Isoluminol as a marker in direct chemiluminescence immunoassays for steroid hormones

We have used isoluminol steroid hormone conjugates in competitive heterogeneous chemiluminescence immunoassays (ClA) for estradiol, progesterone and estriol in body fluids. The assays did not require prior extraction of the steroid hormone with an organic solvent. Polyclonal or monoclonal antibodies, covalently coupled to polyacrylamide microspheres or adsorbed onto the plastic surface of microtritation plates via an intermediate second antibody, were used as solid-phase. The latter solid-phase system allows rapid processing of incubation mixtures. Sensitivie and reliable direct CIAs for estradiol in serum, progesterone in serum and in saliva and for estriol in saliva have been developed and their clinical utility has been assessed. Sensitivities ranged from 0.54 to 0.04nmol/l, and precision (% CV) ranged from 10% to 16%.     

Serological reaction systems using enzyme-labelled immunospecific reagents

Different serological test systems, based on the use of enzyme-labeled immunospecific reagents and intended for testing the material under study for the presence of Yersinia pestis capsular antigen and antibodies to it, are described. Comparative data on the evaluation of their sensitivity to the antigen and antibodies to it in different schemes of enzyme immunoassays (EIA) are presented. As shown in this investigation, EIA systems for the detection of the antigen and antibodies to it can comprise, at the minimum, the following set of reagents: monoclonal antibodies to the capsular antigen, staphylococcal protein A, and the conjugates of the capsular antigen and monoclonal antibodies with horse-radish peroxidase. The authors have come to the conclusion that the use of the serological test systems can essentially increase the reliability of the assay of any individual sample by EIA techniques.     

Preparation of specific antiserum to estriol 3-sulfate 16-glucuronide

The preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Coupling of 6-oxoestriol 3-sulfate 16-glucuronide acetate-methyl ester 6-(O-carboxymethyl)oxime with BSA by the activated ester method followed by removal of the protecting groups with alkali provided the desired conjugate. The antisera raised against the conjugate in rabbits were highly specific to the double conjugate, estriol 3-sulfate 16-glucuronide, discriminating from ring A or D monoconjugated and unconjugated estrogens. The specificity of antisera elicited has been discussed on the basis of stereochemistry of the hapten-[C-6]-BSA conjugate.     

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.     

The detection of antibodies to recombinant interferon alfa-2a in human serum

Three different procedures have been used for detecting antibodies to Roferon-A (recombinant human interferon alfa-2a, rHuIFN alpha-2a) in the serum of patients who received this interferon as part of ongoing clinical trials: an antiviral neutralization bioassay (ANB), the standard method recommended by the World Health Organization (WHO), and the more recently developed radioimmunoassay (RIA) and enzymeimmunoassay (EIA). Although the three tests are based on different principles, the correlation among them was excellent. The assays show differences in sensitivities with the ANB being the least sensitive of the three. The EIA equals the RIA in sensitivity, reproducibility, accuracy and labor and provides the advantage of safety and convenience in the use of non-radioactive materials. Therefore, the EIA has been selected as the most suitable assay for initial screening of the sera of patients receiving Roferon-A for the presence of antibodies to this interferon. EIA positive sera are then tested in the ANB to determine whether or not neutralizing activities are present.     

The use of microdot immunoenzyme analysis with visual detection for the determination of tularemia antibodies

The possibility of using the micropoint enzyme immunoassay (EIA) on a nitrocellulose membrane with the visual evaluation of results for the detection of tularemia IgG antibodies in hamadryas baboons at the postvaccinal period has been studied. The sensitivity of this assay has been compared with that of the passive hemagglutination (PHA) test, the microagglutination (MA) test and EIA with the spectrophotometric evaluation of results in plates. As shown in this study, EIA in the above-mentioned modification can be successfully used for the detection of tularemia antibodies in the blood serum. The sensitivity of micropoint EIA has proved to be not inferior to that of EIA in plates, while exceeding the sensitivity of the PHA test 10- to 20-fold and the sensitivity of the MA test 10- to 1,000-fold. This method is simple, reliable, highly sensitive, economic and requires no special equipment, which makes it highly promising for the diagnosis of tularemia and the evaluation of humoral immunity at the postvaccinal period.     

A retrospective study of the expediency of performing the immunoblot in conducting expert diagnosis at a center for the prevention and control of AIDS

In view of the fact that Russian immunoblot assay systems yield indefinite results, thus making it necessary to carry out prolonged dispensary observations of patients and their multiple examinations, the scheme of the expert diagnostics of HIV infection on the basis of EIA results is proposed. 51 sera which had yielded indefinite results in the immunoblot assay (antibodies to gagl, envl, poll) were handed over for verification to the Regional Center in 1996-1997. Patients from whom the sera had been taken were placed under dispensary observation in the Center for 6-12 months. After the retrospective analysis of these serum samples with the use of the EIA systems 40 samples proved to be negative and 11 samples gave the positive reaction.     

