Periodate or glutaraldyehyde for preparing peroxidase conjugates?

Conjugates of horseradish peroxidase with antibodies (anti-human IgG (H + L)) or their Fab' fragments were prepared according to the newest modification of the periodate (P-) method or the two-step glutaraldehyde (G-) method. The conjugates were analysed by gel chromatography and subsequently tested in three different applications. For tissue immunohistochemistry on sections of lupus erythematosus skin, G-conjugates were preferred to polymeric P-conjugates. In ELISA for detection of human antibodies against penicillin P-conjugates were superior to G-conjugates. For the detection of surface Ig on lymphoid cells both types of conjugate were more or less equally suitable. A scheme for the most suitable combinations of method of preparation and field of application is given.     

Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA.

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.     

A comparison of the ability of beta-galactosidase and horseradish peroxidase enzyme-antibody conjugates to detect specific antibodies.

The ability of two different enzyme-antibody conjugates to detect specific antibodies has been compared. beta-Galactosidase was conjugated to antibodies raised against rabbit Fc fragments using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Horseradish peroxidase (HRP) was conjugated to part of the same batch of antibodies using the periodate method. The beta-galactosidase and HRP labels enabled detection of approximately 8 fmoles and 4 fmoles respectively of human growth hormone (HGH) antibodies, when their enzyme activities were measured spectrophotometrically. The detection limit of the beta-galactosidase label was increased 4-fold when a fluorimetric detection system was employed.     

Intracellular immunoglobulins: A comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates

Formalin fixation followed by routine paraffin embedding allows the demonstration of intracellular immunoglobulin by a sandwich technique using either peroxidase or fluorescein isothyocyanate labelled antibody conjugates. The results compared favourably with those obtained using either the fresh frozen or the Sainte-Marie method for preserving tissue immunoglobulins. The formalin-paraffin method with peroxidase-labelled antibody has advantages for routine use. Morphology is excellent, preparations are permanent, and retrospective studies of stored paraffin-embedded tissue are possible.Some of the problems of labelled antibody studies are discussed.     

False-positive results in diagnostic immunohistochemistry are related to horseradish peroxidase conjugates in commercially available assays

False-positive results in diagnostic immunopathology can lead to unnecessary treatments. The purpose of this study was to do a side-by-side comparison of 10 different antibodies commonly used in the clinical laboratory altering only the horseradish peroxidase (HRP) conjugate. The automated Leica BOND-MAX platform was used to study serial sections from 203 tissues including controls compared in a blinded fashion using the HRP conjugates from Leica (Refine HRP), Ventana Medical Systems (Ultraview HRP), and Enzo Life Sciences (Polyview HRP). False-positive results, defined as signal from cases known to not contain the target, were noted in 23 (13%) of 171 cases with the Leica HRP, 62 (36%) of 171 cases with the Ventana HRP, and no cases with the Enzo HRP. Each data set was performed simultaneously allocating 1 tray for each of the 3 different HRP conjugates. HER2/neu analysis from triple-negative breast cancers were scored as positive by immunohistochemistry in 6 (24%) of 25 cases using either the Refine or Ultraview HRP and in 0 of 25 cases with the Enzo conjugate. It is concluded that false-positive results in a wide spectrum of diagnostic immunopathology tests can occur from 13% to 36% of cases with commonly used commercial assays based on the HRP conjugate.     

A light and electron microscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system

Summary The ability of horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) covalently conjugated with HRP to label retrogradely dorsal lateral geniculate nucleus (dLGN) neurons, subsequent to injections of either marker into rat striate cortex, was assessed by counting labelled neurons in the dLGN. Rats injected with either marker in concentrations ranging from 0.1 to 100g/l of HRP either free or coupled to WGA were perfused 24 h later and their brains incubated using the chromagen tetramethyl benzidine. At high concentrations (2–100g/1), comparable numbers of labelled neurons were observed in the dLGN but at low concentrations (0.1–1.0g/1), WGA-HRP labelled 2–5 times more dLGN neurons than did unconjugated HRP. The sugarN-acetylglucosamine, and free WGA added in excess to WGA-HRP, abolished the retrograde labelling of dLGN neurons.In additional rats, which received striate cortex injections of 100g/1 of either free HRP or HRP coupled to WGA, the injection site was studied with electron microscopy after survivals of 30 min to 24 h. Similar organelles in neuronal perikarya, dendrites and axons were labelled by both markers, with the exception that only rats injected with WGA-HRP had labelled GERL in some of their neurons in striate cortex.It was concluded from these studies that: (1) WGA-HRP is a more sensitive retrograde marker than free HRP at low concentrations in the rat visual system; (2) WGA-HRP binds specifically to moieties with terminalN-acetylglucosamine; and (3) WGA-HRP, but not free HRP, is localized to neuronal GERL of striate cortex subsequent to endocytosis.     

