Studies on the optimal immunization schedule of experimental animals. V. The effects of the route of injection, the content of Mycobacteria in Freund's adjuvant and the emulsifying antigen

The effects of several conditions for the immunization of mice was studied using an aliquot of a viomycin (VM) protein conjugate as the common primary or booster antigen. Responses of the mice were assessed by measuring mouse serum levels of total immunoglobulin G (IgG) and anti-VM antibody responses using the newly improved two assay methods. The choice of route was found to be a very important factor in immunization and intraperitoneal injection was the most optimal among the four routes studied. The effect of the concentration of Mycobacteria in Freund's complete adjuvant (FCA) was also studied, and it was found that a diluted FCA was more effective than a commercial FCA. The effect of the controlled release of the antigen was studied and three important phenomena were observed: The mice immunized by the mini-osmotic pump-aided controlled release of the antigen responded with similar small amounts of both total IgG and anti-VM antibody regardless of the presence or absence of FCA in the antigen; emulsifying the antigen with FCA was a very important condition for the effective elicitation of the specific antibody; a mixture of antigen and FCA without emulsifying produced little specific antibody and a large amount of total IgG. The more effectively immunized mice responded with a larger decrease in body weight soon after the primary injection.     

Studies on the optimal immunization schedule of the mouse as an experimental animal. The effect of antigen dose and adjuvant type

This study was undertaken to establish the optimal immunogen dose for immunization of mice, using a viomycin-protein conjugate as a hapten immunogen. It was found that specific immunoglobulin G (IgG) formation depends on both the dose of antigen and the type of adjuvant: the optimal antigen dose for an immune response is quite different depending on whether the mice are being treated with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FICA). The total IgG amount depends mainly upon the type of adjuvant used. FCA gave the double the level of IgG compared to that obtained with FICA. The antigen dose was found to have little influence on the total production of IgG. Mice given a primary immunization with 10 micrograms of antigen emulsified in FCA and then given a booster with the same amount of antigen emulsified in FICA produced a strikingly high level of specific anti-viomycin antibody of over 2.5 mg/ml of the antiserum. It was also found that decreases in the weight of the mice were related to the kind of adjuvant used as well as to the level of the specific antibody formed.     

Studies on the optimal immunization schedule of experimental animals. VI. Antigen dose-response of aluminum hydroxide-aided immunization and booster effect under low antigen dose

The dose-response relationships of a viomycin (VM) immunogen for total immunoglobulin (Ig) G and anti-VM antibody response of mouse using aluminum hydroxide as adjuvant was studied. The condition required to absorb a protein on aluminum gel was first established. The effective immunogen dose for total and specific IgG response of mouse using aluminum hydroxide as the adjuvant was found to be in the narrow range of 5 to 20 micrograms, and 10 micrograms per mouse was optimal. The most effective number and intervals of booster injections were studied; when mice were immunized with a lower antigen dose than the optimal, both the number and interval period of booster injections greatly affected the immune response; the more boosters were given, the higher was the response level of specific IgG. The results are contrary to those obtained by immunizing with the optimal or a higher antigen dose.     

High sensitivity microgravimetric biosensor for qualitative and quantitative diagnostic detection of polychlorinated dibenzo-p-dioxins

A piezoelectric immunosensor system was developed for the rapid detection of polychlorinated dibenzo-p-dioxins (PCDDs). The system uses a competitive inhibition enzyme immunoassay (EIA) based on a mouse monoclonal antibody that is specific for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and a conjugate of a dioxin-like competitor coupled to the enzyme horseradish peroxidase (HRP). The anti-dioxin antibody was deposited on a 10 MHz AT-cut quartz crystal resonator modified with a self-assembly monolayer of dithiobis-N-succinimidyl propionate. PCDDs at different concentrations in the range 0.001-10 ng mL-1 were mixed with a constant amount of HRP-conjugated competitor. The frequency responses due to the adsorption of the mixed samples on the biosensor surface were measured. The results show that 2,3,7,8-TCDD can be quantitatively detected with the developed immunosensor in the concentration range 0.01-1.3 ng mL-1. Cross-reactivities of the biosensor to various PCDD congeners were also investigated. The sensitivity and selectivity of the quartz crystal microbalance (QCM) biosensor is comparable to EIA and ELISA methods in the detection of polychlorinated dibenzo-p-dioxins. The developed QCM immunosensing system offers significant improvements in speed, sample throughout and cost for the qualitative and quantitative detection of PCDDs compared with GC-MS.     