The diagnostic effectiveness of an immunoenzyme test system for determining diphtheria toxin antigens in the blood serum in a clinical laboratory trial

The data on the approbation of the diagnostic value of the enzyme immunoassay (EIA) system for the determination of diphtheria toxin in the blood sera of diphtheria patients and persons suspected for diphtheria are presented. The EIA system was prepared on the basis of F(ab)2 fractions of purified antidiphtheria antibodies. 240 serum samples from diphtheria and tonsillitis patients and from healthy persons were studied. Diphtheria toxin was determined in all patients with the toxic form of diphtheria and in 41.3% of patients with its localized forms. Blood was taken mainly of the first week of the disease. In healthy persons the results of EIA were negative. Thus, the trial of the assay system in a clinical laboratory showed its good diagnostic effectiveness. The use of this EIA system in medical practice is believed to be quite promising.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Immunoenzyme analysis with visual demonstration for detecting antibodies to the tick-borne encephalitis virus

Good prospects for the use of enzyme immunoassay (EIA) with the simple visual indication of results have been shown with the detection of specific antibodies to tick-borne encephalitis virus in blood serum used as an example. When compared with such highly sensitive method as radioimmunoassay, visual EIA is inferior in both sensitivity and selectivity, but its special advantage is that it requires no instrument for evaluating the result.     

Production of highly specific polyclonal and monoclonal antibodies using estradiol-3-O-carboxymethyl ether as hapten

The preparation of high affinity and high specificity polyclonal and monoclonal antibodies to estradiol is described. Monoclonal antibodies were derived from BALB/c mice hyperimmunized with estradiol-3-O-carboxymethyl ether conjugated to bovine serum albumin. Spleen cells were hybridized with mouse myeloma cells. Quite a few monoclonal antibodies showed very good affinity for estradiol. Extended immunization and hyperimmunization were essential for producing a greater number of positive clones secreting high affinity antibodies. Binding constants of the antisera and their cross-reactivities with related steroids were calculated. Both polyclonal and monoclonal antibodies showed very high affinity for estradiol exhibiting little or no cross-reactivities with structurally related steroids indicating that this site of linkage is a good choice for discriminating between differences at the 16-17 position in the D-ring. This monoclonal antibody (44.28.6), having negligible cross-reactivity with estriol and estrone, can be used for diagnostic purposes.     

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.     

Production of ultrasensitive antibodies against aflatoxin B1

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.     

Use of cooperative monoclonal antibodies in immunoenzyme analysis for determining human tumor necrosis factor

A cooperative sandwich enzyme immunoassay (EIA) based on the newly produced pair of cooperative monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was developed and characterized. It was found that, when used simultaneously, cooperative mAbs was capable to bind TNF from its preformed complexes with soluble TNF receptors (sTNF-R), thus providing the effective TNF detection in ex vivo samples by the respective one-step cooperative EIA. While demonstrating typical analytical characteristics regarding variability, dynamic range and specificity, a cooperative EIA offers an advantageous combination of high sensitivity (< 2 pg/ml) and short-time TNF capture protocol (1 hour). Application of cooperative EIA for TNF detection in clinical samples has demonstrated an increased serum TNF levels in patients with the mixed connective disease and infectious endocarditis that positively correlated with severity of systemic inflammatory reactions. Production and EIA application of cooperative mAbs would be promising in development of standardized and clinically applicable immunoassays for cytokines.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.     

The development and use of an immunoenzyme test system for detecting and the species identification of the monkey pox virus]

An enzyme immunoassay (EIA) system for the species-specific diagnosis of monkeypox, based on the use of monoclonal antibodies (McAb) to monkeypox virus, has been developed. Immunoglobulins, isolated from McAb-containing cultural and immune ascitic fluids, have been conjugated with horse-radish peroxidase and used as detector antibodies. For immunosorption, rabbit polyclonal antibodies to the vaccine virus have been used. The specificity and sensitivity of the EIA system thus obtained have been tested on animals and humans having monkeypox and confirmed by traditional diagnostic methods (the isolation of the virus on chick embryo chorioallantoic membranes and in cell culture).     

Patterns in the immunoenzyme analysis of bacterial cells

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

The use of a nitrocellulose-enzyme immunoassay for the rapid screening of monoclonal antibodies to human enteroviruses

A simple, indirect enzyme immunoassay using purified enterovirus adsorbed on to nitrocellulose has been developed for screening monoclonal antibodies to enteroviruses. The sensitivity of the assay ranged from 10 ng to 1 microgram of viral protein and was 10- to 50-fold more sensitive than conventional EIA on microplates. This simple, sensitive and specific assay proved to be a useful and practical tool for detecting monoclonal antibodies which would not be found in a conventional EIA screening procedure.     

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.