Epidermal growth factor in serum, urine, submandibular glands and kidneys of diabetic mice

Levels of epidermal growth factor (EGF) in serum were significantly decreased in streptozotocin (STZ)-diabetic mice (446 +/- 168 pg/ml after 1 week and 423 +/- 52 after 4 weeks vs 766 +/- 162 pg/ml in controls, P.002 and less than .001. respectively) and in genetically diabetic ob/ob mice (455 +/- 285 vs 962 +/- 453 pg/ml in nondiabetic ob/+ controls, P.043). The urinary excretion of EGF was significantly increased in STZ mice (104 +/- 53 vs 51 +/- 23 ng/h, P.013) but unchanged in ob/ob mice (33 +/- 9 vs 45 +/- 16 ng/h, P.134). However, when expressed per mg creatinine it was decreased in both cases: in STZ mice to 680 +/- 250 ng/mg at 1 week and 684 +/- 211 at 4 weeks vs 1250 +/- 303 ng/mg in controls (P less than .01); and in the ob/ob mice to 552 +/- 117 vs 1237 +/- 300 ng/mg in ob/+ controls (P less than .01). EGF content of the submandibular glands of STZ mice remained unchanged at 1 week (13.1 +/- 2.9 vs 11.0 +/- 1.8 micrograms/mg protein, P.170) but dropped by 4 weeks (4.7 +/- 1.2 micrograms/mg, P less than .001); in the ob/ob mice it was less than 20% that of controls (2.1 +/- 0.8 vs 12.2 +/- 3.6 micrograms/mg protein). In kidneys, the EGF content was not altered in either ob/ob (524 +/- 50 vs 571 +/- 33 pg/mg protein) or STZ mice (652 +/- 183 vs 665 +/- 80 pg/mg). The preproEGF mRNA level in STZ-treated mice was reduced after 4 weeks in submandibular glands but not in kidneys. The results show that diabetes affects EGF production, utilization and/or excretion in mice and that kidneys are spared from suppression of EGF synthesis that is pronounced in the submandibular glands.     

A new, highly sensitive assay for 1,25-dihydroxyvitamin D not requiring high-performance liquid chromatography: application of monoclonal antibody against vitamin D receptor to radioreceptor assay.

A new, highly sensitive radioreceptor assay, which does not require high-performance liquid chromatography, has been developed for the determination of 1,25-dihydroxyvitamin D3 (1,25-(OH2)D3) in serum. The assay involves rapid extraction of serum, Sep Pak silica purification, and addition of 1,25-dihydroxyvitamin D3 receptor, radiolabeled 1,25-dihydroxyvitamin D3, bovine serum albumin, and monoclonal antibody to specifically precipitate the receptor. This method is sensitive to 0.3-0.6 pg/tube, with B50 occurring at 5.8 pg/tube. This sensitivity combined with overall recovery of 1,25-dihydroxyvitamin D3 (81.5 +/- 5.2%, n = 50, mean +/- SD) allows the measurement of serum 1,25-(OH)2D3 in duplicates with only 0.5 ml of serum. Intra- and interassay coefficient of variation were 9.5 and 14.6%, respectively. Dilution analysis, analytical recovery of added 1,25-dihydroxyvitamin D3, and comparison with a standard method using HPLC have been used to validate the assay. Serum 1,25-dihydroxyvitamin D3 level was for normal adults, 36.6 +/- 10.5 pg/ml (n = 14); in primary hyperparathyroidism, 98.9 +/- 19.9 pg/ml (n = 16); in chronic renal failure, 17.8 +/- 5.1 pg/ml (n = 12). This method allows large numbers of samples to be processed at once. Further, the method is rapid and provides an accurate assay using small amounts of serum.     

A highly sensitive enzyme immunoassay (EIA) system for mouse epidermal growth factor (mEGF)

A highly sensitive two-site enzyme immunoassay system for mouse epidermal growth factor (mEGF) was developed, based on the sandwiching of an antigen between anti-mouse EGF IgG antibody-coated on a polystyrene bead and anti-mouse EGF Fab' antibody-linked peroxidase (horseradish peroxidase, EC. 1.11.1.7). The procedure is simple and rapid compared to a bioassay. Also, the Fab' antibody-peroxidase complex is more stable than the 125I-labeled antibody. Purified mEGF is detectable at a concentration as low as 3 pg/ml. The detection range was 0.3 to 680 pg/sample with 0.1 ml samples. Levels of immunoreactive mEGF in extracts from adult male mice well agreed with those determined by a radioimmunoassay and a radioreceptor assay. The submaxillary gland contained an extremely high concentration of EGF, while other tissues had low levels of EGF.     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.     

Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain

Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha.     

Highly Sensitive Immunoassay for Rat Brain-Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.     

Tissue specificities of protease A-like esteroprotease in male mice

An esteroprotease hydrolyzing p-tosyl-L-arginine methyl ester (TAME) has been purified to homogeneity from male mice submandibular glands by the ammonium sulphate precipitation, Sephadex gel chromatography and DEAE-cellulose chromatography. The enzyme was shown as a single chain acidic protein (pI = 5.7) with the molecular weight of 27.5 K and evidence was obtained to reveal that it was similar to protease A. Using this enzyme as antigen we prepared anti-TAMEase antibody. The immunoblotting studies on tissue specificity using 20 different tissues from male mice revealed that cross-reactivities with anti-TAMEase antibody were observed in the crude extract from the sublingual gland, parotid gland and pancreas. The species specificity studies with the submandibular glands of 7 different species indicated that only the crude extract from rat submandibular glands reacted against anti-TAMEase antibody but it exerted a low TAMEase activity.     

Cytokine responsiveness in germfree and conventional NMRI mice

We have investigated the proliferative response of thymocytes from different mouse strains to cytokines in vitro. Interleukin 2 (IL-2), IL-4 and IL-7 induced proliferation of thymocytes from NMRI/KI (a locally bred NMRI mouse strain), NMRI/H ('traditional' NMRI mice), C3H/HeJ and C3H/HeN mice. NMRI/KI thymocytes showed the most prominent proliferation in response to IL-1 alpha and IL-1 beta. IL-3, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), inhibin and lipopolysaccharide (LPS) induced no thymocyte proliferation. Germfree NMRI/KI mouse thymocytes showed a significantly lower proliferation in response to IL-1 alpha and IL-1 beta than conventional mice. Rat tissues, previously shown to contain lymphocyte activating factors (LAFs), were also tested. Skin, tongue, esophagus, proventricular stomach, testis and placenta were all positive in the LAF assay utilizing NMRI/KI thymocytes, whereas none of the tissue extracts could induce proliferation in NMRI/H thymocytes. The higher cytokine responsiveness in conventional mice compared with germfree might suggest that exposure to microflora induces a higher state of activation of the immune system. The LAF assay, utilizing NMRI/KI thymocytes, is a highly sensitive IL-1 bioassay with a detection level of 1 pg/ml for IL-1 beta and 2 pg/ml for IL-1 alpha. The specificity of the assay is increased by utilizing NMRI/H mice to exclude the presence of IL-2, IL-4 and IL-7.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain

Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column. Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined. The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.     

Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17β

A sensitive test system has been developed for estimation ofestradiol-17β (E₂) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E₂ is estimated by displacement of biocytinyl-E₂, that was produced by ligation of estradiol-17β, d-glucuronic acid and biocytin. Bound biocytinyl-E₂ is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to ≈120fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E₂ were found in bovine plasma: male or female calves <2.7pg/ml, cycling cow 0.5–7 pg/ml, cow during last month of pregnancy 9–310 pg/ml, mature bull 5–30 pg/ml. However, up to 1110 pg E₂/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sentitive estimation ofestradiol-17β in plasma from all type of cattle and for control of improper use of E₂ after commitment of a threshhold level.     

