A method for the quantification of a colored or fluorescent signal in enzyme immunoassays by photodensitometry.

A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of either a spectrophotometer or a fluorometer. In this study we compared readings obtained by spectrophotometry, fluorometry and photodensitometry in 96-well ELISA plates and in Terasaki plates. In ELISA plates, it is possible to detect 1220-300,000 femtograms (fg) of peroxidase by spectrophotometry and 4800-125,000 fg by photodensitometry. In Terasaki plates, we were able to measure between 3.8 and 8000 fg of beta-galactosidase per sample by spectrofluorometry, and from 30 to 8000 fg by photodensitometry. Using a sandwich procedure in Terasaki plates we were able to measure between 100 and 10,000 fg of IgE per sample by spectrofluorometry and from 500 to 10,000 fg by photodensitometry. Photodensitometry is the least expensive technique for the reliable detection of enzyme or enzymatic marker in small sample volumes treated with a fluorogenic substrate.

Production of highly specific monoclonal antibodies to monensin and development of a microELISA to detect this antibiotic.

Monensin, a polyether antibiotic of molecular weight 671 Da, was converted into a hemisuccinate and covalently linked to bovine serum albumin via the mixed anhydride method. Using this immunogen, polyclonal anti-monensin antibodies were raised in rabbits and monoclonal antibodies were prepared from mice. The specificity of the anti-monensin antibodies was examined by using several structural analogues as the immunogen and by performing direct binding and competitive microELISA assays on Terasaki plates. Rabbit polyclonal antibodies had a dissociation constant (KD) of 5.5 x 10(-8) M for monensin and reacted with nigericin, an antibiotic structurally related to monensin. In contrast, a mouse monoclonal antibody, 2H8, reacted only with monensin and had a much lower KD = 3 x 10(-8) M for monensin. Monoclonal antibody 2H8 was used to develop a competitive microELISA able to detect as little as 5 ng/ml of monensin in solution which corresponds to 75 pg or 110 fmol of this hapten per Terasaki well.

A highly sensitive microtitre plate enzyme immunoassay for oestradiol-17 beta.

A specific and sensitive solid-phase microtitre plate enzyme-linked immunosorbent assay for oestradiol-17 beta (E2) is described. After coating with an IgG anti-E2 fraction, we used E2-6-carboxymethyl-oxime-beta-galactosidase in a competitive binding assay and revealed the bound activity with a fluorogenic substrate. Two methods for the competitive binding assay were tested: (1) a classical one (method A) defined as a 'two-step competition' because the E2 sample was first incubated alone, and then E2-beta-galactosidase conjugate was added; (2) and a new one (method B) also performed in two steps but in which the E2 sample was evaporated to dryness. The detection limit of method A was 100 pg/ml (9 pg/well). Method B was more sensitive since 1 pg/ml (0.3 pg/well) was statistically different from 0 pg/ml. Specificity was equivalent with both methods while precision was better in B. Thus, this new method may be able to measure very low levels of oestradiol-17 beta in, for example, the blood of domestic mammals.

Recycling of ELISA plates for the serological diagnosis of tuberculosis using a Mycobacterium tuberculosis-specific glycolipid antigen.

A Mycobacterium tuberculosis-specific glycolipid antigen (diacyl-trehalose-DAT) and a battery of defined sera from tuberculous patients were used to evaluate the reliability of recycling ELISA microplates for serodiagnosis of tuberculosis. The reuse of plates was evaluated by the same ELISA technique before and after recycling with an acid buffer to dissociate and release antibodies from antigen-antibody complexes fixed on used plates. The correlation coefficients for comparisons between several new plates used on different days were always higher than r = 0.90, and the variation between tests was always lower than 12%. For comparisons performed systematically between new plates and recycled plates, the values were variable. Up to the fifth recycling, the correlation coefficient was higher than r = 0.60. From the sixth recycling, correlation coefficient values fell to r = 0.381. The results obtained with recycled plates washed manually were much poorer than those obtained after automatic washing. The results described in this report demonstrate that an ELISA test can be performed on recycled plates under the conditions described.

Miniaturization of beta-galactosidase immunoassays using chromogenic and fluorogenic substrates.

Enzyme immunoassay techniques are widely use to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the beta-galactosidase immunoassay. Using microtitration plates (150 microliter samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli beta-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic (o-nitrophenyl-beta-D-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-beta-D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075-0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzyme immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 microliter and finally to 0.3 microliter a progressive decrease from 7 x 107 molecules of IgE to 2.9 x 107 molecules and to 1.5 x 106 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.

