Simultaneous immunoenzymometric assay for antibodies against human interleukin-2 and human serum albumin in rat serum

A simultaneous immunoenzymometric assay for anti-human interleukin-2 antibody and anti-human serum albumin antibody in rat serum was developed. Two antigen-immobilized polystyrene balls were immersed in a diluted serum sample in an assay tube and then the antibodies on the balls were made to react with a horseradish peroxidase labelled anti-rat IgG antibody in the same tube after washing. The enzyme activity of each ball was measured by fluorometry. Not only were the sensitivity (70 ng/ml each), assay recovery (100-101%), and precision (C.V. = 5-13%) comparable to those of conventional immunoenzymometric assays using one antigen-immobilized ball but the assay was also much more feasible for mass routine assays. Thus, conventional immunoassays can be replaced by this convenient simultaneous method.     

Enzyme immunoassays for the detection of sulfamethazine,sulfadiazine, sulfamethoxypyridazine and trimethoprim in milk

Polyclonal antibodies against sulfamethazine, sulfadiazine, sulfamethoxypyridazine and trimethoprim were prepared by using bovine serum albumin conjugates of the respective substances as antigens for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested by using the respective drug coupled to horseradish peroxidase as the labelled antigen in a competitive assay. The antiserum against sulfamethazine showed relative cross‐reactivities of 100 and 56% with sulfamethazine and sulfamerazine, respectively. The antiserum against sulfadiazine showed relative cross‐reactivities of 100, 12, 11 and 10% with sulfadiazine, sulfasalazine, sulfamerazine and sulfathiazole, respectively. The assays for sulfamethoxypyridazine and trimethoprim showed no relative cross‐reactivity above 1%. The detection limits (in buffer solution) for sulfadiazine, sulfamethazine, sulfamethoxypyridazine and trimethoprim were at 1.1, 1.3, 2.8 and 6.0 ng/ml, respectively. The recovery of the sulfonamides from milk samples, artificially contaminated at levels of 5, 10, 50 and 100 ppb, was between 68 and 99%. Recovery of trimethoprim from artificially contaminated milk samples was about 90% at concentrations of 50 and 100 ppb.     

Improved group determination of tetracycline antibiotics in competitive enzyme-linked immunosorbent assay

Seven tetracycline (TC) antibiotics were used as haptens for synthesis of conjugated antigens and as standards in indirect competitive enzyme-linked immunosorbent assay (ELISA). Seven conjugates were prepared by formaldehyde condensation (f) and another seven antigens were synthesised by linking TCs with periodate-oxidised transferrin (TF(pi)). Two gelatine–lymecycline (LC) conjugates were the products of glutaraldehyde and activated esters reactions. To estimate the influence of solid-phase antigen on assay specificity, all the conjugates were absorbed on polystyrene plates and using anti-bovine serum albumin–TC(f) sera, the ELISAs were developed and compared. The variant with immobilised TF–chlortetracycline (CTC(pi)) showed the best group specificity: recognition of seven TCs differed only in 5.3 times. This assay displayed sensitivity 0.1 ng/ml and cross-reactivity indexes: TC (100%), CTC (105%), oxytetracycline (19.8%), methacycline (23.6%), minocycline (70%), doxycycline (25%) and LC (50%). The dependence of ELISA specificity on immobilised antigen spatial structure was demonstrated. Solid-phase conjugate selection is a useful tool for modifying assay specificity.     

Production and characterization of a monoclonal antibody to chloramphenicol

A monoclonal antibody to chloramphenicol (CAP) was produced. After immunization of BALB/c mice with CAP base coupled to human serum albumin and incubation of the stimulated splenocytes in vitro in the presence of antigen for three days, these splenocytes were hybridized with X63‐Ag8·653 myeloma cells. The antibody, designated 6A10, proved to be IgG2b, and it had a detection limit for CAP of 10 ng/ml (0·5 ng/assay) in the direct enzyme immunoassay using horseradish peroxidase‐labelled CAP. The cross‐reactivities with CAP base, p‐nitrobenzyl alcohol, and p‐nitrophenol were 5·0, 0·94, and 0·007%, respectively. No cross‐reactivities were observed with penicillin, tetracycline and thiamphenicol, respectively.     

Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis.     

Enzyme-linked immunosorbent assay (ELISA) of penicillin amidases

Non-competitive, sandwich enzyme immunoassay for both penicillin amidases from Escherichia coli is described. The assay involves the use of monospecific antibodies and their conjugates. The amidases inactivated by heating and by acid- or alkali-treatment cannot be assayed. Reproducible results for each amidase were achieved within 5-6 hours in the range of 3-500 ng/ml (0.25-40 mU/ml) and coefficient of variation was 13%.     

Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat anti-human immunoglobulin G peroxidase, goat anti-rabbit immunoglobulin G alkaline phosphatase, rabbit anti-human immunoglobulin G, and other enzyme conjugates contained endotoxin. Furthermore, the staphylococcal protein A, horseradish peroxidase, and bovine alkaline phosphatase used to prepare enzyme conjugates also contained endotoxin. Commercially obtained bovine alkaline phosphatase contained as much as 1.0 microgram of endotoxin per ml of enzyme solution. Both commercially prepared enzyme conjugates and those prepared by us contained endotoxin as determined by their absorption to immobilized monoclonal antibody to lipid A or to immobilized Limulus amoebocyte lysate. The results of this study further suggest that the endotoxin was associated with the enzyme component of the conjugate. In a competitive inhibition enzyme immunoassay, 10 micrograms of lipid A per ml inhibited binding of the enzyme conjugate to adsorbed Limulus amoebocyte lysate, thereby confirming that endotoxin mediated the binding of the conjugate in that system. The potential significance of endotoxin bound to enzyme conjugates may be far reaching because of the ubiquity of endotoxin in conjugates and the prevalence of antibodies to endotoxin in mammalian serum.     

Detection by ELISA of low transferrin levels in bronchoalveolar secretions of preterm infants

Transferrin levels in bronchoalveolar secretions (BAS) are very low compared to serum levels in humans. For the exact measurement of transferrin concentrations in BAS a very sensitive assay was developed as a double sandwich enzyme immunoassay using the combination of a polyclonal and a monoclonal antibody against human transferrin. The measurable range of the assay was 1.5 to 100 ng/ml of human transferrin. The lowest measurable value was 0.84 ng/ml and the sensitivity of the assay was 0.88 ng/ml. The coefficient of variation was 14.1% for 25 ng/ml (intra-assay) and 11-20% (inter-assay). The levels measured in 123 samples of BAS of preterm infants ranged between 0.03 and 8.93 (microgram/microgram secretory component (SC)). The determination of transferrin in BAS of preterm infants is helpful in determining oxidative damage, e.g. the availability of free iron, in the neonatal lung. The transferrin concentration in BAS of neonates who recovered from respiratory distress syndrome (RDS) in the first six days of life was 0.48 compared to 0.52 ((microgram/microgram SC), median range) for infants who developed chronic lung disease.     

Simultaneous use of poly- and monoclonal antibodies as enzyme tracers in a one-step enzyme immunoassay for the detection of hepatitis B surface antigen.

A one-step third generation enzyme immunoassay (EIA) was developed for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma using a polyclonal (Pab-HBsAg) and two monoclonal antibodies to HBsAg (Mab1-HBsAg and Mab2-HBsAg). In this assay, the solid phase is coated with Mab1-HBsAg and the specimen is incubated simultaneously with peroxidase labelled Pab-HBsAg and Mab2-HBsAg. If HBsAg is present in a specimen it will form sandwich complexes with capture and tracer antibodies. The hook effect, observed in some HBsAg detection tests when a high concentration of HBsAg is present, was minimized in this assay by increasing the concentration of the peroxidase-labelled Mab2-HBsAg. The sensitivity of this assay for HBsAg/ay and HBsAg/ad subtypes in a standard (2 h incubation) procedure was 0.6 and 0.3 ng/ml and in an overnight (16-22 h incubation) procedure 0.2 and 0.15 ng/ml, respectively. Strong elimination of the hook effect was observed with specimens containing high levels of HBsAg compared with test results using peroxidase-labelled Pab-HBsAg alone as enzyme tracer. This EIA offers a procedure, with a high specificity and wide range of sensitivity for the detection of HBsAg in human sera or plasma.     

