A rapid, simple immunofluorometric assay: development and characterization

A novel two-site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small-sized beads (0.5 mum diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid-phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti-IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30-minute incubation was 0.2 to 40 mug/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 mug/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 mug/mL of BSA and intra-assay variability was 2.3% at 3.2 mug/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. (c) 1992 John Wiley & Sons, Inc.     

Binding characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides

The present paper describes the isolation and characterization of a clone of hybrid myelomas (3-E7) secreting a mouse monoclonal antibody to beta-endorphin. An examination of its specificity against a series of human beta-lipotropin fragments and other opioid peptides revealed that the N-terminus portion of beta-endorphin is the determinant. Complete or almost complete cross-reactivity was obtained to methionine- and leucine-enkephalin, beta-lipotropin 60-65, and BAM 22; partial cross-reactivity was seen to dynorphin1-13 and alpha-neo-endorphin, whereas beta-lipotropin, alpha-N-acetyl-beta-endorphin, Des-Tyr1-beta-endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross-reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for beta-endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of beta-endorphin iodinated with its N-terminal tyrosine protected by coupling to an antibody. A solid-phase RIA was developed involving the internally 3H-labeled monoclonal antibody, which resulted in a 10-fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.     

An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).

An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.     

Measurement of glial fibrillary acidic protein by a two-site immunoradiometric assay

The two-site immunoradiometric assay (two-site IRMA) for the brain-specific glial fibrillary acidic protein (GFA protein) is carried out by reaction of the GFA protein solution with a solid-phase anti(GFA) followed by a second reaction in which the insoluble product is incubated with purified, radioactive anti-(GFA). Unreacted labeled antibodies remain in solution and are washed away. As the amount of GFA increases, the radioactivity in the solid-phase increases. The most significant assay variables include (a) stability and reactivity of the solid-phase antibody, (b) washing the solid-phase, (c) nonspecific interference by serum proteins, and (d) a paradoxical fall in tube radioactivity which occurs at high dose (the “high-dose hook effect”). The assay becomes more sensitive and precise and the serum effect is minimized when the solid-phase antibody is separated from the matrix by an immunoglobulin “spacer-arm”. For a triplicate determination, the minimal detectable dose averaged $\text{73}\text{pg}\text{200 μ}\text{l}$ incubation. The assay precision enables a 500-fold assay range. GFA activity found in aged crude tissue or tissue-culture extracts, CSF, and used tissueculture media, often did not appear to be immunologically identical to the purified standard GFA protein. This may be explained by the known tendency of GFA protein to aggregate. The assay does not cross-react significantly with other common CNS proteins. Assay of various rat tissues confirms the localization of GFA protein only to the CNS.     

Development of a non-extracted ''two-site'' immunoradiometric assay for corticotropin utilizing extreme amino- and carboxy-terminally directed antibodies.

The development of a 'two-site' immunoradiometric assay (i.r.m.a.) for the direct estimation of human corticotropin-(1-39)-peptide in plasma is described. The assay is based on the simultaneous addition of 125I-labelled sheep anti-(N-terminal corticotropin) IgG (immunoglobulin G) antibodies and rabbit anti-(C-terminal corticotropin) antiserum to standards and unknowns (0.5 ml) followed by 18h incubation. The use of solid-phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of corticotropin-bound from free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) antiserum, which precipitates bound labelled antibody by complex-formation with rabbit anti-corticotropin antibodies, which are also hormone-bound. Several 125I-labelled sheep anti-(N-terminal corticotropin) IgG preparations were assessed in the i.r.m.a. Although each was derived from antisera raised to a thyroglobulin conjugate of synthetic corticotropin-(1-24)-peptide (Synacthen), purification of immunoglobulins before iodination by selective immunoadsorption resulted in preparations with distinct specificities which demonstrated marked differences in binding to intact human corticotropin-(1-39)-peptide. These preparations are compared in combination with two rabbit anti-(C-terminal corticotropin) antisera. A 'two-site' assay based on the use of 125I-labelled sheep anti-[ corticotropin-(2-16)-peptide] IgG and rabbit anti-[corticotropin-(34-39)-peptide] antiserum was optimized, since steric inhibition of antibody binding was avoided with this combination and because the measurement of only intact human corticotropin-(1-39)-peptide and not fragments was assured by the use of terminal antibodies. This i.r.m.a. is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 4% over the range 30-2200 pg/ml) and high sensitivity [8 pg of corticotropin/ml (95% confidence interval 3.7-12.0) (4 pg minimal detectable mass) can be detected directly in plasma].     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

