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.

The 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.     

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

Summary The 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 -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.     

Immunohistochemistry of acid phosphatase in the human prostate: normal and pathologic. Cytochemistry and biochemistry of acid phosphatases II

Three different antisera against human prostatic acid phosphatase were used for direct and indirect immunohistochemical demonstration of acid phosphatase in paraffin sections of infantile and adult normal, hyperplastic and carcinomatous prostatic tissue. All antisera were prepared in rabbits. Antiserum A was prepared from highly purified acid phosphatase extracted from autopsy specimens. Antiserum B was a concentrate of a commercial antiserum used in radioimmunoassay and was prepared from purified extracts of human seminal fluid. Antiserum C was a peroxidase-conjugated antiserum prepared from purified extracts of human seminal fluid. The specificity of the three antisera was compared using different immunohistochemical methods and tissues. It was comparably high in all three antisera which gave only slightly different staining results in prostatic tissue. The staining results in prostatic carcinoma were only dependent on the titer of the respective antiserum. Carcinomas with a cribriform growth pattern showed variable staining, but always had a positive immunoreactions, provided the titer of the antiserum was sufficiently high. Striking differences were observed in metaplastic, atrophic and hyperplastic prostatic epithelium. The most intense reaction was observed in atrophic glands: it was much less intense in hyperplastic and normal epithelium and negative or slightly positive in metaplastic epithelium.     

Fine needle aspiration cytology of prostatic adenocarcinoma metastatic to the lung confirmed by the immunoperoxidase technique

Two cases of prostatic adenocarcinoma metastatic to the lung are presented. The prostatic origin of the metastatic carcinomas was confirmed by immunocytochemical demonstration of prostate-specific antigen and prostatic acid phosphatase in cellular samples obtained by fine needle aspiration of the pulmonary lesions.     

Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique

The use of immunoperoxidase techniques was investigated in 21 fine needle aspiration (FNA) cytology smears that had been previously stained by the Papanicolaou technique. The retrospectively selected slides were destained before applying the immunostain, utilizing antisera to calcitonin, prostatic acid phosphatase (PrAP), prostate-specific antigen (PSA), alpha-lactalbumin (AL), S-100 protein (S-100), carcinoembryonic antigen (CEA), common leukocyte antigen (LA), epithelial membrane antigen (EMA) and alpha-fetoprotein (AFP). Positive results were obtained with six of nine small-cell carcinomas of the lung stained with EMA, all three colonic carcinomas stained with CEA, one of two prostatic carcinomas stained with PSA and PrAP, one of two lymphomas stained with LA and the one medullary thyroid carcinoma stained with calcitonin. Negative staining results were observed in the one melanoma stained with S-100, the two breast carcinomas stained with AL and the one hepatocellular carcinoma stained with AFP. These results indicate that immunostaining can be a helpful diagnostic tool in diagnosing some fine needle aspirates using smears previously stained with the Papanicolaou stain.     

The localization of enzymes in tissue sections by immuno-histochemistry. Conventional antibody and mixed aggregation techniques

Synopsis Methods for detecting enzymes in tissue sections by antibody techniques are reviewed. In all these techniques, sections are first incubated with antibody. The bound antibody is visualized in one of four ways: identifying a label such as fluorescein linked to the antibody; using a labelled anti-antibody; employing complement and labelled anti-complement; or making use of a mixed aggregation immuno-cytochemical method.The last technique consists of three steps. A section is first incubated with antiserum, and secondly with the soluble enzyme under investigation. Thirdly the desired enzyme is stained using a conventional cytochemical method. The method is specific since, for example, the soluble enzyme used in the second step can bind only to antigenic determinants which are identical to those of the enzyme localized in the tissue. Thus purification of antigen and antibody sources is simplified, and chemical modifications of the antigen and antibody are avoided.Antibody also acts as a selective fixative for tissue antigen. It will inhibit the catalytic activity of its antigen and, in this way, permit the enzyme activity arising after the reaction of tissue enzyme-antibody complex with soluble enzyme to be amplified selectively. The mixed aggregation immuno-cytochemical technique has been used successfully with membrane-bound enzymes and cytoplasmic enzymes and for the demonstration of catalytically inactive enzyme precursors.     

Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.     

Differentiation of epithelial cells in human jejunum: Localization and quantification of aminopeptidase,alkaline phosphatase and aldolase isozymes in tissue sections

Summary Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.     

Prostatic acid phosphatase immunoperoxidase staining of cytologically positive effusions associated with adenocarcinomas of the prostate and neoplasms of undetermined origin

An immunoperoxidase staining technique was employed in an effort to demonstrate prostatic acid phosphatase in sections of the effusion cell blocks in a retrospective investigation of the incidence of malignant prostatic cells in body cavity effusions in 33 patients with histologically confirmed prostatic cancer. An attempt was also made to identify the prostate as a possible anatomic site of origin in 26 patients with an unknown primary but with cytologically positive fluids. Neoplastic cells were identified in the effusion specimens in 21.2% of the patients with confirmed prostatic cancer; the sources, however, were either primary or metastatic carcinomas of nonprostatic origin. None of the cytologic specimens in this study demonstrated a positive prostate-specific acid phosphatase staining reaction, as did the prostatic metastases to the lungs used as controls.     

Isolation of anti-carcinoembryonic antigen (CEA), specific immunoglobulins and their use in immunocytochemical and enzyme-immunoassays (ELISA)

Anti-carcinoembryonic (CEA) polyclonal antibodies in sheep and rabbits were raised using purified CEA from acid extracts of human colon adenocarcinoma. CEA was purified by gel filtration on Sepharose 4B CL and chromatography on DEAE-Sephadex A50. The antiserum was adsorbed with human serum and perchloric acid extract from normal colon. Anti-CEA IgG was purified from monospecific antiserum by ion-exchange chromatography and its specificity was tested on cryostat sections from colon adenocarcinoma by the indirect immunoperoxidase technique. The specific reaction was compared with that obtained by using a similar technique and two CEA specific monoclonal antibodies. An anti-CEA IgG peroxidase conjugate was obtained allowing to establish a "sandwich" ELISA-CEA system with two antibodies. CEA determinations were made in a group of 15 normal controls (mean value 4.8 +/- 0.4 ng/ml) and in 30 colorectal tumor patients (mean value 26.6 +/- 2.15 ng/ml). The anti-CEA antibodies are proven useful in immunocytochemical and ELISA techniques and may be further used in radioimaging of tumors.     

Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique

A simple direct enzymeimmunoassay (EIA) on microtiter plates for plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label (EIA-HRP) is described and compared with an identical EIA procedure which employed alkaline phosphatase (AP) as the enzyme label (EIA-AP). The assays used antiserum raised against progesterone-7-carboxyethlthioether-BSA in rabbits. Both systems were further compared with the conventional direct progesterone radioimmunoassay (RIA) in regular use. The enzymes HRP and AP were coupled to progesterone-6 beta-hydroxy-hemisuccinate by a mixed anhydride method. While the precision of EIA-HRP was comparable to RIA, the sensitivity in terms of the lowest detection limit obtained in EIA-HRP was about 10 times better than that seen in RIA. Progesterone estimates from plasma samples in EIA-HRP showed good correlation (r = 0.94) with the RIA values and the levels measured in the two systems were identical. Progesterone estimates from plasma samples in EIA-AP were at least three times higher than those obtained by either EIA-HRP or RIA. Thus, only the EIA-HRP but not the EIA-AP was suitable for the reliable direct measurement of progesterone in plasma.     

Immunological characterization of human acid phosphatase gene products

The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10–20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not cross-react with the erythrocyte enzyme. (10–20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.This study was supported by DHHS Research Grant GM 27003 from the U.S. National Institute of General Medical Sciences and by Grant SFB-104 from the Deutsche Forschungsgemeinschaft.     

Antigenic analysis of Chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-LGV-specific antigen.

Two-dimensional immunoelectrophoresis was utilized to study precipitins in hyperimmune rabbit serum made against chlamydiae and from patients with chlamydial infections. An antigen of Triton X-100-solubilized L2/434/Bu organisms with an electrophoretic mobility of 0.65 relative to bovine serum albumin at pH 8.6 was excised from the agarose gel of electrophorograms as antigen-antibody complexes and used to immunize rabbits. A monospecific antiserum to antigen 0.65 was obtained that reacted with Trachoma-LGV strains L2/434/Bu, B/TW-5/OT, and K/UW-31/Cx, but not with the mouse pneumonitis (Nigg) strain or the psittacosis strain meningopneumonitis (Cal-10). The Trachoma-LGV specificity of antigen 0.65 was further shown by indirect immunofluorescence straining with the monospecific antiserum of chlamydial inclusions in infected HeLa cells. Precipitins with a specificity for antigen 0.65 were indentified in 15 of 18 sera from patients with diagnosed Chlamydia trachomatis infections LGV, trachoma, nongonococcal urethritis, and nongonococcal cervicitis by using monospecific antiserum to antigen 0.65 in the peak suppression test. Thus, antigen 0.65 appears to be a Trachoma-LGV-specific antigen that has considerable promise for serodiagnosis.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

In situ embedding method of cells grown in beem capsules for immunoelectron microscopic studies of oncornaviruses.