Toxicological biotest on Diptera larvae to detect ciguatoxins and various other toxic substances.

A toxicological test was developed using a new animal sensitive to various toxins, especially ciguatoxin (CTX), which poisons humans who eat tropical fishes. The larvae of Parasarcophaga argyrostoma (Diptera, Sarcophagidae) were selected for their marked ability to consume spontaneously large quantities of various proteins; meat, fish flesh or liver, mussels, etc. Larvae grown on meat overnight can easily be observed without requiring optical instruments. Ten larvae were placed on 5 g of the sample to be tested, and left for 3-24 hr. Samples containing > 1 ng of CTX/g of flesh (moray.eel CTX) killed the larvae in about 3 hr; samples with lower concentrations inhibited larval growth, which can be noted with the marked eye. These readings were rendered objective by weighing the larvae before and after their being placed on the test sample and comparing the result to that of the control. The reading with the naked eye seems to be satisfactory up to 0.2 ng of CTX/g, while that by weighing was acceptable up to 0.10-0.15 ng/g. The coefficient of variation in the sensitive zone (approximately 50% of normal growth) was about 25% for a series of 11 experiments. P. argyrostoma larvae are sensitive to various other toxic substances, such as tetrodotoxin, okadaic acid, aflatoxin B1 and amanitin. The test is extremely simple in its theory and practice. It does not require complex materials or equipment and it is very inexpensive. It could be used to detect ciguatera toxins in the monitoring of fishing areas and for the commercialization of big-sized fish.

Development of a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite.

We describe a new ELISA which allows the measurement of the concentration of venom antigens in whole blood. The assay can be performed in less than 20 min and requires a 200 microliters sample of blood. It allows the accurate evaluation of concentrations of Vipera ammodytes venom in quantities smaller than 1 ng/ml of blood. Using this ELISA, we were able to follow in rabbits the kinetics of experimental envenomation with non-lethal doses of venom. This ELISA was also used to measure post mortem the level of venom antigens in various tissues such as liver, kidney, muscles and abdominal serosity of a rabbit. The method, which might be adapted to measure envenomation by other snake species, seems to be sufficiently rapid and sensitive to allow routine evaluation of the gravity of a snake bite in humans and to estimate the efficacy of immunotherapy.

Preliminary results towards ciguatoxin immunodetection.

Due to the lack of purified ciguatoxin (CTX), monensin was used as a model for developing an enzyme immunoassay to detect CTX. Specific antibodies directed against monensin have been produced in rabbits and mice using a monensin-protein immunogen obtained in bulk quantities. Rabbit polyclonal and mouse monoclonal (MAb) antibodies of high specificity and affinity have been produced. Using MAb 2H8, in a competitive micro-ELISA performed in Terasaki plates, the detection limit for free monensin was 75 pg. No cross-reactivity was detected against CTX but a procedure requiring only 100 micrograms of hapten is under current investigation with a brevetoxin (PbTx-3), another marine toxin with a polyether backbone structure similar to CTX and recently commercially available.

Effects of pH or ionic strength on the reactivities of mono- and polyspecific IgG antibodies.

The reactivities of mono- and polyspecific mouse monoclonal antibodies (mAb) were compared by ELISA using immobilized antigens under different conditions, varying the pH or the NaCl concentration. The monospecific group was composed of 6 IgG directed against Staphylococcus aureus capsular polysaccharides and of 3 IgG specific to actin, myosin or tubulin. These antibodies were compared with 6 polyreactive mAb also of the IgG isotype. Marked differences were noted between the reactivities of the mono- and polyreactive IgG. pH variations had little or no effect on the reactivity of monospecific mAb to polysaccharides or to proteins. In contrast, the binding of polyreactive mAb was dependent on the pH, and the profile differed for each antigen. The NaCl concentration had opposite effects on mono- and polyreactive mAb: the binding of almost all the monoreactive mAb was increased at high NaCl concentration, while it was decreased for polyreactive mAb. In contrast, the effects of varying the pH or ionic strength on the coated antigens were negligible. The variation coefficients calculated for the pH and NaCl concentration were higher for the polyreactive mAb under study, which seems to indicate that electrostatic interactions and charged residues might be more important for these mAb than for the monoreactive ones. This characteristic might be one explanation for the particular properties of these polyreactive antibodies.

Antiactin and antitubulin antibodies in canine visceral leishmaniasis.

