COMPARISON of ANALYTICAL SENSITIVITIES of THREE ENZYME IMMUNOASSAY PROCEDURES FOR the DETECTION of SALMONELLA LIPOPOLYSACCHARIDE

The analytical sensitivities of three different enzyme linked immunoassays (ELISA), two competitive and a capture format were assessed. the assay systems employed monoclonal antibodies to Salmonella lipopolysaccharide (LPS) outer core epitopes to detect crude LPS antigens from Salmonella typhimurium. the most sensitive ELISA was the capture procedure, being capable of detection 1.3 ng/ml of LPS. This technique, however also gave the greatest between-test variation and as a result, the lowest amount that could be detected with a 95% confidence limit was actually 12.8 ng/ml and it took the longest time to perform (3 h, 30 min). A competitive ELISA using limiting monoclonal antibody to compete between solid phase antigen and soluble antigen in the sample, ranked second in sensitivity, and can detect 2.8 and 3.8 ng/ml of LPS when tested with two different monoclonal antibodies. However, because of the slight between test variation, the actual sensitivities that could be detected with a 95% confidence limit were 3.1 and 4.6 ng/ml, respectively. This test takes approximately 1 h and 30 min to perform.
The classical type of competitive assay, employing a labelled antigen, was the least sensitive being capable of detecting 5.8 ng/ml if the LPS was conjugated with horseradish peroxidase and 16.0 ng/ml if alkaline phosphatase was used as a label. to account for the between-test variation, the sensitivities with a 95% confidence limit were 8.6 and 18.7 ng/ml for the respective assays, which take 2 h and 15 min to perform.
These sensitivities compare favorably with those published for similar assays, but all of the procedures were judged insufficiently sensitive for direct use on food samples to be tested for the presence of Salmonella species. However, the assays would be quite suitable for demonstration of Salmonella sp. after an enrichment procedure.     

Further characterization of cooperative interactions of monoclonal antibodies

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.     

Properties of monoclonal antibodies interacting with determinants of hepatitis B virus surface antigen

Fourteen hybridoma clones have been isolated producing the monoclonal antibodies to the surface antigen of the hepatitis B virus (HBsAg). Monoclonal antibodies have been shown to react in high titres with HBsAg in the reactions of PHA, PH and ELISA. The specificity of monoclonal antibodies to two antigenic determinants has been found by the competitive solid phase ELISA technique. Monoclonal antibodies from nine clones react with one determinant while monoclonal antibodies from the rest five clones react with the other nonoverlapping determinant.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive ELISA and for antigen capture in a sandwich ELISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by Western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from Egyptian or South African lineages. These monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive eLISA and for antigen capture in a sandwich eLISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from egyptian or South African lineages. these monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.Key words: RVF, nucleoprotein, competitive ELISA, RVFV lineages     

Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.     

Monoclonal antibodies for use with 125iodine-labeled radioligands in progesterone radioimmunoassay

The interactions of heterologous and homologous 125I-iodinated radioligand with polyclonal and monoclonal antibodies directed against 11-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin were compared. Our data show that, with a polyclonal antibody, the use of the same bridge in the tracer and the antigen results in a low sensitivity while a heterologous tracer can decrease markedly the titer of antibody but increases the sensitivity of the radioimmunoassay. Due to the high specificity of monoclonal antibodies, the interaction with heterologous and homologous iodinated tracers is extremely variable from one antibody to another but the present results demonstrate that radioimmunoassays of very high sensitivity can be obtained with homologous and heterogeneous tracers. However, an appropriate tracer has to be selected to meet the requirement of such an assay.     