O-antigen biotin conjugates. Preparation and use in direct competitive enzyme immunoassays.

Bacterial O-antigens coupled to biotin were shown to function well as labelled antigens in direct enzyme immunoassay. O-polysaccharides released from lipopolysaccharides by mild acid hydrolysis were oxidized by sodium periodate at sites located within the lipopolysaccharide inner core region and the generated aldehyde groups were subjected to reductive amination with 1,3-diaminopropane to yield per-aminated O-polysaccharide derivatives. Biotin was coupled to the introduced amino groups by way of a N-hydroxy-succinimide ester derivative. Biotinylated polysaccharides were used in direct enzyme immunoassays, that employed monoclonal antibody coated microtitre plates and detection of bound biotinylated antigen by streptavidin/horseradish peroxidase reagent. The assay format was designed for rapid estimation of the affinity constants of monoclonal antibodies for small oligosaccharide inhibitors, but the assay is also well suited to fast and sensitive detection of bacterial O-polysaccharides at concentrations within the range 1-10 ng/ml.     

Quantitation of mouse monoclonal antibody and human anti-mouse antibody response in serum of patients

The assays required to study pharmacokinetics of monoclonal antibodies and human anti-mouse antibody response in man, utilizing enzyme linked immunosorbent assay, have been at best semi-quantitative. We have developed and well characterized a quantitative assay for measurement of murine monoclonal antibodies and human anti-mouse antibody in circulation of patients injected with murine monoclonal antibodies. The sensitivity of mouse IgG assay is approximately 1 ng/ml and cross reactivity of IgG's from other species were less than 0.01%. The pre-therapy samples from 30 patients treated with monoclonal antibody CO17-1A as well as serum from 54 individuals had no detectable human anti-mouse antibody indicating specificity. The sensitivity of this assay was approximately 10 mg/ml. The utility of these assays was demonstrated in a group of patients receiving 400 mg of monoclonal antibody CO17-1A. The half-life and the degree of immune response can be measured using these assays.     

The use of N-(aminobenzoyloxy) succinimide as a two-level heterobifunctional agent for the preparation of hapten-protein conjugates. Daunomycin as a model hapten with an amino group

Three geometric ortho-, meta-, and para-isomers of N-(aminobenzoyloxy)succinimide (ABS) were synthesized, and their usefulness as a two-level heterobifunctional cross-linking agent in the preparation of hapten-protein conjugates was evaluated. The conjugation was based on the principle that ABS reacts immediately with an amino group of a hapten, and an aminobenzoyl group incorporated into the hapten is then activated by diazotization to a functional diazobenzoyl group acting on tyrosine or histidine residues of the protein. Using the anti-tumor antibiotic daunomycin (DM) as a model hapten, the three isomers of ABS were compared for their ability to conjugate DM with bovine serum albumin (BSA); DM incorporation onto a BSA molecular was found to occur to the highest degree with m-ABS, followed by p-ABS. while o-ABS completely failed to conjugate under the same coupling conditions. Using m-ABS it was possible to introduce more than 10 molecules of DM per BSA molecule. One of the DM-BSA samples was used as the immunogen for the production of anti-DM serum in a rabbit. The antibody specificity was shown to be direct to DM but not to other anti-cancer drugs (bleomycin, mitomycin C, actinomycin D and 5-fluorouracil) by the double antibody enzyme immunoassay (DEIA) using DM-beta-galactosidase conjugate as a label. An enzyme-linked immunosorbent assay (ELISA) for anti-DM IgG was developed using a DM-human serum albumin (DM-HSA) conjugate similarly prepared with m-ABS and horseradish peroxidase-conjugated goat anti-rabbit IgG as the solid-phase antigen and the labelled second antibody, respectively. This ELISA permitted us to measure accurately as little as 50 ng of anti-DM IgG per ml using a standard anti-DM IgG which had been purified from the anti-DM serum using an affinity column of Sepharose 4B with DM-HSA as the ligand. Using this ELISA as well as a sandwich enzyme immunoassay (SEIA) for total IgG, serum levels of anti-DM IgG and total IgG levels were easily monitored in a rabbit following immunization with DM-BSA. These results indicate that the use of DBS provides a novel method for preparing hapten-protein conjugates which will be useful in biochemistry and immunochemistry.     