Detection and characterization of monoclonal antibodies to platelet membrane proteins

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.     

Solid-Phase Radioimmunoassay of Total and Influenza-Specific Immunoglobulin G

An antigen-antibody system of polystyrene tubes coated with immunoglobulin antibody was used for quantitating immunoglobulins. A similar radioimmunoassay method was adapted for a viral antigen-antibody system. The viral system can be used for quantitating viruses and for measuring virus-specific antibodies by reacting with (125)iodine-labeled anti-immunoglobulin G (IgG). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay method were investigated. Comparison of direct and indirect methods of assaying for immunoglobulins or viral antibody indicates that the indirect method is more sensitive and can quantitate a minimum of 0.037 mug of IgG per ml. Results of solid-phase radioimmunoassay for influenza antibody correlate well with hemagglutinin antibody titers but not with complement-fixing antibody titers. Radioimmunoassay results for influenza antibody by solid phase are likewise in agreement with results by the carrier precipitate radioimmunoassay method. The simplicity, reproducibility, and versatility of the solid-phase procedure make it diagnostically useful.     

Extension of dynamic range of sensitive nanoparticle-based immunoassays

Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000 ng/ml, and with cTnI from 25 to 1000 ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07–0.11) and a y-intercept of −7.79 (−17.87–2.29) ng/L (n = 65, r = 0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.     

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

A liquid-phase two-site immunoradiometric assay for human prolactin.

The development of a 'two-site' immunoradiometric assay for human prolactin (hPrl) is described. The assay is based on the addition of radio-iodinated sheep anti-hPrl immunoglobulin G (IgG) and rabbit anti-hPrl serum to standards and unknowns followed by 3 h incubation. The use of solid phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of hPrl-bound and free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) serum which precipitates bound labelled antibody by complex formation with rabbit anti-hPrl antibodies which are also hPrl-bound. Varying the order of addition of specific antibodies had a pronounced effect on the 'operating range' and sensitivity of resultant assays. This was attributed to competition between labelled and unlabelled antibodies for binding sites on the hPrl molecule. The immunoradiometric assay employing 'simultaneous addition' of specific antibodies was compared to a 'simultaneous addition' hPrl radioimmunoassay developed using the same sheep antiserum as that used to prepare the radioiodinated sheep anti-hPrl IgG. This immunoradiometric assay is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 10% over the range 8-10000 mU/l), and high sensitivity (2.6 mU/l, 13 pg). In contrast, the hPrl radioimmunoassay required an incubation of 18 h, demonstrated a much reduced 'operating range' (the precision of dose estimates was less than 10% only over the range 25-1500 mU/l) and reduced sensitivity (9.8 mU/l, 49 pg).     

Comparative evaluation of whole blood D-dimer test to plasma D-dimer test for diagnosis of disseminated intravascular coagulation

Three rapid D-dimer test methods were compared for the diagnosis of acute disseminated intravascular coagulation (DIC). These were (a) SimpliRED, an autologous red cell agglutination assay. (b) DIMERTEST latex agglutination assay, containing monoclonal antibody DD-3B6/22(6), and (c) D-DI latex agglutination assay containing mouse anti-human D-dimer monoclonal antibodies. The D-DI latex method having higher sensitivity (100%) and specificity (81%) in clinically acute DIC was postulated as the gold standard and compared with the other two methods. The results suggest that D-DI latex agglutination assay containing mouse anti-human D-Dimer monoclonal antibodies are the better assay methods amongst all the three kits analyzed. It is advisable to look for the nature of the antibody used to coat the latex particles in plasma based kits. In emergency setting RBC kits may be of some use as rapid diagnosis is advantageous.     