Motor innervation of the mole and shrew snout muscles by means of horseradish peroxidase technique

The axonal transport method of the horseradish peroxidase (HRP) was used for defining the anatomical background of the snout movements in 54 Japanese shrew-moles, Urotrichus talpoides, 4 Temminck's moles, Mogera wogura, and 21 big-clawed shrews, Sorex unguiculatus. Either snout muscle or proximal stump of the facial nerve on one side was exposed to HRP. The results obtained from this experimental study were as follows: HRP-containing cells were found either ipsilaterally or bilaterally in the facial motor nucleus in both the moles and shrews. The HRP-containing cells were grouped into 4 subnuclei: ventrolateral, ventromedial, dorsal and accessory. In case of the snout muscle being exposed to the HRP were the HRP-containing cells found in the ventral groups. A HRP-positive fiber bundle was found at the genu of the facial motor root extending toward the median raphe, when the proximal stump of the facial nerve was exposed to the HRP.     

Novel horseradish peroxidase substrates for use in immunohistochemistry

New chromogens expand the colour palette for horseradish peroxidase chromogens used in immunohistochemistry. Tissue staining of cytokeratin with three new cyanine-based chromogens is described. Their use as fluorescent reporters is demonstrated.     

快速溶胶-凝胶法制备纳米级羟基磷灰石

目的以四水硝酸钙和五氧化二磷为原料，无水乙醇为溶剂采用溶胶-凝胶法合成纳米羟基磷灰石。方法利用TG—DTA曲线分析凝胶的特性，利用XRD和FTIR研究干凝胶烧结后的组成变化，并采用TEM观察合成粉体的形貌与尺寸。结果经700℃焙烧可得到最大粒径25nm左右，分散良好的纳米羟基磷灰石。加入聚乙二醇(PEG)作络合剂，可得到粒径25nm左右，尺寸分布均匀的纳米羟基磷灰石。结论采用该溶胶一凝胶法可以合成颗粒大小均匀，分散性好的纳米羟基磷灰石粉体。该法的优点是无需调节pH值，且无需剧烈搅拌和长时间的水解即可成胶，HA的合成周期较短，所得纳米粉体的稳定性好。     

快速溶胶-凝胶法制备纳米级羟基磷灰石

目的以四水硝酸钙和五氧化二磷为原料,无水乙醇为溶剂采用溶胶-凝胶法合成纳米羟基磷灰石.方法利用TG-DTA曲线分析凝胶的特性,利用XRD和FTIR研究干凝胶烧结后的组成变化,并采用TEM观察合成粉体的形貌与尺寸.结果经700℃焙烧可得到最大粒径25nm左右,分散良好的纳米羟基磷灰石.加入聚乙二醇(PEG)作络合剂,可得到粒径25nm左右,尺寸分布均匀的纳米羟基磷灰石.结论采用该溶胶-凝胶法可以合成颗粒大小均匀,分散性好的纳米羟基磷灰石粉体.该法的优点是无需调节pH值,且无需剧烈搅拌和长时间的水解即可成胶,HA的合成周期较短,所得纳米粉体的稳定性好.     