抗登革病毒NS1单抗的制备及检测NS1方法的建立

制备抗登革病毒NS1蛋白单克隆抗体,建立检测NS1的ELISA方法。表达1~4型登革病毒NS1蛋白,将1型NS1蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术制备单克隆抗体。经ELISA、Western blotting、间接免疫荧光筛选和鉴定单克隆抗体,进行纯化和HRP标记。通过鉴定每两株单抗之间是否存在竞争作用,选择非竞争单抗组合并建立NS1捕获法ELISA。结果获得7株高滴度抗NS1单抗,捕获法ELISA可以检出10ng/mL NS1。原核表达登革病毒NS1蛋白制备的单抗可以和天然病毒抗原反应,NS1捕获法ELISA可以用于登革病毒感染检测。     

Evaluation of monoclonal antibodies against Plasmodium vivax sporozoites for ELISA development

Nine monoclonal antibodies (MAbs) developed against Plasmodium vivax (Grassi & Feletti) salivary gland sporozoites were evaluated for use in an enzyme-linked immunosorbent assay (ELISA), using sporozoites developed in Anopheles dirus Peyton & Harrison An. gambiae Giles and An.maculatus Theobald. Four of the antibodies were unsuitable due to the low sensitivity of the resulting assays or the requirement for high concentrations of capture antibody. An additional two MAbs were rejected because they resulted in assays with high background absorbance, attributed to self-binding. Of the three remaining MAbs, the use of Navy vivax sporozoite (NVS) 3 resulted in an ELISA with the highest sensitivity and the lowest concentration requirement for capture antibody. Assay sensitivity varied with sporozoite strain indicating possible quantitative epitope heterogeneity. None of the MAbs cross-reacted with the heterologous sporozoites tested by immunofluorescence antibody assay (IFA). The IFA activity was not an indicator of ELISA sensitivity. The use of MAb NVS 3 in a standardized ELISA method resulted in an assay 10 times more sensitive than reported previously for P. vivax sporozoites, with a detection limit of fewer than 100 sporozoites per mosquito.     

Configuration and optimization of a common two-site immunoassay for human prolactin using a chemiluminescent tracer and an enzymatic tracer

We have developed a two-site immunoassay for human prolactin using two monoclonal antibodies: one of them immobilized on the solid phase and the other labelled with biotin. The serum is incubated simultaneously with the antibody-coated bead, the biotinylated antibody and the tracer (streptavidin–isoluminol or avidin–peroxidase complex). Our experimental work has been directed towards a common set of reagents to capture the prolactin and then, with different tracers, towards obtaining on the calibration curve the same results for unknown samples. On the basis of the positive results we obtained, we have developed a kit that can be used by the customer or as an enzyme-immunoassay or as a chemiluminescent immunoassay, depending on instrumentation available, spectrophotometer or luminometer.     

Modification of the ELISA for the estimation of tetanus antitoxin in human sera

The use of indirect ELISA for the quantitation of tetanus toxin neutralizing antibodies in human sera is limited by marked overestimations in low titered sera. The reasons for the discrepancy between the results obtained by ELISA and by in vivo assay and modifications of the ELISA to overcome the problem were investigated. Catching ELISA and indirect ELISA using trays coated with the contaminant proteins in toxoid preparations indicated that antibodies to contaminants were only partly responsible for the discrepancy and the introduction of these modifications did not solve the problem. In ELISA competition experiments with toxin neutralizing monoclonal antibodies, the human immunoglobulins irrelevant in toxin neutralization, but detectable in indirect ELISA, were found to be difficult to inhibit in their binding to the solid antigen phase. These might represent antitoxins bound bivalently to the solid phase but with affinities in monovalent binding insufficient for toxin neutralization or other coupled antibodies due to conformational changes of the antigen. A competition ELISA with toxin in solution was therefore developed to assess selectively the antitoxin capable of binding the antigen in solution and by this approach the in vivo activities of even low titered sera were accurately predicted. This antigen competition ELISA may be easily introduced into routine tetanus serology and the principle may also be of value for the in vitro detection of functional antibodies to other antigens.     