Solid-phase extraction of arginine vasotocin and isotocin in fish samples and subsequent gradient reversed-phase high-performance liquid chromatographic separation

Gradient high-performance liquid chromatography (HPLC) preceded by a solid-phase extraction (SPE) step is described for determining arginine vasotocin and isotocin, the neurohypophysial nonapeptides, in fish plasma samples. The combination of these two methods significantly improves the separation and increases the sensitivity of the assay. The proposed assay may be a useful alternative for analysis of similar nonapeptides in plasma without the use of radioisotopes, while taking into consideration a difference in detection sensitivity.     

Development of a direct microplate enzyme immunoassay for the determination of pregnanediol-3 alpha-glucuronide in urine

A sensitive direct enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was developed. The assay system involves the use of an antiserum against pregnanediol-3 alpha-glucuronide and an enzyme-labelled antigen chemically prepared by linking beta-D-galactosidase to 20 alpha-hydroxy-5 beta-pregnane 3(O-carboxymethyl)oxime. Free from antibody-bound antigen was separated by a solid-phase double antibody method, using a microplate coupled with goat anti-rabbit gamma-globulin. This solid-phase enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was validated in terms of specificity, accuracy and sensitivity. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid phase enzyme immunoassay and conventional radioimmunoassay methods agreed well (n = 30, r = 0.922). This assay system has an advantage over radioimmunoassay, because it does not require the use of radioisotopes. The procedure of this method is very simple, since it does not require purification steps of the biological samples.     

Development of an open sandwich fluoroimmunoassay based on fluorescence resonance energy transfer

We have developed a sensitive, one-step, homogeneous open sandwich fluoroimmunoassay (OsFIA) based on fluorescence resonance energy transfer (FRET) and luminescent semiconductor quantum dots (QDs). In this FRET assay, estrogen receptor beta (ER-beta) antigen was incubated with QD-labeled anti-ER-beta monoclonal antibody and Alexa Fluor (AF)-labeled anti-ER polyclonal antibody for 30 min, followed by FRET measurement. The dye separation distance was estimated between 80 and 90 A. The current method is rapid, simple, and highly sensitive, and it did not require the bound/free reagent separation steps and solid-phase carriers. A concentration as low as 0.05 nM (2.65 ng/ml) receptor was detected with linearity. In addition, the assay was performed with commercial antibodies. This assay provides a convenient alternative to conventional, laborious sandwich immunoassays.     

Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains

Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (S---CH₂CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP–antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP–antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP–antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.     

Production of Monoclonal Antibodies to Guinea Pig Liver Transglutaminase

Monoclonal antibodies are now a powerful tool in biology and medicine. Transglutaminase has been implicated in diverse biological functions, and the characteristics of its catalytic action are suitable for applied enzymology. In this study, we produced hybridoma cells which synthesize monoclonal antibodies against guinea pig liver transglutaminase by fusing mouse myeloma cells with spleen cells of mouse immunized with the enzyme protein. Eight hybridoma clones (coded 2F, 4B, 7C, 8B, 8D, 8E, 9F and 11C) were selected to produce monoclonal antibodies. The subclass of IgG produced by clone 9F was IgG_2a and those from the seven other clones were all IgG₁ The 9F antibody inhibited transglutaminase activity, but the other antibodies did not. A solid-phase antibody-binding assay showed that of these antibodies, 8D antibody has the highest affinity to the antigen. Transglutaminase protein in crude liver extract was identified with Western blotting analysis using 8D antibody as the probe.