Characterization of antibody labelled colloidal gold particles and their applicability in a sol particle immuno assay (SPIA)

This study describes the characterization of antibody labelled colloidal gold particles and their applicability in a sol particle immuno assay (SPIA) to quantify murine monoclonal antibodies of all IgG isotypes. Two physical methods (transmission electron microscopy (TEM) and dynamic light scattering (DLS], were used to obtain information about particle size and morphology of the gold sols, but only with DLS antibody labelling could be detected. In addition, electrophoretic methods like agarose electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed also that antibody labelling was successful. The biological activity of the antibody gold conjugates was determined by using them in a SPIA and as an electrondense marker in an immunogold labelling procedure to visualize meningococcal surface exposed outer membrane proteins labelled with monoclonal antibodies. The SPIA was applicable to determine murine monoclonal antibodies of all IgG isotypes with a sensitivity of 20-80 ng/ml and a co?ffici?nt of variation of 6.7 +/- 2.2%.     

Highly sensitive biotin-avidin sandwich ELISA for the rapid detection of pneumococcal capsular polysaccharide antigens

The immunological detection of soluble pneumococcal polysaccharide antigens in pathological products is of importance in the direct diagnosis of meningitis or pulmonary infections. We have developed a double antibody sandwich ELISA method using a biotin-avidin system using antibodies constituted with a mixture of IgGs from pooled and/or monospecific antipneumococcal sera provided by the Danish Statens Seruminstitut. The sensitivity of this rapid ELISA method was optimized with purified capsular polysaccharides of the 24 main pneumococcal serotypes. With incubation steps of 30 min at 37 degrees C for the antigens and the conjugates, the detection limit was close to 1 ng/ml for 75% of the purified polysaccharides. A retrospective study of 46 CSF samples established the validity of the assay. This type of modified ELISA system represents a specific, sensitive and rapid procedure for the potential detection of capsular soluble antigens of all pneumococcal serotypes.     

Radioimmunologic assay of alpha-fetoprotein in various normal and pathological conditions]

AFP can be measured by radioimmunoassay in 52 hours using an incubation at 18 degrees C for 48 hours and the double antibody solid phase method to separate the free labelled AFP from the labelled AFP bound to the antibodies. In these conditions, the sensitivity and precision of the assay are very satisfactory and the results are directly in correlation with the results obtained using the classical double antibody method. In the serum of normal people, except pregnant women, the concentration of AFP is not detectable or is less than 20 ng/ml. In the serum of pregnant women, AFP increases from the 8th. week of gestation to the 32nd., then stabilises or gradually decreases.     

Dextran, a hapten carrier in immunoassays for s-triazines. A comparison with ELISAs based on hapten-protein conjugates.

A conjugate of 2-aminohexylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (AHA), a derivative of the herbicide atrazine, with dextran as carrier has been synthesized and used as the coating antigen in ELISA procedures. The quantification of terbutryn, atrazine and prometryn in ELISA formats using monoclonal antibodies and the AHA-dextran conjugate was at least as sensitive as ELISAs using protein conjugates as immobilized antigens (sensitivity at 50% B/B0 was 0.4-0.6 micrograms/l for terbutryn). Formats with immobilized antibody and enzyme labelled AHA proved to be less sensitive (1.5 micrograms/l for terbutryn). The observed differences in sensitivity do not apparently result from structural effects of the carrier bound hapten since all conjugates were prepared with one form of the hapten, 2-aminohexylamino-atrazine, which was covalently linked via its amino function to the carriers or enzymes.     

