Horseradish Peroxidase Complex Research Articles

Cerebellar cortical and nuclear afferents from the feline locus coeruleus complex

The origin and distribution of cercbellar cortical and nuclear afferents from the locus coeruleus complex (the nucleus locus coeruleus. the nucleus subcoeruleus. the medial and lateral subdivisions of the parabrachial nucleus and the Ko¨lliker Fuse nucleus) have been studied by means of retrograde transport of the wheat germ agglutinin horseradish peroxidase complex in the cat. Cerebellar cortical depositions of the tracer were made by pressure injections, while nuclear depositions were made by implanting the tracer in crystalline form. The projection is bilateral with an ipsilateral preponderance. It reaches all the cerebellar nuclei as well as vermal, intermediate and lateral parts of the cerebellar cortex. The highest cell counts were made after tracer depositions in vermal and intermediate parts of the cerebellum. The projection in the cat has a more widespread origin than previously reported. It originates mainly within the nucleus locus coeruleus and the parabrachial nucleus (especially in its lateral subdivision), hut retrogradely labelled neurons were also found in nucleus subcoeruleus. the Ko¨lliker Fuse nucleus and the A4 cell group. The cells of origin were of different shapes, but usually had a maximum diameter of the cell body between 15 and 30 μm. Cerebellar efferent axons passing through the locus coeruleus complex were anterogradely labelled following implants in all the cerebellar nuclei, but definite terminal labelling was not observed within the nuclei of the locus coeruleus complex.

Simultaneous detection of antibody to the human immunodeficiency virus and of the surface antigen of hepatitis B virus in human serum.

The human immunodeficiency virus (HIV) is the etiologic agent of AIDS. This retrovirus destroys selected classes of T lymphocytes, which results in progressive, severe immunodeficiency. Populations at high risk for AIDS are also at risk for hepatitis B [1]. ELISAs that detect the surface antigen of hepatitis B virus (HBsAg) and antibody to HIV are routinely used to monitor donated blood and blood products to prevent transmission of these serious infections [2, 3]. A single test that could detect HBsAg and antibody to HIV simultaneously would be desirable for the clinical laboratory. In this preliminary report, we describe a singlewell immunoassay with specificity for these two markers. The HIV component of the assay uses synthetic oligopeptides unique to the env region of HIV [4,5]. The antibody bound to the env oligopeptides (anti-HIV env) on the solid phase is then detected by a complex of env peptide, biotin, streptavidin, and horseradish peroxidase (HRPO). The env peptide assay was combined with a sandwichtype immunoassay to detect HBsAg. HBsAg bound to a monoclonal antibody on the solid phase is detected with a noncompeting HRPO-conjugated monoclonal antibody to HBsAg. This combination assay accurately detected HIV antibody and HBsAg in several panels of sera that had been characterized with separate licensed procedures for each of these components. Using antigen standards to quantitate sensitivity, the combination assay detected 1 ng of HBsAg.

Low-temperature magnetic circular dichroism studies of the photoreaction of horseradish peroxidase compound I.

Horseradish peroxidase (HRP) compound I is photolabile at all temperatures between room temperature and 4 K. The photoredox reaction has been studied in frozen glassy solutions by using optical absorption and magnetic circular dichroism spectra following photolysis of HRP compound I with visible-wavelength light at 4.2 and 77 K. The photochemical process is characterized as a concerted two-electron transfer reaction which results in the conversion of the Fe(IV) heme pi-cation radical species of HRP compound I into a low-spin Fe(III) heme species. This reaction occurs even when photolysis is carried out at 4.2 K. Spectra recorded between 4.2 and 80 K for the low-spin ferric hydroxide complex of HRP closely resemble the data measured for the photochemical product. The proposed mechanism for the photoreaction is (formula; see text) No evidence is found for the formation of an Fe(II) heme at these temperatures.