Studies on the optimal immunization schedule of experimental animals. IV. The optimal age and sex of mice, and the influence of booster injections

To establish the optimal condition for preparing mouse antiserum specific to a drug, the optimal age and sex of mice for the immune response were studied by measuring the mouse serum levels of total immunoglobulin G (IgG) and specific antibody to viomycin, as well as the changes in weight of mice immunized with a viomycin immunogen. It was observed that age was a more important factor than sex, and strongly affected productions of both total and specific IgGs of mice. The mice aged 8 weeks yielded the highest levels of both total IgG and the specific antibody. In the study on the influence of booster schedule, the number of boosters given had a larger influence on the immune response than the interval between priming and boosters. The greater the number of booster shots given, the less was the production of total and specific antibodies. The decrease in the weight of mice after immunization was also studied in more detail; it was found that it only occurred in the first week after priming but not after a booster injection. The mice aged eight weeks showed the largest weight loss.     

New highly sensitive enzyme immunoassay for the determination of pravastatin in human plasma

New highly sensitive enzyme immunoassay (EIA) has been developed and validated for the determination of pravastatin (PRV) in human plasma samples. PRV was coupled to keyhole limpt hemocyanin (KLH) and bovine serum albumin (BSA) via its terminal carboxylic acid group by carbodiimide reagent. PRV-KLH conjugate was used as an immunogen for raising anti-PRV polyclonal antibody in rabbits. The generated anti-PRV antibody recognized PRV with high affinity and selectivity. PRV-BSA conjugate was immobilized onto microwell plates and used as a solid phase. The assay involved a competitive binding reaction between PRV, in plasma sample, and the immobilized PRV-BSA for the binding sites on a limited amount of the anti-PRV antibody. The anti-PRV antibody bound to the plate wells was quantified with horseradish peroxidase-labeled anti-immunoglobulin second anti-rabbit IgG antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of PRV in the sample was quantified by its ability to inhibit the binding of the anti-PRV antibody to the immobilized PRV-BSA and subsequently the color development in the assay wells. The conditions of the proposed EIA were investigated and the optimum conditions were employed in the determination of PRV in plasma samples. The assay limit of detection was 0.2 ng mL⁻¹ and the effective working range at relative standard deviation (RSD) of ≤5% was 0.5-20 ng mL⁻¹. The mean analytical recovery of PRV from spiked plasma was 100.9 ± 2.98%. The precision of the assay was satisfactory; RSD was 2.61-3.70 and 3.96-4.17% for intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA has a great value in the routine analysis of PRV in plasma samples for its therapeutic monitoring and pharmacokinetic studies.     

A highly sensitive and specific polyclonal antibody-based enzyme immunoassay for therapeutic monitoring and pharmacokinetic studies of atorvastatin

This study describes the development and validation of a highly sensitive and specific enzyme immunoassay (EIA) for therapeutic monitoring and pharmacokinetic studies of atorvastatin (ATR). The assay employs a polyclonal antibody that recognizes ATR with high specificity and affinity, and ATR conjugated to bovine serum albumin (ATR-BSA) immobilized onto microwell plates as a solid phase. The assay involved a competitive binding reaction between ATR and the immobilized ATR-BSA for the binding sites on a limiting amount of the anti-ATR antibody. The bound anti-ATR antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin secondary antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of ATR in the sample was quantified by its ability to inhibit the binding of the anti-ATR antibody to the immobilized ATR-BSA and subsequent color development in the assay wells. The conditions for the EIA were investigated and optimized for the determination of ATR in plasma samples. The limit of detection was 0.04 ng mL^?1 and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1–10 ng mL^?1. Mean analytical recovery of ATR from spiked plasma was 99.3?±?2.8%. The precision of the assay was satisfactory; RSD were 2.7–4.6 and 3.3–5.7% for intra- and inter-assay precision, respectively. The reliability of the EIA was confirmed by HPLC. The EIA is convenient, and one can analyze ～ 200 samples per working day, facilitating the processing of large-number of samples of ATR.     