Nerve growth factor (NGF) is present in human placenta and semen, but undetectable in normal and Paget's disease blood: measurements with an anti-mouse-NGF enzyme immunoassay using a recombinant human NGF reference

An enzyme immunoassay (EIA) originally developed for mouse beta-nerve growth factor (NGF) and commercially available was validated for human NGF. Cell culture medium containing bioactive recombinant human NGF was used as a reference, and mouse 2.5S beta-NGF as a standard. One of three human placentas contained measurable NGF (70 pg/g of tissue of mouse beta-NGF equivalents), a second detectable, and a third undetectable NGF. In three human semen samples NGF content ranged from 0.13 - 1.4 ng/ml. NGF could not be detected in normal human serum and in plasma from patients with Paget's disease, although mouse 2.5S beta-NGF added to human blood could be completely recovered from the serum.     

Radioreceptor assay for epidermal growth factor.

An established cell line of human lung fibroblasts with a high number of surface receptorsfor mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. ¹²⁵I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.     

Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species.

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta'-component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)'(2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed.     

神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

Highly sensitive second-antibody enzyme immunoassay for determination of estradiol-17beta concentration in blood plasma of the mithun (Bos frontalis)

The objective of the present study was to develop and validate a simple, sensitive, quick and economic enzyme immunoassay (EIA) for estradiol-17beta (E2) in mithun (Bos frontalis) plasma on microtiter plates using a second-antibody coating technique and hormone-horseradish peroxidase as a label. For the assay, the wells of microtiter plates were coated with affinity-purified goat anti-rabbit IgG that binds the hormone-specific antibody. One milliliter of mithun plasma was extracted using benzene and 50 microl of 300 microl volume reconstituted with assay buffer was run in the assay along with standards ranging from 0.10-100 pg/well prepared in assay buffer. The sensitivity of the assay was 0.72 pg/ml. The intra- and inter-assay coefficients of variation were below 10%, and the extraction efficiency was >93%. Linearity of recovery of the added hormone concentrations was recorded. The assay developed was further validated biologically by estimating the hormone concentrations in six female and five male mithun calves, 12 cyclic mithuns for the entire reproductive cycle, and four pregnant mithun cows. The EIA developed can estimate low concentrations of E2 (2.2-5.2 pg/ml) in growing calves as well as very high concentrations of the hormone during pregnancy (E2=85.6-143.5 pg/ml). Apart from being non-radioactive, the assay developed has several advantages over conventional radioimmunoassays: it is more sensitive, less labor intensive, simpler to perform, and less time consuming. In conclusion, the EIA procedure described herein is sufficiently reliable, economic, safe, quick and sensitive to estimate the hormone at all physiological levels in bovine plasma.     

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Summary A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.     

Radioimmunoassay for the detection of hepatitis e antigen (HBeAG) and antibody (anti-HBe).

A solid phase micro-immunoradiometric assay (micro-SPIRA) for the detection of hepatitis e antigen (HBeAg) and antibody has been developed. Chimpanzee anti-HBe/2 was developed by repeated immunizations with purified antigen containing HBeAg/1 and HBeAg/2. An anti-HBe/2 titer of 1:4 was determined by immunodiffusion (ID) analysis. Anti-HBe/1 was not detected. The anti-HBe IgG used in the assay was purified from plasma by a combination of DEAE-cellulose and affinity chromatography. The sensitivity of the micro-SPIRA for antigen and antibody was 193 ng/ml and 65 ng/ml, respectively. By comparing relative endpoint titers obtained by ID to micro-SPIRA, it was determined that micro-SPIRA for antigen and antibody is 320 and greater than 1300 times more sensitive, respectively, than ID. The specificity of the assay was ascertained by the examination of various non-B specimens. The application of the assay to a panel of 50 hepatitis B surface antigen (HBsAg)-positive specimens resulted in an increase in positivity of 18% for antigen and 22% for antibody.