Identification of 19-nor-deoxycorticosterone in normal rat serum by enzyme immunoassay and gas chromatography-mass spectrometry

Recent reports on the isolation and identification of 19-nor-deoxycorticosterone from the urine of rats with adrenal regeneration hypertension give rise to the possibility of a role of this steroid in the pathogenesis of low renin essential hypertension. The present study was undertaken to investigate the presence of 19-nor-deoxycorticosterone in normal rat serum both by a sensitive enzyme immunoassay and by gas chromatography-mass spectrometry (GC/MS). 19-Nor-deoxycorticosterone in rat serum was separated from other steroids prior to enzyme immunoassay by using high performance liquid chromatography (HPLC). The average concentration of 19-nor-deoxycorticosterone in normal rat serum was 137 +/- 62 ng/dl (mean +/- SD, n = 32). Pooled normal rat serum (50 ml) was purified by HPLC and the purified sample was acetylated with acetic anhydride for GC/MS measurement. The retention time and m/z ions (358, 285, and 257) on the resulting mass fragmentogram coincided in position with those of authentic 21-acetoxy-19-nor-deoxycorticosterone and acetylated normal rat serum extract. The combined characteristics of HPLC elution, antigen-antibody reaction, GC retention and selected ion responses provided strongly evidence supporting the presence of 19-nor-deoxycorticosterone in normal rat serum.     

Diagnosis of invasive candidosis by enzyme immunoassay of serum antigen.

Enzyme immunoassay was used to measure Candida albicans-related antigen in sera of rabbits and mice infected with C albicans. A range of heterologous antigens tested gave no cross reactions. Of the three patients who were investigated and gave positive reactions, two were confirmed as having deep candida infections. Enzyme immunoassay is efficient, simple, and sensitive, and may prove valuable in the early diagnosis of invasive candida infections.     

Homogeneous enzyme immunoassay of chenodeoxycholate conjugates in serum.

It is shown that biased answers are given by the mathematical method used by Stein and his colleagues (Hankin B. L. Hankin, W. R. Lieb, and W. D. Stein (1972)Biochim. Biophys. Acta288, 114–126) to calculate $\text{K}{}_{21}{}^{\mathrm{<mtext>ic</mtext>}}$, the half-saturation concentration for the entry of glucose into erythrocytes in infinite-cis conditions. A method for calculating $\text{K}{}_{21}{}^{\mathrm{<mtext>ic</mtext>}}$ accurately is described and tested. The published estimates of $\text{K}{}_{21}{}^{\mathrm{<mtext>ic</mtext>}}$ are low; nevertheless, even when they are revised upwards, the asymmetrical carrier model of glucose transport still fails to satisfy the “rejection criteria” of Hankin et al. (1972).     

Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma

We have developed and validated a sensitive, simple and direct (i.e. without extraction) enzyme immunoassay (EIA) system for the measurement of progesterone in bovine milk and blood plasma. Progesterone (P) has been analysed by a microtiterplate EIA, employing polyclonal antibodies against P-7α-carboxyethylthioether-BSA as the antigen. The enzyme used as a label is horseradish peroxidase (HRP) and the chromogen is tetramethylbenzidine (TMB). Sensitivity of the EIA has been greatly improved by introduction of a heterologous tracer, in which progesterone is coupled to HRP at the 6β position. Compared to the radioimmunoassay (RIA) in which the same antiserum has been used, the sensitivity is 20 times greater. The detection limit of the assay is 0.4 pg per well. The working range of the standard curve is 0–20 pg per well (i.e. 0–40 ng per ml), and 50% reduction of the initial binding is obtained with 2.5-5.0 pg. Results can be obtained either by spectrophotometric measurement at 450 nm, or by naked eye. Total time needed for the assay of 40 replicate samples is approximately 3 h. Comparison of the EIA system with a previously validated RIA system gave a regression line EIA = 0.85 RIA + 2.11 (r = 0.93, n = 400 milk samples). Application of the milk-progesterone EIA to pregnancy testing (n = 66) gave an accuracy of 79.6% for positive diagnoses and 100% for negative diagnoses.     