从构建的噬菌体抗体库中筛选抗甲肝人单抗

用固相化甲肝抗原,对所构建的噬菌体抗体库进行了3轮淘筛。第3轮淘筛后洗脱下来的噬菌体,较第l轮增加了近100倍。含有抗体重链基因和轻链基因的重组克隆．也由淘筛前的25％增至100％。说明甲肝抗原对抗体库的淘筛,富集了表面呈现甲肝人单抗的噬菌体。经夹心ELISA法筛选到抗甲肝病毒噬菌体抗体,并以竞争抑制实验进一步证实了这些抗体的特异性。     

Immunoassays based on Penicillium marneffei Mp1p derived from Pichia pastoris expression system for diagnosis of penicilliosis

Background

Penicillium marneffei is a dimorphic fungus endemic in Southeast Asia. It can cause fatal penicilliosis in humans, particularly in HIV-infected people. Diagnosis of this infection is difficult because its clinical manifestations are not distinctive. Specialized laboratory tests are necessary to establish a definitive diagnosis for successful management. We have demonstrated previously that a cell wall mannoprotein Mp1p, abundant in P. marneffei, is a potential biomarker for diagnosis of P. marneffei infections. In the present study, we describe immunoassays based on Mp1p derived from the yeast Pichia pastoris expression system.

Methodology/Principal Findings

We generated monoclonal antibodies (MAbs) and rabbit polyclonal antibodies (PAbs) against Mp1p expressed in P. pastoris. Subsequently, we developed two Mp1p antigen capture ELISAs which employed MAbs for both the capture and detecting antibodies (MAb-MAb pair) or PAbs and MAbs as the capture and detecting antibodies (PAbs-MAb pair) respectively. The two Mp1p antigen ELISAs detected Mp1p specifically in cultures of P. marneffei yeast phase at 37–40°C and had no cross-reaction with other tested pathogenic fungi. The sensitivities and specificities of the two antigen assays were found to be 55% (11/20) and 99.6% (538/540) for MAb-MAb Mp1p ELISA, and 75% (15/20) and 99.4% (537/540) for PAbs-MAb Mp1p ELISA performed using 20 sera with culture-confirmed penicilliosis, and 540 control sera from 15 other mycosis patients and 525 healthy donors. Meanwhile, we also developed an anti-Mp1p IgG antibody ELISA with an evaluated sensitivity of 30% (6/20) and a specificity of 98.5% (532/540) using the same sera. Furthermore, combining the results of Mp1p antigen and antibody detection improved the sensitivity of diagnosis to 100% (20/20).

Conclusions/Significance

Simultaneous detection of antigen and antibody using the immunoassays based on Mp1p derived from P. pastoris greatly improves detection sensitivity. The procedures should be useful for the routine diagnosis of penicilliosis.     

The detection of Rickettsia prowazekii in the vectors of louse-born typhus Pediculus humanus by using monoclonal antibody-based preparations]

The results of this investigation indicate that preparations obtained on the basis of monoclonal antibodies have proved to be suitable for the detection of R. prowazekii antigens in the natural carrier of typhus when used in all types of the enzyme immunoassay; of these, the assay made by the capture method has been found to possess the highest sensitivity. The testing of the sensitivity limits has shown that this method known as ELISA-mu-capture, i.e. ELISA carried out with the use of antibodies to the mu chain of human immunoglobulin, is capable of detecting the antigen in a dose of 0.5-1 ng. Preparations based on monoclonal antibodies to species-specific R. prowazekii antigen permit the identification of the causative agent of typhus in its natural carrier within 24 hours.     

The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus

On the basis of an epitope model, capture enzyme immunoassay systems using monoclonal antibodies have been devised for the detection and quantification of Tick-borne encephalitis virus and compared with a reference system employing polyclonal sera. Monoclonal antibodies were used both as capture and detector antibodies, their suitability depending primarily on their avidity and intrinsic background activity. A considerable increase in sensitivity was achieved by combining antibodies to different non-overlapping epitopes. Biotinylation of the detector antibodies allowed the construction of multiple site simultaneous binding assays. Furthermore the use of monoclonal antibodies of defined serological specificity made virus type identification possible. This assay can therefore be used as a rapid 'test of identity' as required during the manufacture of viral vaccines.     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.     

The Binding of Bispecific Monoclonal Antibodies to the Solid Phase-Adsorbed Antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-human IgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (K _a) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than K _afor Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90–95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5–10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.