A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes.

A highly specific and sensitive enzyme-linked immunosorbent assay, using a heterologous combination of antiserum raised against testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin and penicillinase-labeled testosterone-11 beta-carboxymethyl ether, was developed for measuring testosterone in human plasma. Immunoassay plates (96 wells) provided a sensitivity of 2.5 pg/well. This was achieved by maintaining the molar ratios of steroid to enzyme between 10 and 20. The assay was very specific for testosterone and did not show any cross-reaction with the related C19 steroids tested. Replacement of immunoassay plates with the locally available polypropylene tubes raised the detection limits to 25 pg/tube, but improved the range of doses of testosterone that could be measured up to 10,000 pg. The antiserum to testosterone derivative was linked to both immunoassay plates and polypropylene tubes through immunochemical bridges. Comparison of testosterone values of 52 plasma specimens obtained by both solid phase methods with those of radioimmunoassay showed good correlation.     

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.     

A sensitive solid phase enzymeimmunoassay for testosterone in plasma and saliva.

A sensitive, solid phase enzymeimmunoassay suitable for determining testosterone concentrations in samll aliquots of plasma (20 microliter) and saliva (200 microliter) has been developed. A solid phase antiserum raised against a testosterone-11 alpha-hemisuccinate/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. The "enzyme label" was a covalently linked testosterone/horseradish peroxidase conjugate. The assay had a lower limit of sensitivity of 4pg/assay tube and satisfied accepted criteria of specificity and precision. Testosterone concentrations determined by enzyme-immunoassay were in excellent agreement not only with a gas liquid chromatography/mass spectrometry procedure (r=0.96, n=12) but also with the radioimmunoassay in routine use (r=0.95, n=12). The EIA can therefore replace RIA in both the small clinical laboratory and high throughput service centres for determining plasma and salivary testosterone concentrations. In normal males salivary testosterone concentrations reflected circulating steroid levels and indicated the possibility of assaying saliva rather than plasma in clinical studies.     

Enzyme immunoassay of testosterone in plasma, with use of polyethylene glycol to separate antibody-bound and free hormone.

We describe an enzyme immunoassay for testosterone in which we use a testosterone-3-(carboxymethyl)oxime horseradish peroxidase conjugate as the label and an antiserum, raised in rabbits, to testosterone-3-(carboxymethyl)oxime-bovine serum albumin conjugate. Polyethylene glycol (Carbowax 6000) is used to separate antibody-bound and free steroid. The assay has a sensitivity of 12 pg/assay tube and satisfies the usual criteria of specificity, precision and accuracy. The results agree well with those obtained with a comparable radioimmunoassay.     

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.     

An enzyme-linked immunosorbent assay for quassin and closely related metabolites

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% ($\text{v}\text{v}$) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.     

A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (50O fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an llα-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an llα-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the “enzyme label” and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by “extracting” testosterone from samples with a solid-phase anti testosterone-3-¦O-carboxymethyl¦-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r > 0.98, n = 28) and saliva (r > 0.99, n = 28). In saliva samples collected at 2 hourly intervals by normal healthy women (n = 5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n = 7), mean salivary testosterone concentrations in samples collected daily throughout one complete cycle ranged from 5O to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50% and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.     

脱落酸的酶标免疫测定

使用国产辣根过氧化物酶(HRP)合成酶标记物,用竞争法进行了植物内源激素脱落酸(ABA)的酶标免疫测定研究。测定范围为0.0125ng—100ng,在此范围内logitB/B。与ABA浓度的对数之间呈较好的线性关系。检测的灵敏度达到5×10~(-14)克分子。比较了两种酶标记方法对于测定的影响,结果发现直接使ABA共价结合到HRP上形成ABA-HRP酶标记物比先将ABA与牛血清白蛋白(BSA)结合后,然后进一步再与HRP反应形成ABA—BSA—HRP复合物灵敏度高,非特异性吸附小,而且合成步骤较少。酶标记物的稀释度直接影响测定的灵敏度和浓度对数与logit B/B。之间的线性关系好坏;在以每毫升3—6微克免疫球蛋白包埋免疫吸附板进行测定时,用每毫升5微克酶标记物的浓度获得了最佳结果。     

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.     