A comparative study for ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates

Various experimental conditions were tried to stain intracellular IgG globulins within popliteal lymph node cells of rats and rabbits by the use of specific antibodies and their Fab fragments labelled with peroxidase following two different procedures.Fixation with 4% formaldehyde/24 hr, 1% formaldehyde and 1% glutaraldehyde/1–1.5 hr and 1–1.5% glutaraldehyde/1–1.5 hr provided satisfactory ultrastructural conservation of the tissues. None of these fixatives appeared to denature IgG globulins, but formation of cross-linkages prevents conjugates from reaching antigenic sites. Weak aldehyde fixation may enhance penetration of conjugates; however, ultrastructural detail is poorly preserved and proteins will leak out from insufficiently fixed tissue, thus increasing the possibility of staining artifacUnder our conditions, best results were obtained by using frozen sections prepared from well-fixed specimens. Apparently, the most important points are tissue fixation and sampling, above all other experimental steps. Conjugates with molar ratios of antibody or Fab fragment to peroxidase being one, seem more effective, although no significant differences were noted between peroxidase conjugates of whole antibody molecules and Fab fragments.     

Afferents to the cerebellar cortex of turtles studied by means of the horseradish peroxidase technique

Summary Origins of afferents to the cerebellar cortex from the brainstem were explored in turtles by means of the horseradish peroxidase (HRP) technique. Following relatively large injections involving all cortical layers, HRP label was observed in neural perikarya of the following structures: 1) contralateral reticular formation just lateral and ventral to the hypoglossal nucleus; 2) a few cells in the central gray of the cervical spinal cord; 3) neurons scattered in the dorsolateral, ventromedial and descending vestibular nuclei, mainly ipsilaterally; 4) a few solitary cells in the mesencephalic and medulalry tegmentum; 5) the nucleus isthmi magnocellularis caudalis on the ipsilateral side; 6) a group of small cells in the isthmic tectum; 7) the ipsilateral nucleus of the optic tract; 8) a prominent group of small cells in the isthmic region just rostral to the vestibular complex ipsilaterally. Most of these cells were localized within the so called nuclei gustatorius secundarius,-lemnisci lateralis and-isthmi parvocellularis. This parvocellular isthmic complex (PIC) was the only region containing labelled cells when small injections restricted to the molecular layer were achieved. We interpret the PIC as a source of climbing fibers, possibly corresponding to the mammalian inferior olive which migrates from the alar plate to its' ventral destination during ontogenesis. Connecting axons were sometimes homogeneously stained which permitted the tracing of connecting pathways. Contorted axon branches stained by anterograde HRP transport were found concentrated in cerebellar and superior vestibular nuclei and sparsely distributed in other vestibular nuclei.     

The rapid anterograde transport of horseradish peroxidase

Intravitreal injections of horseradish peroxidase in the rat consistently resulted in the labelling of contralateral and ipsilateral efferents of the retina. Simultaneous intravitreal injections of horseradish peroxidase and tritiated amino acids yielded identical projection patterns. Intravitreal injections of colchicine and pentobarbital reversibly blocked this transport of horseradish peroxidase from the eye to the brain. Furthermore, silver impregnation procedures indicated that this transport occurs in the absence of significant neurol damage. The anterograde transport of horseradish peroxidase along the optic pathways occurs at a rate between 288 and 432 mm per day.These observations show that the anterograde migration of horseradish peroxidase is subserved by fast axonal transport and that its occurrence does not depend on neurol injury or passive diffusion. The anterograde transport of horseradish peroxidase thus offers a valid and reliable anatomical method for tracing efferent connections in the nervous system.     

Pallido-cortical projections in the cat studied by means of the horseradish peroxidase retrograde transport technique

Using a multi-microelectrode, in 5 animals, orientation tuning was measured simultaneously in 30 closely spaced parallel penetrations perpendicular to the surface of the striate cortex. Actual penetration angles were determined by three-dimensional track reconstruction. Above and below layer IVc, two columnar systems were found whose orientation angles were independent.     