A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels

Pre-beta1-HDL, a putative discoid-shaped high density lipoprotein (HDL) of approximately 67-kDa mass that migrates with pre-beta mobility in agarose gel electrophoresis, contains apolipoprotein A-I (apoA-I), phospholipids, and unesterified cholesterol. It participates in the retrieval of cholesterol from peripheral tissues. In this study we established a new sandwich enzyme immunoassay (EIA) for measuring plasma pre-beta1-HDL using mouse anti-human pre-beta1-HDL monoclonal antibody (MAb 55201) and goat anti-human apoA-I polyclonal antibody. MAb 55201 reacted with apoA-I in lipoprotein [A-I] with molecular mass less than 67 kDa, and with pre-beta1-HDL separated by nondenaturing two-dimensional electrophoresis, whereas it did not react with apoA-I in alpha-HDL. Pre-beta1-HDL levels measured by this method declined when incubated at 37 degrees C for 2 h, whereas this decrease was not observed in the presence of 2 mM lecithin:cholesterol acyltransferase inhibitor 5,5'-dithiobis (2-nitrobenzoic acid). To clarify the clinical significance of measuring pre-beta1-HDL by this method, 47 hyperlipidemic subjects [male/female 22/25; age 55 +/- 14 years; body mass index 25 +/- 4.5 kg/m(2); total cholesterol (TC) 245 +/- 64 mg/dl; triglyceride (TG) 232 +/- 280 mg/dl; HDL cholesterol (HDL-C) 51 +/- 23 mg/dl] and 25 volunteers (male/female 15/10; age 36 +/- 9.3 years; body mass index 23 +/- 3.5 kg/m(2); TC 183 +/- 28 mg/dl; TG 80 +/- 34 mg/dl; HDL-C 62 +/- 15 mg/dl) were involved. Plasma pre-beta1-HDL levels were significantly higher in hyperlipidemic subjects than in volunteers (39.3 +/- 10.1 vs. 22.5 +/- 7.5 mg/ml, P < 0.001) whereas plasma apoA-I levels did not differ (144.2 +/- 28.4 vs. 145.3 +/- 16.3 mg/dl).These results indicate that this sandwich EIA method specifically recognizes apoA-I associated with pre-beta1-HDL.     

An enzyme immunoassay of estrone in swine serum

An enzyme immunoassay for estrone in swine serum was established. For this, beta-galactosidase from E. coli was conjugated through estrone-17 (O-carboxymethyl)oxime using a mixed anhydride reaction. The percentage of immunoreactive estrone-17 (O-carboxymethyl)oxime-beta-galactosidase conjugate was estimated to be about 70%. The recovery rate of estrone (25-500 pg) added to 0.05 ml of swine serum averaged 91.4%. The sensitivity of the present enzyme immunoassay was 5 pg/tube. The coefficients of variation (CV) were 5.9-8.2% (within assays) and 4.1-5.9% (between assays), respectively. Estrone values determined by the present enzyme immunoassay were highly correlated with those determined by radioimmunoassay (r = 0.99, P less than 0.005). This method of enzyme immunoassay was determined to be suitable for the routine assay of serum estrone.     

Analytical performance of a sandwich enzyme immunoassay for pre beta 1-HDL in stabilized plasma

We have established an immunoassay for pre beta 1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre beta 1-HDL is unstable during storage, fresh plasma must be used for pre beta 1-HDL measurements. In this study, we describe a method of stabilizing pre beta 1-HDL, and evaluate the analytical performance of the immunoassay for pre beta 1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Pre beta 1-HDL concentration was measured by immunoassay. In nonpretreated samples, pre beta 1-HDL decreased significantly from the baseline after 6 h at room temperature. Although pre beta 1-HDL was more stable at 0 degrees C than at room temperature, it increased from 30.2 +/- 8.5 (SE) to 56.5 +/- 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 +/- 1.2 to 37.9 +/- 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at -80 degrees C, pre beta 1-HDL increased from 29.0 +/- 4.0 to 38.0 +/- 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, pre beta 1-HDL concentration did not change significantly under any of the above conditions. Moreover, pre beta 1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of pre beta 1-HDL.     

Development of an enzyme immunoassay for cryptococcal antibody

An enzyme immunoassay (ELISA) for measurement of cryptococcal Ig G antibody in human serum is described. Clinical studies indicate that the assay is a useful addition to the currently available techniques for measuring antibodies in cryptococcosis. IgG-specific antibody (titers 4 to 1,024) was detected in the serum of 78 % of the cryptococcosis patients tested and in 61 % of the serum from healthy individuals with positive delayed skin hypersensitivity to cryptococcin. The micro-ELISA for cryptococcal antibody is of potential value in patient management, and in epidemiological studies.     

A versatile enzyme immunoassay for the determination of progestogens in feces and serum

The ability of zoos to monitor the reproductive status of their animals can vastly improve the effectiveness of husbandry/management practices, and noninvasive methods such as fecal steroid analysis are the easiest to apply in a zoo setting. Furthermore, enzyme immunoassay (EIA) is preferred to radioimmunoassay (RIA) as the method of quantifying hormones because EIAs do not involve the use, storage, and disposal of radioactive materials. However, progesterone is excreted in the feces as predominantly unconjugated metabolites (progestogens) and, until recently, antibodies able to cross‐react with a variety of progestogens were used primarily in RIAs. An EIA using a broad‐spectrum progestogen antibody is described and applied to serum and/or fecal samples from female African elephants, black rhinoceros, white rhinoceros, okapi, and hippopotami. The clear progestogen profiles generated in these species suggest that the described EIA would be as versatile as the RIA using the same antibody and could be a practical and economical alternative to RIAs for monitoring gonadal function via progestogen analysis in zoo species. Zoo Biol 20:227–236, 2001. © 2001 Wiley‐Liss, Inc.     