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF)

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.     

A sensitive and specific enzymeimmunoassay for serum testosterone.

A very sensitive enzymeimmunoassay for testosterone was developed using testosterone-penicillinase conjugate and an antibody to testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin. The specificity of the assay was demonstrated by the fact that estradiol-17 beta, estrone, estriol, progesterone, 17 alpha-hydroxy-progesterone, dehydroepiandrosterone, androstenedione, cortisol and cortisone were ineffective in crossreacting with testosterone while dihydrotestosterone was 8 times less crossreactive as compared to testosterone. The minimum detectable amount of testosterone was 10-15 pg per assay tube. Intra-assay and inter-assay coefficients of variation for samples containing 0.3-6ng/ml of testosterone were 6-8% and 8-10%, respectively. A high degree of correlation (r = 0.97) was observed between serum testosterone values obtained by enzymeimmunoassay and radioimmunoassay. The levels of testosterone in the sera of normal men and women and those in hypogonadal males following stimulation with human chorionic gonadotropin determined by this enzymeimmunoassay appear similar to those reported by other investigators.     

Production of monoclonal antibodies and ELISA for thebaine and codeine

The ratio of hapten and bovine serum albumin in antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. Monoclonal antibodies against thebaine and codeine were produced by hybridoma fused with the sprenocytes immunized with thebaine- and codeine-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. No cross-reaction of anti-thebaine antibody against morphine was observed. Very small cross-reaction appeared in codeine (0.004%). The cross-reaction of anti-codeine antibody against morphine and thebaine was 2.97 and 5.98%, respectively. The full measuring range of the assay extends from 60 pg mL to 1 ng mL for thebaine and 1 ng mL to 100 ng mL for codeine.     

Synthesis and application of organomercury haptens for enzyme-linked immunoassay of inorganic and organic mercury

Two organomercury haptens were synthesized via the classical oxymercuration reaction. An intramolecular oxymercuration reaction was the strategy employed to prepare a structurally simple, but chemically robust, organomercury hapten that was conjugated to chicken immunoglobulin G (IgG). The resulting immunogen afforded mouse anti-mercury antibodies that were evaluated in an enzyme-linked immunosorbent assay (ELISA). Antibodies demonstrating high titers were obtained, and various immunoassay parameters were investigated. The sensitivity and selectivity of the resulting antibodies were evaluated by exploring different cross-coupling chemistries and solid-phase synthetic variations. A second hapten was prepared with the intermolecular oxymercuration reaction, and the resulting compound, once coupled to carrier protein, afforded a solid-phase conjugate that revealed the versatility of the mouse anti-mercury antibody. The anti-mercury antibody developed in this study was capable of detecting both mercury(II) salts and organomercury compounds.     

Production and characterization of aflatoxin B2a antiserum.

The specificity and sensitivity of antiserum elicited from rabbits against aflatoxin B2a-bovine serum albumin conjugates were characterized with a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA). Aflatoxin B1 was first converted to aflatoxin B2a and then conjugated to bovine serum albumin and horseradish peroxidase by a reductive alkylation method. The antiserum was developed in New Zealand white rabbits by multiple-site injection with the aflatoxin B2a-bovine serum albumin conjugate. Antibody titers were determined by both RIA and ELISA. Competitive RIAs with various aflatoxin analogs indicated that the antiserum was most reactive with aflatoxin B1 and slightly cross-reactive with aflatoxins B2a, B2, and M1. Competitive ELISAs showed the antiserum to be equally specific for aflatoxins B2a and B12 and less reactive with aflatoxins B2 and M1. The relative sensitivities of RIA and ELISA for aflatoxin B1 quantitation were 100 and 10 pg per assay, respectively.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum

A new time-resolved fluoroimmunoassay (TR-FIA) of testosterone in serum is described, using a biotinylated testosterone tracer, with a long spacer arm between biotin and testosterone, coupled to the C3 of the testosterone: a biotinylaminodecane carboxymethyloxime testosterone. This tracer affords a great sensitivity of the standard curve, because a amount of 0.3 pg of testosterone can be significantly measured on the testosterone standard curve. The "functional" sensitivity is at least equal to 21 pg/ml of serum. The specificity of the assay is insured by a celite chromatographic step on new minicolumns before immunoassay. The variation coefficient of inter-series reproducibility measured on low and normal testosterone levels in untreated and testosterone treated hypogonadal men were between 2.17 and 5.07%. The accuracy test, (overload and dilution tests) gave satisfying results. Moreover, in a comparison with GCMS, it appeared that the correlation coefficient was 0.992 and no significant difference could be exhibited between the two methods. Consequently, this specific, sensitive reproducible and easy to use method is well suited to the measurement of testosterone in clinical and pharmacological conditions.     

Improved sensitivity of the mouse interstitial cell testosterone assay with the addition of forskolin.

Modification of the mouse interstitial cell testosterone assay by the addition of 1.5 microM forskolin to the incubation medium has improved the sensitivity of this luteinizing hormone bioassay from approximately 100 to 3 pg/tube of NIH rLH RP-2. Luteinizing hormone can be clearly detected in 1 microL of serum from rats castrated 1 week previously and 5 microL of serum from intact rats. Parallelism was noted between dilution curves of serum from intact and castrated rats, and the luteinizing hormone standard curve. Luteinizing hormone detected in serum samples from 30 intact rats by the improved bioassay and by radioimmunoassay was significantly correlated (r = 0.85). Secretion patterns of circulating luteinizing hormone in individual rats were similar when detected by either bioassay or radioimmunoassay. Thus, with the addition of forskolin, the mouse interstitial cell testosterone assay has been improved so that luteinizing hormone can be detected in small volumes of serum or plasma from male rats.     

A comparison of alkaline phosphatase, beta-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtitre plate enzymeimmunoassay

Assays for alkaline phospatase, beta-galactosidase, penicillinase and peroxidase were optimised for quantitation in microtitre plate wells. Their value as labels in microtitre plate enzymeimmunoassay (EIA) for progesterone was assessed following coupling with 11 alpha-hydroxyprogesterone 11-glucuronide using an active ester procedure. Bridge-heterologous antiserum (11 alpha-hydroxyprogesterone 11-hemisuccinate-bovine serum albumin as immunogen) was used to minimize bridge recognition. The limits of detection of the enzymes were in the order penicillinase greater than peroxidase greater than alkaline phosphatase greater than beta-galactosidase. Under appropriate conditions it was possible to achieve greater than 50% displacement of label with 50 pg of progesterone for all four labels.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

A sensitive sandwich enzyme-linked immunosorbent assay of rat apolipoprotein A-I: effect of various sample treatments on apolipoprotein A-I immunoreactivity and an application to young and aged rat sera.

A highly sensitive sandwich enzyme-linked immunosorbent assay for rat apo A-I was developed. Samples and standards were added to each well of microtiter plates precoated with immunoaffinity-purified IgG. Bound apo A-I was detected with immunoaffinity-purified Fab'-horseradish peroxidase conjugate by a colorimetric method. The sensitivity reached 2.5 pg/well, and the working range for the measurement of serum apo A-I concentration was 0.1 to 1.0 ng/well. The mean intra- and interassay coefficients of variation were 2.8 and 4.1%, respectively. The epitopes of apo A-I in serum were effectively exposed by the use of 6 mol/liter guanidine.HCl. Serum apo A-I concentrations in 36- to 40-week-old rats (62.3 +/- 8.6 mg/dl, mean +/- SD, n = 16) were significantly higher (P less than 0.05) than those in 8- to 12-week-old rats (55.1 +/- 4.3 mg/dl, n = 9). But the age-related change of serum apo A-I was much smaller than that of serum apo E. Apo A-I was contained in smaller HDL particles (or HDL2) in normal rat serum.