A new method for the determination of steroid sulphatase activity in leukocytes in X-linked recessive ichthyosis

The diagnosis of X‐linked ichthyosis can now be reliably established by using a non‐radioactive method to detect steroid sulphatase deficiency in leukocytes. This new method yields the same results with leukocytes as with cultured fibroblasts. The second type of microsomal arylsulphatase previously described in cultured fibroblasts is also present in leukocytes.

The nature of placental steroid sulphatase deficiency in man

Human placental steroid sulphatase was partially purified from microsome suspensions of control and steroid sulphatase deficient placentae. After polyacrylamidegel electrophoresis, staining for protein and enzymatic activity revealed that steroid sulphatase from control placenta migrates at Rf = 0.44. In steroid sulphatase deficient microsomes no protein band and only a very faint sulphatase activity band could be detected at Rf = 0.44. Immunoelectrophoresis employing antisera raised against both steroid sulphatase preparations, only gave a protein precipitation line with sulphatase activity when using control placenta microsomes and the antiserum against steroid sulphatase from control placenta. All other placental microsomes-antisera combinations appeared to be negative. Our results strongly suggest that the nature of X-linked steroid sulphatase deficiency is a decreased amount of steroid sulphatase protein.

Comparative investigation of the mixed aggregation immunocytochemical technique and the indirect peroxidase technique for the detection of prostate specific acid phosphatase in paraffin or paraplast sections

SummaryThe results obtained with the indirect peroxidase technique for the identification of prostate specific acid phosphatase in formalin fixed, paraffin or paraplast embedded autopsy material are compared with the results obtained with the mixed aggregation immuno-cytochemical technique. When using a monospecific antiserum the former technique is prefered. However, when a monospecific antiserum is not available, one has to balance the advantages of the mixed aggregation immuno-cytochemical technique against the disadvantages of having to prepare a monospecific antiserum, necessary for the indirect peroxidase technique. Both methods appeared positive in 20 prostatic carcinomas and in 36 metastases of prostatic carcinomas. In the epithelium of the seminal vesicles and in osteoclasts no acid phosphatase could be detected with the antiserum. A comparison of both techniques, as well as different types of preincubation to diminish nonspecific background staining are discussed.

Studies on cross-reacting material to steroid sulphatase in fibroblasts from patients affected by different types of steroid sulphatase deficiency

SummaryImmunologically cross-reacting material to antibodies against steroid sulphatase has not been found in fibroblasts from patients with steroid sulphatase deficiency.

Detection of Antibodies Against Herpes Simplex Viruses in Sera of Patients with Cervical Cancer by Using the Western Blotting Technique

Abstract Routinely prepared nitrocellulose replicas (“western blots”) of electrophoretically separated denatured antigens from uninfected and in vitro herpes simplex virus type 1 or type 2 (HSV-1, −2) infected human fetal lung cells are incubated with reference rabbit antisera, with sera of persons with an established HSV-2 infection and with sera from cervical carcinoma patients. Immobilized immune complexes are visualized by means of autoradiography after incubation with 125I labelled protein A from Staphylococcus aureus. In many cases the western blotting technique provides a rapid means to detect HSV infections and to discriminate between HSV-1 and HSV-2 as the infectious agents. The method seems much faster and reliable than the laborious virus neutralization test.