Fc? receptors on keratinocytes in psoriasis

Epidermis in cryostat sections of skin biopsy specimens from patients with psoriasis and from healthy individuals bound bovine erythrocytes (E) sensitized with rabbit IgG antibodies (A)(EA). No binding occurred using E or E sensitized with IgM or F(ab')2 fragments of IgG. The binding of EA was inhibited by human IgG and by Fc fragments of IgG, whereas human IgA, IgM, albumin, and F(ab)2 fragments of IgG did not inhibit the binding, indicating the presence of receptors for the Fc part of IgG (FcR). EA bound mainly to stratum spinosum and most strongly above FcR-positive cell infiltrates in dermal papillae. The binding of EA to sections from patients with active psoriasis was stronger than to sections from patients with stationary psoriasis vulgaris. Sections of unaffected skin from patients with psoriasis and healthy individuals also bound EA, but the binding was weaker than to sections of psoriatic lesions. The receptors were sensitive to periodic acid, formaldehyde, and heat. Using immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP, the receptors were localized to the outer aspect of the keratinocytes and to the inflammatory cells in the microabscesses. The strongest binding occurred in the same areas which adhered EA most strongly. FcR on dendritic epidermal cells could not be demonstrated in situ. A monoclonal antibody against FcR also stained the outer aspect of most keratinocytes throughout the epidermis. FcR on keratinocytes support the assumption that these cells contribute to immune reactions in the skin.

Synthesis and hybridization of a series of biotinylated oligonucleotides.

A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin substituents were subsequently attached to the allylamino sidearms by reaction with N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester. These oligomers were hybridized to target DNA immobilized on microtiter wells (ELISA plates), and were detected with a streptavidin-biotinylated horseradish peroxidase complex using hydrogen peroxide as substrate and o-phenylenediamine as chromogen. We found that the sensitivity of detection of target DNA by biotin-labeled oligonucleotide probes was strongly dependent upon the position of the biotin label. Oligonucleotides containing biotin labels near or off the ends of the hybridizing sequence were more effective probes than oligonucleotides containing internal biotin labels. An additive effect of increasing numbers of biotin-dUMP residues was found for some labeling configurations.

Electroblotting and immunohistochemical staining for identification of platelet antibodies.

The Western blot procedure has been adapted for use with a biotinylated antiglobulin reagent and a horseradish peroxidase complex (Vectastain ABC) to determine specific sites of antigen binding and permit discrimination between various types of platelet antibodies. Monospecific anti-PlA1 antisera or sera containing mixtures of multispecific HLA and unidentified platelet specific antibodies were tested with PlA1 phenotyped platelets. Using monospecific anti-PlA1, one intense band with relative molecular weight (Mr) of 88,000 and corresponding to glycoprotein IIIa was seen with the PlA1+ platelets. With mixtures of antibodies, reactions were seen with platelet specific antibodies without interference from the HLA antibodies; with one serum a band of Mr approximately 135,000 was identified with Baka+, but not Baka- platelets, indicating the presence of an anti-Baka in the serum. The location of the Baka antigen corresponded to the area for GP IIb. With another serum, a band of Mr 88,000 was revealed with PlA1- and some PlA1+ platelets suggesting the presence of an anti-PlA2. Two additional bands of Mr 160,000 and 200,000 were present on all preparations including autologous controls, probably due to the presence of non-specific IgG. Thus, immunohistochemical staining is readily adaptable to the Western blot technique, and antibodies to platelet-specific antigens can be easily differentiated from HLA antibodies.

Substance P-immunoreactive nerves in the rat kidney

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize substance P-immunoreactive (SPI) nerves in the rat kidney. Rats were perfused with 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24–48 h, the kidneys were sectioned with a Vibratome at 200 or 300 μm and incubated in the primary antiserum for 18 h at room temperature. A dense plexus of SPI nerves innervates the rat renal calyx. A small proportion of intrarenal SPI axons innervates interlobular arteries and afferent arterioles, but most perivascular SPI axons terminate on interlobar and arcuate arteries. The densest plexuses are located on segments of interlobar arteries near the hilus of the kidney. Some of these axons probably are nociceptive; others may be chemo- or baroreceptive.

Vasoactive intestinal peptide-immunoreactive nerves in the rat kidney.