Visceral leishmaniasis, a chronic and often fatal disease, is caused by the protozoan parasite Leishmania donovani. Both specific and nonspecific antibodies are produced in the course of the disease, and autoantibodies may be involved in pathogenesis. Tubulin and actin have been found to be associated with L. donovani. To learn whether antiactin and antitubulin antibodies are present in visceral leishmaniasis, we tested sera from 263 infected dogs by enzyme-linked immunosorbent assay for antibodies to the antigens L. donovani, actin, and tubulin. All samples reacted positively with L. donovani, and a high percentage reacted positively with all three antigens. Sera from 202 uninfected dogs were also tested, none reacted with L. donovani antigen, although positive reactions were observed for 8 of the samples with actin or tubulin. It was found that the antibody-antigen reaction occurred at the Fab portion of the immunoglobulin molecule. Competitive enzyme immunoassays showed that the reaction was inhibited if the positive serum was first incubated with L. donovani antigen, actin, or tubulin and then tested by enzyme-linked immunosorbent assay. These results suggest that antiactin and antitubulin antibodies are present in the sera of dogs infected with visceral leishmaniasis.

Direct evaluation of class-specific anti-DNA antibodies by an immunoenzymatic technique.

Antibodies against native DNA have been evaluated by an immuno-enzymo assay (ELISA) using glucose oxidase labelled Ig class-specific antibodies. Results of the ELISA test were significantly correlated with those of the Farr test performed with native DNA as well as with the degree of clinical activity. ELISA is proposed as a simple method for the direct evaluation of class-specific anti-native DNA antibodies in systemic lupus and related diseases.

Effect of temperature on the reactivities of polyreactive and monospecific monoclonal IgG antibodies.

In this study, the reactivities of two groups of murine monoclonal antibodies (mAbs) of the IgG isotype were compared by ELISA at various temperatures (Ts) (range: 4-56 degrees C). The first group was constituted of 4 polyreactive mAbs that reacted with various antigens (Ags), such as actin, myosin and tubulin. The second contained 3 commercially available monoreactive mAbs specific to actin, myosin or tubulin. The binding of the monospecific mAbs to their Ags was modified only slightly at the other Ts compared with binding at 37 degrees C. In contrast, the activities of the polyreactive IgGs were considerably modified depending upon the T during incubation on Ag. In a second series of experiments, the effects of the T of the washes and conjugate incubation on the mAb/Ag interaction were evaluated. In these experiments, all steps and incubations were carried out at 37 or 4 degrees C. The values were then compared to those obtained when the washes and conjugate incubation were performed at room temperature. These two protocols generated very little difference in terms of monospecific mAbs. For polyreactive IgG, the values were generally lower when the incubations were carried out at 4 degrees C. However, on the whole, the effect of the T of the washes and conjugate incubation was negligible when the mAb/Ag complex had already formed on the polystyrene plate. Furthermore, at 4 degrees C, 2 of the polyreactive mAbs behaved like monospecific antibodies, while the other 2 remained polyspecific. It can be concluded from these experiments that the reactivities of polyreactive mAbs are more T-sensitive than those of monoreactive mAbs. This seems to indicate that they may possess a more plastic structure and thus may more easily undergo deformation when subjected to non-physiological conditions. In addition, the fact that the polyreactive mAbs showed the same variations for the 3 Ag tested suggests that the same paratope could be involved in all reactions.

Visualization of natural autoantibody polyreactivity by rotary metal-shadowing electron microscopy.

Immune complexes formed by mouse polyspecific natural autoantibodies and various structurally different antigens, such as DNA, tubulin and myosin, were analysed by rotary-shadowing electron microscopy. Each of the four natural IgM autoantibodies studied (E7, D23, 3C3 and M2-9) recognized multiple epitopes on the myosin molecule. These results, confirmed by immunoblotting experiments using myosin subfragments as antigens, strikingly contrasted with those obtained with an induced myosin-specific IgG antibody which interacted with a single myosin antigenic site. Based on the measurements of the antibody position on the antigen, made on a series of electron micrographs, two negatively charged myosin peptides were prepared by solid phase synthesis. Polymeric forms of one of the two peptides interacted with the positively charged CDR part of E7 and inhibited the binding of E7 and M2-9 to myosin. The importance of charge in the observed cross-reactivities was further supported by enzyme immunoassays showing that most, but not all, antigen/natural autoantibody interactions were sensitive to increasing concentrations of NaCl.

Fatty acid acylated peroxidase as a model for the study of interactions of hydrophobically-modified proteins with mammalian cells.

Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.