The somatotopic organization of the cat trigeminal ganglion as determined by the horseradish peroxidase technique

The somatotopic organization of the cat trigeminal ganglion has been investigated in the present study by using the horseradish peroxidase (HRP) technique. In separate animals, the corneal, supraorbital, infraorbital, inferior alveolar, or mental branches of the trigeminal nerve have been transected and then soaked in concentrated solutions of HRP. Retrogradely labeled corneal and supraorbital neurons have been found, with extensive overlap between the two cell populations, in the anteromedial region of the trigeminal ganglion. Inferior alveolar and mental neurons have been found to possess similar distributions within the posterolateral part of the trigeminal ganglion. Infraorbital cells have been localized in a central position. The cell bodies of any given nerve are found in at least minimal numbers in all dorsoventral levels of the trigeminal ganglion. However, cell bodies of origin of the supraorbital nerve and the lateral branch of the infraorbital nerve, innervating more posterior or lateral areas of the head and face, are found in greater numbers dorsally. Conversely, cell bodies of origin of the medial branch of the infraorbital nerve, the inferior alveolar nerve, and the mental nerve, supplying more rostral or intraoral areas of the orofacial region, are present in greater numbers ventrally. In contrast, corneal neurons are distributed uniformly in the dorsoventral axis. The ophthalmic and maxillary regions of the trigeminal ganglion appear to be well segregated, whereas the maxillary and mandibular regions exhibit a somewhat greater degree of overlap. Cell bodies of corneal afferent neurons range from 20 to 50 μm in diameter, whereas those of supraorbital, infraorbital, inferior alveolar and mental neurons measure from 20 to 85 μm. It is concluded from the findings of the present work that much of the cat trigeminal ganglion is organized somatotopically in not only the mediolateral axis but also in the dorsoventral axis.     

Heterogeneity in anticatalytic activity of antibody specific for horseradish peroxidase

Rabbit antibodies specific for horseradish peroxidase are heterogeneous in their ability to inhibit enzyme activity. Heterogeneity was demonstrated by fractionation of the total antiperoxidase pool by differential ammonium sulfate precipitation and differential elution of antibody from enzyme affinity columns. Both fractionation methods yielded antibody subpopulations that differed in anticatalytic activity. Some antibody subpopulations decreased enzyme activity almost completely at low molar ratios of antibody to peroxidase. Other subpopulations were not effective inhibitors even at great molar excess. Admixture experiments demonstrated that inefficient antibody pools decreased the anticatalytic effect of highly inhibitory antibody. The degree of inhibition observed with unfractionated antiserum is a reflection of the interaction of various antibody subpopulations with the enzyme. No correlation was found between the immunoglobulin class of antiperoxidase and anticatalytic efficiency in analyses of numerous antisera. The determinant specificity of an antiperoxidase molecule determines its anticatalytic ability. A peptide fragment (mol. wt 22,500) of peroxidase prepared by partial tryptic digestion bount 60 to 70% of the total antiperoxidase in a number of antisera. However, the peptide did not bind inhibitory antibodies.     

Afferent and efferent connections of brainstem locomotor regions: study by means of horseradish peroxidase transport technique

Afferent and efferent connections of the hypothalamic and mesencephalic locomotor regions and also the bulbar locomotor strip were studied in cat using retrograde (horseradish peroxidase) transport technique. To study the sources of afferent projections, the enzyme microinjections were performed exactly into the same brain sites eliciting treadmill locomotion by means of electrical stimulation. When studying efferent projections horseradish peroxidase labeled neurons were revealed within locomotor regions after enzyme microinjections into different brain structures. Experimental data have shown that the hypothalamic and mesencephalic locomotor regions have mutual afferent and efferent projections with numerous brain areas including interconnections. Apart from the entopeduncular nucleus, the great number of different sensory nuclei are noted: among the sources of afferent projections are the nucleus tractus spinalis nervi trigemini, nucleus cuneatus, nucleus tractus solitarius and vestibular nuclei. In addition, after horseradish peroxidase injection into the mesencephalic locomotor region labeled neurons were found in the cochlear nuclei. Direct descending neuronal projections of the hypothalamic and mesencephalic locomotor regions are distributed mainly in the ipsilateral brainstem. Only a few of them reach the lumbar spinal cord. The most marked efferent projections of given regions are those to the brainstem reticular formation. After horseradish peroxidase injection into a functionally identified bulbar locomotor strip, labeled neurons were revealed in different stem regions mainly caudal to the enzyme injection site. The existence of a locomotor strip as an independent structural formation is called into question. When studying the locomotor region connections, the structural heterogeneity of these regions is revealed. Transitory fibers of ascending tracts are presumably within their limits side by side with neurons. The role of these fibers in stepping initiation by electrical stimulation of locomotor regions remains uncertain.