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 μg mL⁻¹). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5–20 μL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system. Sang Youn Hwang and Yoichi Kumada have same rights on this paper.     

A new enzyme immunoassay for a solid Chinese crude drug, pinellia tuber

A new immunoassay for a solid Chinese crude drug was studied. An antiserum specific for Pinellia tuber was elicited in two rabbits. Using the antiserum and powdered Pinellia tuber-coated microtiter plate as the immunological reagents, and beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G (IgG) as the tracer, a new enzyme immunoassay for a solid Pinellia tuber with a working range between 0.1 and 1000 micrograms/ml was developed. The assay was specific for a solid Pinellia tuber and showed low cross-reaction values on other Chinese crude drugs and the extract of Pinellia tuber. The specificity of the assay was compared with the selected antibody enzyme immunoassay (SAEIA) for the extract of Pinellia tuber recently developed. Both methods utilized the same immunological reagents such as the serum and the enzyme-labeled goat anti-rabbit IgG, and the only difference between them was the solid-phase antigen used. The assay results of several antigens determined by them were quite different, showing that selective measurements of different antigens, either solid or the extract of Pinellia tuber, were possible using the same antiserum, when the tracing reaction in the immunoassay was adequately selected.     

Regulation of the humoral immune response by polyspecific natural autoantibodies

Two different BALB/c IgMk polyspecific monoclonal natural autoantibodies E7 and D23 were administered to neonatal BALB/c mice. When adults, these mice were immunized and challenged with calf myosin, BALB/c actin, human transferrin, calf thymus DNA or TNP-coupled bovine serum albumin (TNP/BSA), in complete Freund's adjuvant. The levels of serum antibody were evaluated by enzyme immunoassay.No differences in anti-actin, anti-transferrin and anti-DNA antibody titres were noted between control and antibody-treated mice. However, anti-myosin antibody titres significantly increased in mice treated with either the E7 or D23 antibody, and anti-TNP antibody titres significantly decreased in mice treated with E7 but not with D23. These differences persisted after antigenic challenge and involved only the IgG response of treated mice. These results suggest that polyspecific natural autoantibodies may be involved in the regulation of the humoral immune response.     

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.     

Method for the synthesis of multi-epitopic Streptococcus pyogenes lipopeptide vaccines using native chemical ligation

The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.     

Rapid methods for detection and enumeration of Campylobacter spp. in foods

Campylobacter spp. are the most commonly reported bacterial cause of acute diarrheal disease in humans throughout the world. Traditional cultural methods for the detection and quantitation of Campylobacterspp. are slow and tedious; therefore, specific, sensitive, and rapid methods for campylobacters are needed to collect sufficient data for risk assessment and food safety policy development. We developed several rapid methods based on polymerase chain reaction (PCR), DNA hybridization, hydrophobic grid membrane filters (HGMFs), and enzyme immunoassays (EIAs). A PCR assay targeting C. jejuni, combined with a simple sample preparation procedure, detects as few as 0.3 most probable number (MPN)/mL C. jejuni in naturally contaminated chicken rinses after 20-24 h enrichment. An HGMF-EIA method using a commercial polyclonal antibody for Campylobacter detects and enumerates thermophilic Campylobacter spp. from spiked chicken rinse and milk, and naturally contaminated chicken rinses. A C. jejuni-specific probe in an HGMF-DNA hybridization protocol specifically detects and quantitates C. jejuni in food samples. A dot-blot EIA combined with an MPN procedure quantitates thermophilic campylobacters from samples that might be difficult to filter through HGMFs.     

Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.     

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.     

Enzyme immunoassay of staphylococcal enterotoxins in dairy products with cleanup and concentration by immunoaffinity column