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize vasoactive intestinal peptide-immunoreactive (VIPI) nerves in the rat kidney. Rats were perfused with 4% paraformaldehyde or 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24-48 hours, the kidneys were sectioned with a Vibratome at 200 or 300 micron and incubated in the primary antiserum for 18 hours at room temperature. A sparse plexus of VIPI nerves innervates the rat renal calyx. Some VIPI nerves innervate interlobar arteries and each succeeding segment of the arterial tree including afferent arterioles, but most innervate arcuate and interlobular arteries. VIPI axons do not innervate each arcuate artery or each interlobular branch of an arcuate artery with equal density. Although some axons follow each interlobular branch, most form a dense plexus on only one or two branches. Therefore, most VIPI nerves in the rat kidney innervate a restricted segment of the renal arterial tree. These nerves may be efferent and may selectively dilate arcuate and smaller arteries, or they may be afferent and may sense local changes in mechanical or chemical parameters.

Transport of immune complexes from the subcapsular sinus into the lymph node follicles of the rat

To study the mode of transport of immune complexes from the subcapsular sinus into the follicles of draining popliteal lymph nodes, horse radish peroxidase (HRP)-anti HRP was injected in rat footpads. Within six minutes, complexes were already present in the subcapsular sinus freely or attached to the plasma membrane of different types of cell including cells forming the stroma. A few minutes later, complexes were also seen in the deeper part of the outer cortex, after two hours they had reached the periphery of the follicles. They were always seen scattered between lymphoid and non-lymphoid cells. After one day, complexes were present on well-developed follicular dendritic cells. After injection of HRP, no localization of this antigen was observed in the deeper part of the outer cortex including the follicles. These results strongly suggest that HRP-anti HRP complexes are passively transported through the outer cortex into the follicles where they are trapped and retained by follicular dendritic cells.

Demonstration of Fcγ receptors on human peripheral nerve fibres

Cryostat sections of human peripheral nerves adsorbed sheep erythrocytes (E) sensitized with rabbit IgG antibodies (A) (EA). No adsorption occurred using unsensitized E or E sensitized with IgM or F(ab′) 2 fragments of IgG. The binding of EA was inhibited by IgG and by Fc fragments of IgG, but not by IgA, IgM, F(ab′) 2 fragments of IgG or albumin, and thus indicated the presence of receptors for the Fc part of IgG (FcR). IgG 1 and IgG 3 subclasses inhibited the haemadsorption. IgG 4 was less inhibitory than IgG 1 and IgG 3, whereas IgG 2 did not inhibit the binding of EA. The receptors were sensitive to periodic acid, formaldehyde and heat. FcR were present in sections of both unmyelinated and myelinated nerves, indicating that the binding was not to myelin. FcR were also demonstrated in sections of fetal peripheral nerves at a gestational age of approximately 21 weeks. Using immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP and a monoclonal antibody against FcR, the receptors were shown on the nerve fibres, probably on the Schwann cell membrane (neurilemma).

Two types of conformers with distinct Fe-C-O configuration in the ferrous CO complex of horseradish peroxidase. Resonance Raman and infarared spectroscopic studies with native and deuteroheme-substituted enzymes.

The presence of at least two types of conformers in the ferrous CO complex of horseradish peroxidase has been demonstrated with the use of native and deuteroheme-substituted enzymes. Type I conformers, predominant in acidic pH, exhibited both an Fe-CO stretching and an Fe-C-O bending Raman line together with an infrared C-O stretch band below 1920 em-1. On the other hand, type II conformers, dominant species in alkaline pH, showed only an Fe-CO stretching Raman line with the C-O stretch above 1930 cm-1. They were interconvertible either by the changes in pH or by the binding of benzhydroxamate, a substrate for the enzyme. The pKa value for the pH-dependent interconversion of CO complex of deuteroheme-substituted enzyme was 8.3. These findings were interpreted to mean that the bound CO molecule in type I conformers was more tilted over the heme-plane than that in type II conformers. A steric hindrance by the bound substrate or the protonated form of a distal amino acid residue, presumably of histidine, is considered to be the cause for the isomerization. By summarizing present and previous data on the vibrational frequencies of heme-carbonyl complexes, we found that there are inverse-linear relationships between the square of Fe-CO and that of C-O stretching frequencies, while squares of Fe-CO stretching and Fe-C-O bending frequencies were linearly correlated with each other. Also found is that the dissociation rate constant of CO molecule from heme-carbonyl complexes is a linear function of the Fe-CO stretching frequency. The significance of these results is discussed.

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase.

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.

The oscillator strengths of hemoproteins. Their relation to the coordination structure and magnetic susceptibility.

The oscillator strengths of hemoproteins in the light frequency range of 1.11 X 10(4) to 3.23 X 10(4) cm-1 (wavelength range of 900 to 310 nm) were measured by means of computer-assisted spectrophotometry. The obtained values of oscillator strength per molar heme ranged from about 1.4 to 2.2. By comparing the oscillator strength values of the ferric and ferric cyanide-bound forms of hemoproteins and also the values of low molecular weight ferric heme complexes, it was found that the oscillator strength was lower for those hemoproteins whose heme was coordinated with strong field ligands. It was also found that the hemoproteins showing a smaller pH-dependent change in the carbon monoxide-difference spectrum had lower oscillator strengths. The following linear relation was observed, with various ligand complexes of bovine methemoglobin, horse metmyoglobin, and ferric horseradish peroxidase, between the oscillator strength (f) determined in the present study and the respective magnetic susceptibility (10(6) X chi 20 degrees M) values in the literature: f = A (10(6) X chi 20 degrees M) + B. The values of constants A and B in the equation were estimated for horseradish peroxidase, methemoglobin, and metmyoglobin. On varying the temperature in the range of 0 to 40 degrees C, the oscillator strength of the metmyoglobin-azide complex changed in parallel with the change in the spin state. Taking advantage of the fact that fluoride complexes of many hemoproteins show 10(6) X chi 20 degrees M values close to 14,500 and also that the values of intersection B are around 86.4% of the respective values of the fluoride complexes of ferric horseradish peroxidase, methemoglobin, and metmyoglobin, an empirical equation was evolved for the calculation of an approximate 10(6) X chi 20 degrees M value from the f value of a given complex (fobs) and that of the fluoride complex (fF) of a hemoprotein. The approximate magnetic susceptibilities of various ligand complexes of bovine lactoperoxidase could be thus calculated with the equation. The oscillator strengths of ferrous hemoproteins were also investigated and ligand-dependent regular changes were found.

Immunohistochemistry of primary gastrointestinal lymphomas: a study of 76 cases.

A retrospective study of 76 primary gastrointestinal lymphomas utilizing an avidin: biotinylated horseradish peroxidase complex (ABC) technique demonstrated 22 B-cell lymphomas, including two associated with alpha-heavy chain disease. Seven cases were classified as true histiocytic lymphomas based on a positive reaction for one or more of three histiocytic enzyme markers utilized, predominantly alpha-1-antitrypsin and alpha-1-antichymotrypsin. However, in 20 cases, an intense admixture of reactive histiocytes was noted and these cells stained preferentially for the enzyme, lysozyme. Twenty cases, which stained for both kappa and lambda light chains and positively or negatively for albumin, could not be classified and 27 cases failed to stain with any of the antisera utilized.

Comparison of heme environments and proximal ligands in peroxidases and other hemoproteins through carbon-13 nuclear magnetic resonance spectroscopy of carbon monoxide complexes

Carbon-13 nuclear magnetic resonance signals for the carbon monoxide ligand in ferrous complexes of horseradish peroxidase, lactoperoxidase, and chloroperoxidase are located respectively at 209.1, 208.3, and 200.8 parts per million from the tetramethylsilane reference. On the basis of previous hemoprotein and model compound studies these resonance positions are consistent with coordination of a proximal histidine ligand in horseradish peroxidase and lactoperoxidase, and coordination of a cysteinyl mercaptide ligand in chloroperoxidase. Carbonyl chemical shift values for acidic and basic horseradish peroxidase isoenzymes are very similar.

Improved immunocytochemical staining through the use of Fab fragments of primary antibody, Fab-specific second antibody, and Fab-horseradish peroxidase.

A modification of the unlabeled antibody method of immunocytochemistry is described here that offers increased immunoreagent penetration and greatly reduced background staining. The method involves the following alterations to the conventional technique; the use of Fab fragments of primary antibody, rather than whole immunoglobulin G (IgG) or serum; the use of a second, or link, anti-rabbit IgG serum that is Fab fragment-specific, rather than directed against the whole rabbit IgG molecule; the use of the Fab--horseradish peroxidase complex described by JR Slemmon, PM Salvaterra, and K Saito (J Histochem Cytochem 28:10, 1980), rather than peroxidase--antiperoxidase (PAP). Steps 2 and 3 alone brought about a significant reduction in background staining, but did not increase the depth of immunostaining, as compared to the PAP technique. When all three steps were combined, however, background staining was further reduced, and there was a five- to tenfold increase in the depth of immunostaining. These readily made changes should be useful in preembedding immunocytochemistry whenever enhanced reagent penetration is required.

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears.

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).

Fluid‐phase and adsorptive endocytosis in ciliated epithelial cells of the rat ductuli efferentes

Ciliated cells of the ductuli efferentes show at their apex a discrete endocytic apparatus composed of small vesicles connected to or subjacent to the apical plasma membrane, small apical membranous tubules, and pale multivesicular bodies. Deeper in the cytoplasm, there are acid phosphatase-positive denser, multivesicular bodies and secondary lysosomes showing an electron-dense cortex and a crystalline, paler stained core. Cationic ferritin and concanavalin A-ferritin used to demonstrate adsorptive endocytosis, when injected into the rete testis, rapidly reached the lumen of the ductuli efferentes. At 1 min after injection, these tracers were seen bound to the apical plasma membrane of ciliated cells and within small endocytic vesicles and by 5 min in narrow apical tubules. At 15 and 30 min after injection, the tracers appeared in pale multivesicular bodies while at 1 hr they were found within dense multivesicular bodies. At 2 hr and longer time intervals these tracers accumulated within secondary lysosomes. Native ferritin, concanavalin A-ferritin in the presence of alpha-methyl-D-mannoside, and horseradish peroxidase or albumin-colloidal gold complexes were used to analyze fluid-phase endocytosis. At various intervals following their injection into the rete testis, these tracers presented a distribution identical in all respects to that described for cationic ferritin and concanavalin A-ferritin. In the present work, none of the above tracers were transported to the abluminal aspect of the ciliated cells. These cells, like the nonciliated epithelial cells of the ductuli efferentes are thus involved in adsorptive as well as in fluid-phase endocytosis.

Role of immunocytochemistry in diagnosis of prostatic neoplasia by fine needle aspiration biopsy

The efficacy of immunocytochemical staining for prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) was studied in aspiration biopsy specimens from 19 patients. Eighteen patients had prostatic carcinoma and one had hyperplasia of prostate. Specimens were obtained from both the primary tumors and metastatic sites. Immunoperoxidase staining was performed on alcohol-fixed cytology smears (some prepared up to 9 years previously) using appropriate antisera followed by an avidin-biotinylated horseradish peroxidase complex. Results were scored according to the percentage and intensity of positively stained malignant cells. Corresponding histologic specimens were stained and scored in a similar fashion. Correlations were made between the staining characteristics of the tumor markers and grade of tumor, using the University of Texas M.D. Anderson Hospital classification of prostate carcinoma. Overall there was good correlation between cytologic and histologic specimens for the presence of PSA and PSAP, although metastases tended to show fewer positively stained cells than the primary tumor. There was no relationship between tumor grade and percentage of positively stained cells. Ninety-three percent of aspirated primary and secondary prostatic tumors stained positively for PSAP compared with 81% for PSA. In one of 3 patients, negative staining of neoplastic cells by both PSAP and PSA was helpful in confirming the existence of a second primary tumor.

Substance P-immunoreactive fibers in the trigeminal sensory nuclei of snakes with infrared sensitivity

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize substance P-immunoreactive (SPI) nerves in the rat kidney. Rats were perfused with 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24–48 h, the kidneys were sectioned with a Vibratome at 200 or 300 μm and incubated in the primary antiserum for 18 h at room temperature. A dense plexus of SPI nerves innervates the rat renal calyx. A small proportion of intrarenal SPI axons innervates interlobular arteries and afferent arterioles, but most perivascular SPI axons terminate on interlobar and arcuate arteries. The densest plexuses are located on segments of interlobar arteries near the hilus of the kidney. Some of these axons probably are nociceptive; others may be chemo- or baroreceptive.