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay

Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme‐linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity‐purified anti‐C‐terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin‐linked horseradish peroxidase. Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2‐ to 5‐fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or β‐estradiol, in cultured adipocytes reduces resistin protein levels in a dose‐dependent manner. Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.     

Detection of abnormally high amygdalin content in food by an enzyme immunoassay

Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.     

A novel enzyme immunoassay of anti-insulin IgG in guinea pig serum

A novel enzyme immunoassay of anti-insulin IgG in guinea pig serum is described. Guinea pig anti-insulin serum diluted with nonspecific guinea pig serum was incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate and a rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. After washing to eliminate nonspecific guinea pig IgG in the diluted serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex of anti-insulin IgG and the conjugate. The eluate was incubated with an avidin-coated polystyrene ball. Finally, the amount of guinea pig anti-insulin IgG in the complex trapped onto the avidin-coated polystyrene ball was measured by incubation with rabbit (anti-guinea pig IgG) Fab'-peroxidase conjugate. This enzyme immunoassay was 10,000-fold more sensitive than the conventional enzyme immunoassay using insulin-coated polystyrene ball and rabbit (anti-guinea pig IgG) Fab'-peroxidase conjugate.     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

Clotting of citrated plasma by a proteolytic enzyme associated with an antibacterial agent from serum

Two to 4 mg/ml of an antibacterial agent occurring in the serum of humans and animals caused the clotting of citrated rabbit, human, calf, ox, sheep, goat, guinea pig, rat, mouse, horse, chicken, pigeon and swine plasma. Heparinized plasmas of the same species were found resistant to the clotting action of the antibacterial agent. Citrated plasma previously heated at 56 C for 30 min, treated with 640 units/ml tyrosinase for 60 min, or absorbed with 0.2M Ca₃(PO₄)₂ and 0.2M Mg(OH)₂ was also found resistant to the clotting action of the antibacterial agent. The clotting action of the antibacterial agent was not affected by heating at 66 C for 60 min, nor by multiple passage through Seitz filters. The coagulation of citrated plasma proceeded most rapidly with 0.2M Tris(hydroxymethyl)-amino-methane buffer, pH 7 to 9, or distilled water as the antibacterial agent solvent; 0.2M phosphate buffer, pH 6 to 8, reduced the clotting action of the antibacterial agent, while 0.2M citrate-phosphate buffer, pH 4.0, or 0.2M carbonate-bicarbonate buffer, pH 10.0, inhibited entirely the clotting activity of this agent.     

Identification of c-myc coding region determinant RNA sequences and structures cleaved by an RNase1-like endoribonuclease

The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705-1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705-1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.     

The development and application of an enzyme immunoassay for urinary estrone conjugates

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High correlation coefficients for values obtained from each assay indicate that EIA measurements provide information that is comparable to values obtained by radioimmunoassay. EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.     

Development of enzyme immunoassay for serum 13,14-dihydro-15-ketoprostaglandin F2 alpha

An enzyme immunoassay was developed for a convenient and sensitive assay of 13,14-dihydro-15-ketoprostaglandin F2 alpha, a metabolite of prostaglandin F2 alpha appearing in human blood. The compound was chemically conjugated to beta-galactosidase from Escherichia coli. The enzyme-labeled antigen was mixed with a sample containing 13,14-dihydro-15-ketoprostaglandin F2 alpha, and the mixture was allowed to react competitively with the antibody immobilized in a polystyrene tube. The activity of beta-galactosidase bound to the antibody was assayed by fluorometry. The enzyme activity was plotted against the amount of authentic 13,14-dihydro-15-ketoprostaglandin F2 alpha to obtain a calibration curve, and the compound was detectable over a range of 10 fmol to 10 pmol. Prostaglandins were extracted from human serum by the use of an octadecylsilyl silica column, and the extract gave an abnormally high level of 13,14-dihydro-15-ketoprostaglandin F2 alpha by enzyme immunoassay due to the presence of unidentified interfering substance(s), which was removed by high-performance liquid chromatography (HPLC). The purified material gave a value in the order of 0.1 pmol per ml of human serum. Validity of the enzyme immunoassay was confirmed by radioimmunoassay and gas chromatography/mass spectrometry (GC-MS) of a methyl ester n-butoximedimethylisopropylsilyl ether derivative.