A technique of in situ embedding of cells grown in BEEM capsules has been devised for immunoelectron microscopic studies of oncornaviruses. As compared to other immunoelectron microscopic procedures, this technique is less time and reagent-consuming. The quality and specificity of this method were tested on well-characterized mouse mammary tumor virus (type B virus) and murine sarcoma virus (type C virus particles). This method gave good results in labeling of the virus particles with ferritin or peroxidase in the cells of mouse tissue cultures. In an application of this method, peroxidase labeling of type B virus particles was obtained in frozen sections of normal prostatic tissues of C3H/Dm and A/Dm strain mice treated with rabbit antiserum to mouse mammary tumor virus from A/Dm strain mouse milk. These results indicate that this method is useful and reliable for immunoelectron microscopy studies of oncornaviruses in tissue culture cells and also in frozen sections of tissues.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

酶标抗原火箭免疫电泳法及其在蛇毒抗原成分含量测定中的应用

为解决目前蛇毒成份检测困难以及寻找一种可用于蛇伤快速鉴别诊断的方法,本研究建立了酶标抗原火箭免疫电泳法。本法结合了酶标法灵敏度高和火箭电泳操作简单的特点,对待测的同种抗原标记辣根过氧化物酶(酶标抗原),检测时把微量的酶标记抗原掺入被测样品中,在含多价抗血清的免疫电泳板进行电泳。最后用辣根过氧化物底物3.3′-二氨基联苯胺在板上直接显色,显色后观察到的免疫沉淀线高度与被测抗原含量呈正相关(r=+0.98)。采用本方法检测眼镜蛇毒细胞毒素和限镜王蛇毒 L-氨基酶氧化酶,细胞毒素的最低检测浓度为110ng/ml,氨基酸氧化酶为60ng/ml。比单向火箭免疫电泳法灵敏度高30倍。用同一原理的酶标记抗原融合电泳法监测L-氨基酸氧化酶的层析分离时,比酶活性法检测灵敏度高20倍。全部检测只需30分钟。初步观察商品眼镜蛇毒抗血清可对蛇毒15种成分产生免疫沉淀线,眼镜蛇的细胞毒与同科异科蛇毒的细胞毒素无交叉免疫反应。因此,采用本法可以确定不同蛇种蛇毒中所含的特异性抗原,获得蛇伤鉴别诊断的依据。     

Preparation of monospecific antisera to IgA in laboratory animals using cascade immunization without preliminary isolation of the antigen

The proposed method of "cascade" immunization for the preparation of monospecific antisera to IgA of the experimental animals (mice, rats, guinea-pigs) requires no isolation and purification of an antigen, since it is based on using precipitation lines containing IgA. The subsequent steps of the method are the following: 1) preparation of a polyspecific antiserum against one of the secretions or IgA-containing serum fraction; 2) preparation of precipitation lines using this antiserum and some other secretions; 3) immunization of other rabbits with these precipitation lines to obtain monospecific anti-IgA antiserum. The antisera obtained do not require any additional absorption except for the removal of anti-IgA antibodies. The method can be used for the preparation of monospecific antisera to any other serum or secretion proteins.     

Immunocytochemistry of epithelial markers in citral-induced prostate hyperplasia in rats.

Immunocytochemical characterization of several epithelial markers using the PAP technique was analyzed during different stages of induced prostatic hyperplasia in rats. Intact adolescent rats (42 days old) were treated with citral (3,7 dimethyl-2,6 octadienal) for 10, 30 and 100 days and their ventral prostate compared to untreated, matched-age animals. Among the epithelial markers studied the prostatic specific acid phosphatase was present in hyperplastic prostates of rats. The immunoreaction showed a fair correlation with the severity of lesion and duration of treatment. The prostatic specific antigen showed equally immunoreactive in both control and treated rats. The hyperplastic and normal rat prostates did not show immunoreactivity towards the other epithelial cell markers such as epithelial membrane antigen, carcinoembrionic antingen and alpha-fetoprotein antisera. It is concluded that prostatic specific acid phosphatase, and to a lesser extent prostatic specific antigen, might represent valuable markers for comparative studies of prostatic hyperplasia in rodents.     

Effects of primary antiserum dilution on staining of "antigenrich" tissues with the peroxidase antiperoxidase technique.

The effect of primary antiserum dilution on staining results with the peroxidase antiperoxidase method were investigated using frozen sections of perfused rat cerebellum and optic nerve. Results comparable to formalin fixed and paraffin embedded tissue were attainable only when low antiserum concentrations were used. Optimal staining of antigen rich tissue, such as frozen sections, with the peroxidase antiperoxidase method required low antiserum concentrations apparently to minimize the binding of both antigen-binding fragments of the bridging antibody to the tissue bound antiserum. It appears that low antiserum concentration insures that sufficient bridge antibody molecules will be only singly bound and thus free to attach the peroxidase antiperoxidase complex.