Quantitative determination of pegylated consensus interferon in rhesus monkey serum using a competitive enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) with generated mouse anti-consensus interferon (CIFN) antibody was developed for quantitative determination of pegylated consensus interferon (PEG-CIFN) in rhesus monkey serum. The operational concentrations of the original assay were determined using the chessboard method. ELISA working range was 10–5000?ng/ml, corresponding to a limit of quantification of 8.4?ng/ml in rhesus monkey serum. In a precision test, intra-assay CV ranged 1.8–9.6% and inter-assay CV ranged 3.5–12.7%. Relative recovery rate of this ELISA assay ranged from 102.65–115.77%, with RSD values ranging from 2.26–5.44%. Three groups of rhesus monkeys received 1250μg/kg, 300μg/kg, 150μg/kg PEG-CIFN by subcutaneous administration, and blood samples were drawn via the femoral vein at the specified time points. PEG-CIFN in rhesus monkey serum was determined using the competitive ELISA, and the results were compared with antiviral activity assay. In conclusion, the competitive ELISA assay we developed has sufficient sensitivity, precision, and accuracy for the analysis of a rhesus monkey serum sample.     

The use of a specific enzyme‐linked immunosorbent assay to monitor the persistence of penicillin g residues in milk following intramammary antibiotic treatment

Lactating dairy cows were treated with a single intramammary infusion of a commercial penicillin G‐dihydrosteptomycin formulation commonly used for the treatment of mastitis. The levels of penicillin G excretion were measured directly in the raw milk using a specific competitive enzyme‐linked immunosorbent assay. Clearance of penicillin residues (<3–0 ng, 0–005 IU per ml) was seen between four and nine milkings for individual animals with 68% of the injected antibiotic being excreted intact. The dynamic range of the assay was between 3 and 96 ng/ml, the mean intra‐assay variation was 5–3% and interassay variation was 5–4% at the limit of quantification.     

Measurement of small quantities of alpha 2-macroglobulin in bronchoalveolar lavage fluid by enzyme-labelled immunoassay, and its clinical implication in pulmonary disease

We established an enzyme labelled immunoassay for the determination of alpha 2 macroglobulin (alpha 2M). The assay range was from 2 to 140 ng/ml and the within-assay coefficient of variation (CV) were 5.2% at 31.2 ng/ml and 6.4% at 62.5 ng/ml. Between-day CV ranged from 6.9% to 15.4%. Using this method, alpha 2 M was determined in the bronchoalveolar lavage fluid (BALF) from patients with interstitial lung diseases. Those diseases were active and inactive sarcoidosis, hypersensitivity pneumonitis and idiopathic pulmonary fibrosis (IPF, including collagen disease). We divided the IPF patients into two groups, 'acute type' and 'chronic type', judging from the prognosis. alpha 2 M/Albumin ratio in BALF in the active sarcoidosis and acute type IPF groups is significantly higher than that in the inactive sarcoidosis and chronic type IPF. These findings suggest that alpha 2 M in BALF can be a sensitive marker of the interstitial lung disease.     

Enzyme immunoassay for antibodies to thyroxine in human serum using synthesized antibody as a calibrator

We prepared human IgG labeled with rabbit antibodies to thyroxine, and used it for the assay of antibodies to thyroxine in human serum as a calibrator. The assay system consisted of thyroxine labeled with beta-D-galactosidase and a microcolumn containing goat antibodies to human IgG immobilized on Sepharose 4B. Each serum sample or standard serum containing human IgG labeled with anti-thyroxine antibodies was incubated with the enzyme-labeled thyroxine, and the reaction mixture was passed through the microcolumn. The column was washed to remove the unbound label, and enzyme activity of the bound label was assayed. The minimum detectable amount of the anti-thyroxine antibodies was 0.3 ng/assay tube. The analytical recoveries of human IgG labeled with the antibodies added to human serum were from 103% to 108%. We measured anti-thyroxine antibodies in human sera from 187 patients with autoimmune thyroid diseases. The antibodies were detected in 32 serum samples, and the values in positive samples varied from 0.31 to 2.31 micrograms/ml. On the other hand, in 58 sera from patients with non-thyroid diseases, the antibodies were detected in only two samples.