Two different immunoaffinity columns (IACs) were prepared for detection of staphylococcal enterotoxins (SETs) from dairy products. First, a specific IAC for staphylococcal enterotoxin A (SEA), IAC-1, was prepared by coupling monoclonal antibody (mAb) directed against SEA; second, a polyspecific IAC for SEA, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SECs), and staphylococcal enterotoxin D (SED), IAC-2, was prepared by coupling a mixture of mAbs against SEA, SECs, and SED, and rabbit IgG against SEB. These columns were applied for detection of SETs in dairy products, after extraction, immunoaffinity chromatography, and enzyme immunosorbent assay (EIA). Overall recoveries from dairy products spiked with 1 ng SEA/25 g averaged 81.2% (range, 76-85%) on IAC-1. The repeated use of IAC-1 was then determined with good efficiency of 91.5%, in more than 10 runs. On the other hand, a recovery yield of 77% of SETs (SEA, SEB, SEC, and SED) from dairy products spiked with 2.5 ng of each enterotoxin per 25 g, was obtained with IAC-2. IAC-2 was also successfully subjected to the chromatography of naturally contaminated foods implicated in staphylococcal food poisoning outbreaks. This new extraction-concentration-immunoaffinity-chromatography method (ECIC) is very useful for improving staphylococcal enterotoxin detection and eliminating matrix effect in EIA of dairy products.     

Production of a monoclonal antibody and development of enzyme-linked immunosorbent assay for alkyl ethoxylates

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed for the determination of alkyl ethoxylates (AEs) that are the most widely used nonionic surfactants in the world. Three types of hapten, hemi-succinated AEs (C12EO7suc, C16EO23suc, and C18EO10suc), were synthesized and conjugated to bovine serum albumin (BSA) for mouse immunization. The mice immunized with the C12EO7suc-BSA that showed the high immune responses were used for cell fusion. The obtained monoclonal antibody (TFG2-76) was specific to AEs, which had alkyl (C) and ethoxy (EO) chain lengths of C10-12 and EO5-15, respectively. Two types of solid support, namely, a polystyrene tube and a 96-well microplate, were used for antibody immobilization. The working ranges of the tube-type and plate-type ELISAs were 2-100 and 20-1000 μg/L with IC₅₀ values of 12 and 71 μg/L AE (C12EO7), respectively. Moreover, the lowest quantification limit of plate-type ELISA could be lowered to 5 μg/L by decreasing the coated antibody concentration. Cross-reactivities with non-AE surfactants were determined, and the assay proved highly selective for AEs. The application of plate-type ELISA to determine spiked AEs in distilled water, tap water and river water provided good recoveries without matrix effects.     

Enzyme immunoassay of thyrotropin releasing hormone (TRH).

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a thyrotropin releasing hormone (TRH)-like immunoreactive substance has been developed. In order to synthesize TRH-labeled beta-D-galactosidase (beta-gal), a newly devised TRH derivative, pGlu-His-Pro-NH-(CH2)6-NH2 (TRH-Hex), was employed. TRH-Hex was linked to beta-gal by the N-(epsilon-maleimidocaproyloxy) succinimide coupling procedure. For competitive reactions, the TRH antibody was incubated with standard TRH and TRH-Hex-beta-gal (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.8 to 100 pmol/well of TRH.     

Studies on peptides. CLXVII. Solid-phase syntheses and immunological properties of fragment peptides related to human malaria circumsporozoite protein

A glycine-linked tetramer of Asn-Ala-Asn-Pro, a tandem repeated sequence of malaria circumsporozoite (CS) protein, was synthesized by the Boc-based solid phase method, followed by deprotection with 1 M trimethylsilyl trifluoromethanesulfonate-thioanisole in trifluoroacetic acid. In addition, three tetramer-related peptides were similarly synthesized, i.e., a 34-residue peptide [linked with TH, a proposed T-cell epitope of CS, at the C-terminus of the tetramer], a 46-residue peptide and a 59-residue peptide [linked with HA or HA', two proposed T-cell epitopes of influenza hemagglutinin protein, at the N-terminus of the above 34-residue peptide]. Their immunological properties were examined by enzyme-linked immunosorbent assay, for which three different congenic strains of mouse were used to raise the specific antibodies. Despite conjugation of T-cell epitopes to the tetramer, the mice of low-responder strains to the tetramer failed to produce any antibody specific to the tetramer. However, with the aid of recombinant interleukin 2 as an adjuvant, the low-responder mice produced antibody with relatively high titers.     

Development of an enzyme‐linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti‐glycyrrhizic acid monoclonal antibody

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti‐glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid–bovine serum albumin conjugate with the hypoxanthine–aminopterin–thymidine‐sensitive mouse myeloma cell line (Sp2/0‐Ag14). Subsequently, an indirect, competitive enzyme‐linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12–2500 ng/mL. Both intra‐assay and inter‐assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine.