Development of a modified microtiter plate with a concave portion in the center

We have developed a modified microtiter plate which has following advantageous features and functions to both conventional microtiter plate and protein array, such as 1) use of conventional microtiter plate reader and washer, and 2) allowance of simultaneous reaction in the same liquid for all wells. Four proteins of human serum albumin, human C-reactive protein (CRP), human plasminogen and human MIP-1alpha as sample proteins, were measured with the modified microtiter plate. Although the reaction liquid of each wells on the modified microtiter plate shares through concave portion, its antigen/antibody in each well is independent. The independence of the reaction is supported by the result that the above four proteins produced dose-response curves simultaneously. Unlike a conventional protein array, our plate does not need the drying process for antibody adhesion to the plate, preventing inactivation of the antibody. And one can detect the antigen/antibody reaction using the enzymatic amplification reaction (for example utilizing the biotin-streptavidine interaction) like a conventional plate. In addition to these features, our microtiter plate also has the merit of eliminating the so-called "edge effect".     

Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay

Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant (K_aff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure K_aff by solid-phase EIA. Based upon the Law of Mass Action and using serial dilutions of both antigen (coating the plate) and antibody, K_aff has been measured by EIA. A microtiter plate was coated with antigen (Ag) and then incubated with monoclonal antibody (Ab). The plate was sequentially incubated with a second enzyme-antibody conjugate (EAC) and with the enzyme substrate. The amount of Ab adherent to Ag on the plate [Ag Ab] and [Ag₂ Ab] was reflected by the enzyme product measured by OD. The use of serial dilutions of Ab resulted in a sigmoid curve of OD versus logarithm of total Ab added to the well. Comparison of the OD at the upper plateau (OD-100) for different antibodies was a reflection of the relative number of epitopes on the Ag that were identified by the different antibodies, provided excessive EAC was used. [Ab]_t and [Ab′]_t were the measurable total antibody concentrations in the wells at OD-50 and OD-50′ for plates coated with [Ag] and [Ag′], respectively. [Ag] and [Ag′] were not true antigen concentrations, but were a measure of antigen density on the plate. For [Ag′] = [Ag]/2, K_aff = 1/2(2[Ab′]_t − [Ab]_t). Using five different anti-CEA antibodies and different proportions of CEA in the coating solution, K_aff was measured. K_aff determined by EIA correlated well with K_aff measured by soluble phase inhibition assay. This EIA method of estimation of K_aff is simple, rapid, and reliable.     

Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of Pseudomonas aeruginosa.

Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseudomonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of these forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bicarbonate buffer method was as efficient as the other sensitivity-enhancing plate-coating methods compared. The rough LPS, which has an overall hydrophobic characteristic, was shown to adhere effectively, regardless of the coating method used, to only one type of microtiter plate, CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors hydrophobic interactions between the rough LPS and the well surfaces. Dehydration methods were effective for coating microtiter plates with the neutral LPS examined, which is composed predominantly of a D-rhamnan. For the two dehydration procedures, LPS suspended in water or the organic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoating of plates with either polymyxin or poly-L-lysine did not give any major improvement in coating with the various forms of LPS. The possibility of using proteinase K- and sodium dodecyl sulfate-treated LPS preparations for ELISAs was also investigated. Smooth LPS prepared by this method was as effective in ELISA as LPS prepared by the hot water-phenol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA.     

Analysis and application of substrate hydrolysis rates in indirect ELISA of a purified plant virus

The transient colorimetric signal in a microtiter plate is used to quantify a purified plant virus, cowpea mosaic virus (CPMV), over five concentration decades in a single plate. The method involves the coating of the polystyrene microtiter plate wells directly with the CPMV antigen, followed by incubation with a rabbit-derived CPMV-specific antibody, and lastly by incubation with a commercially available antibody against rabbit immunoglobulin which has been pre-labeled with alkaline phosphatase. The rate of p-nitrophenylphosphate hydrolysis, both non-specific and that which was catalyzed by this enzyme, was measured spectrophotometrically at 405 nm. Enzyme-catalyzed hydrolysis rates followed first order kinetics at all antigen coating concentrations, and the 1 degree rate constants, which ranged from 2 X 10(-6) min-1 to 1 X 10(-3) min-1, were found to increase with increasing antigen concentration.     

Use of a Multiwell Assembly for Chemotaxis and Evaluation by Enzyme-Linked Immunosorbent Assay (ELISA)

A multiwell chamber assembly for chemotaxis tests was designed, which integrates the established microtiter system. A microtiter plate is covered with a plastic plate containing up to 96 holes of the diameter of the microtiter wells. Between the plates, a Nucleopore filter sheet (5 µm) and a silicon rubber gasket is placed. As a model system, human monocytes and lymphocyte-derived chemotactic factors were used. As it was observed that monocytes migrate through the membrane and settle on the bottom of the microtiter wells, an ELISA was adapted for quantitation of cells. After washing and incubation with a xenoantiserum against human monocytes, the bound antibody was quantitated using protein-A-conjugated alkaline phosphatase and p-nitrophenyl phosphate as detection system. The plates were read in a multichannel photometer. Cell numbers were determined directly from a calibration curve established before with varying numbers of monocytes. Current experience allows the following conclusions: The chemotaxis test in microtiter plates is simpler, faster and uses less material than conventional Boyden chambers. Evaluation by ELISA is much faster and more accurate than by microscopy.     

A rapid and simple method for efficient coating of microtiter plates using low amounts of antigen in the presence of detergent

Bio-Beads SM-2 have previously been used for the removal of non-ionic detergents from protein solutions. Addition of Bio-Beads SM-2 to detergent solubilized antigen significantly enhanced the immobilization of antigen to microtiter wells. Depending on the incubation time used 35-45% of the applied antigen could be immobilized to the microtiter wells. Using this method and a subsequent ELISA procedure it was possible to detect monoclonal antibodies in hybridoma supernatants after coating microtiter wells with 100 microliters of a solution containing 16 ng antigen/ml in the presence of 0.01% Triton X-100.     

Effect of coating the wells of a polystyrene microtiter plate with xanthorrhizol on the biofilm formation of Streptococcus mutans

Colonization on the surface of tooth by Streptococcus mutans is an important step in the initiation of dental plaque. Polystyrene microtiter plates have been employed to study bacterial colonization and biofilm formation of periodontal bacteria. The objective of this work was to evaluate the effect of coating the wells of a polystyrene microtiter plate with xanthorrhizol isolated from java turmeric (Curcuma xanthorrhiza Roxb.) on Strep. mutans biofilm formation. Our studies demonstrated that coating of a polystyrene microtiter plate with 5 microg/ml of xanthorrhizol resulted in significant (up to 60%) reduction of adherent cells compared to that of cells in uncoated wells. This result suggests that xanthorrhizol displays potent activity in preventing Strep. mutans biofilm formation.     

Rapid dot enzyme immunoassay for the detection of antibodies to cytomegalovirus.

Cytomegalovirus (CMV) antigen was coated onto a white opaque plastic card as small dots inside circles marked in the microtiter plate well pattern. The card with antigen dots could be cut according to the number of test samples to be assayed. Small drops of undiluted serum samples, goat antibodies to human immunoglobulin G labeled with alkaline phosphatase, and finally substrate (5-bromo-4-chloro-3-indolyl phosphate) were sequentially added to the antigen spots and incubated in the open air at room temperature for 5 min each. The antigen dots showed blue color for sera with immunoglobulin G antibodies to cytomegalovirus but no color for those without. The developed antigen dots could be rinsed with water and kept as permanent records. For the assay of a large number of serum samples, a modified procedure with serum diluted 1:10 and longer first two incubations (20 min each) was found to be more comfortable to perform. The results of this assay for 123 undiluted and 256 diluted serum samples revealed very good correlations with those obtained by a commercially available test kit for immunoglobulin G antibodies to cytomegalovirus with 97 and 99% agreement, respectively. This dot test was very reproducible and required no instrumentation. The reagents, including coated antigen dots, are stable at room temperature for at least 2 months and are ready for use.     

Degranulation of eosinophils by IgG antibody to Candida antigen]

The pathophysiological role of IgG antibody to fungi antigen widely distributed in environment such as Candida albicans in bronchial asthma has not been clarified. Wells of microtiter plate were coated with the extract of Candida albicans and then IgG antibody was immobilized on the wells by incubation with patient's serum. After cultivation of eosinophils on the well, degranulation of eosinophils, as assessed by quantitation of EPX in the supernatant, has been observed. Degranulation was completely abrogated after depletion of IgG in the serum and also decreased by incubation of the cells with anti-CD32 antibody, or anti-CD18 antibody, but not anti-CD23 antibody. Immune complex, which had been prepared by incubation of the extract of Candida albicans with patient's serum, also evoked degranulation of eosinophils. We have examined whether degranulation can be induced by two purified antigens of Candida albicans, i.e., mannan A and acid protease. IgG antibody to acid protease was detected at no or minimal levels in most sera and the antigen did not induce degranulation. On the other hand, mannan A induced degranulation. This observation may be due to response for the presence of IgG antibody to mannan A in the sera. These results suggest that immobilized IgG induced degranulation of eosinophils through Fc gamma RII (CD 32) on eosinophils and mannan A is a major allergen associated with IgG-induced eosinophil degranulation.     

Improved antibody coating protocol using a second antibody antiserum. Application to total thyroxin immunoassay

A complete antibody coating protocol for the preparation of dry antibody coated tubes is presented. This protocol is based on a recently described antibody immobilization principle. We modify this immobilization principle in order to improve and simplify the coating procedure. In addition, we propose a drying procedure that provides long-term storage stability of the antibody coated tubes. According to the modified protocol, polystyrene plastic tubes are first coated with rabbit gamma-globulins. The tubes are incubated with a sheep anti-rabbitIgG antiserum dilution. After incubation, antigen-specific antibody antiserum raised in rabbits is added directly into the tubes containing the sheep anti-rabbit IgG antiserum solution (difference from the original protocol). Finally, the tubes are washed, blocked, and dried following the drying procedure developed. The suitability of the modified protocol for the development of immunoassays requiring high loading of antibody was exemplified through the development of a RIA for total thyroxin. The estimated assay characteristics (detection limit 4 microg/L, dynamic range up to 210 microg/L, within-run CV 2.7-5.7%, between-run CV 5.1-7.3%, recovery 84.4-112%, cross-reactivity for T3 1.9%) were comparable with those provided by commercially available RIA kits for the determination of thyroxin.     

Enzyme-linked immunosorbent assay for quantitating the humoral immune response to the colonization factor antigen of enterotoxigenic Escherichia coli

The enzyme-linked immunosorbent assay technique was used to quantitate, in milligrams per milliliter, anti-colonization factor antigen/I (CFA/I) immunoglobulin G (IgG) in acute- and convalescent-phase sera of individuals who experienced diarrhea associated with CFA/I-positive enterotoxigenic Escherichia coli. Purified CFA/I was used as antigen to coat polystyrene Microtiter plate wells for the determination of anti-CFA/I antibody. A reference anti-CFA/I IgG preparation was obtained by affinity chromatography of a high-titered serum with a CFA/I-Sepharose 4B column; IgG was the only class of immunoglobulin detectable in this serum as anti-CFA/I. Goat anti-human IgG conjugated to alkaline phosphatase was used in the enzyme-linked immunosorbent assay. Quantitation of IgG in the reference anti-CFA/I serum was achieved by comparison with a known sample of pure human IgG. Anti-CFA/I in test sera was quantitated by titration with CFA/I-coated Microtiter plate wells in the enzyme-linked immunosorbent assay, using a standard curve obtained with the reference anti-CFA/I serum. Anti-CFA/I IgG in paired sera was determined as percentage of total IgG by using the radial immunodiffusion technique to quantitate total IgG for each test serum. Diarrhea with isolation of CFA/I-positive enterotoxigenic E. coli was associated with a significant rise in serum anti-CFA/I IgG when these values were expressed as either milligrams of IgG per milliliter or as percentage of total IgG, although the response varied quantitatively and nonresponders were detected. None of the matched controls showed an anti-CFA/I IgG response. Further elucidation of the immune response to enterotoxigenic E. coli can now be accomplished by applying these methods to determine the class and specificity of immunoglobulins in external secretions such as saliva and intestinal contents.     

A modified ELISA that selectively detects monoclonal antibodies recognizing native antigen

A modified ELISA procedure is described which permits selective detection of monoclonal antibodies reactive with the native antigen. The essence of the method is the use of immobilization methods that do not result in denaturation of the antigen during adsorption to the microtiter plates. This was accomplished by using polyclonal antibodies, adsorbed to the wells of the microtiter plate, as the immobilizing agent.     

Poly(Vinyl Alcohol) as a Blocking Agent in Enzyme Immunoassays

We examined the blocking ability of poly (vinyl alcohol) (PVA) in enzyme immunoassays by coating polystyrene microtiter wells with PVA of different molecular weights (MW) and percent hydrolysis (%Hyd). Blocking ability was measured by the differences in non-specific binding of an anti-rabbit IgG-horseradish peroxidase conjugate to coated and uncoated wells. PVA with a MW of 124 000–186 000 and >99 %Hyd was the most effective in suppressing the binding of the conjugate. This PVA at 0.5% (w/v) was significantly better at reducing non-specific binding than commonly used blocking agents and did not interfere with the specific binding of the conjugate to antigen-coated microtiter wells.     

Development of coated tubes RIA for serum T3 (tri-iodothyronine) for production scale

A coating procedure that could provide immobilization of antibodies, with increased binding capacity, that is cost effective, simple, robust, and appropriate for production scale application, is described. This coating approach of T3 antibodies to the polystyrene tubes has been systematically investigated to determine its utility for the development of coated tube Radioimmunoassay (RIA) for T3 in human serum. Further, the results obtained by the developed coating procedure are found to be comparable with those obtained by the "gold standard," the liquid phase RIA for T3. The coating procedure is completed in three major steps, each step involving an overnight incubation. The normal rabbit gamma-globulins are physically adsorbed onto the polystyrene tubes and incubated. After washing, a second antibody (goat anti-rabbit antiserum) is added and incubated. To this antigen specific antibody is added (T3 antibody produced in rabbit) and further incubated. Finally, the non-specific sites on the tubes are saturated by the blocking solution. The concentration of normal rabbit globulin, titers of second antibody and T3 antibody, and time required for coating are optimized to arrive at a suitable coating protocol. The coated tubes were evaluated for precision, reproducibility, and stability. Various parameters such as total reaction volume, incubation time and temperature, total number and volume of washings, concentration of 8-anilino-1-naphthalene sulfonic acid (ANS), and quantity of tracer per tube are optimized to arrive at a suitable standard curve. The optimized assay is validated for the quality control parameters such as intra- and inter-assay variations, recovery, and parallelism. The developed coated tubes assay had an assay range of 0.3-4.8 ng/mL with a sensitivity of 0.3 ng/mL at 90% B/B0. Batch to batch variation in coating was < 10%. The coated tubes were stable up to 1 year, which is adequate for production scale.     

Adherence of Pseudomonas aeruginosa to human tracheobronchial mucin.

A microtiter plate assay was developed to study the adherence of Pseudomonas aeruginosa to purified human tracheobronchial mucin. The wells of the plates were treated with silicon to minimize nonspecific binding of bacteria and then coated with a solution of purified human tracheobronchial mucin. Bacteria were added to the wells, and the plates were incubated at 37 degrees C. The wells were washed 15 times in an automated microtiter plate washer, and the bacteria bound to wells were desorbed with Triton X-100 and plated for enumeration. Scanning electron microscopy verified bacterial adherence to the mucin-coated wells and desorption of bacteria by Triton X-100. Adherence of P. aeruginosa increased as the concentration of mucin used to coat the wells was increased, with saturation occurring at 0.5 microgram of mucin protein per ml. Other parameters that affected adherence included the time of incubation and concentration of bacteria. Similar studies with strains of Escherichia coli and Klebsiella pneumoniae indicated a relative lack of binding of these bacteria to mucin. In comparing different strains of P. aeruginosa, there were small differences in binding between strains. It is inferred that there may be specific sites on human tracheobronchial mucin which facilitate this preferential binding.     

Microsticks as solid-phase carriers for enzyme-linked immunosorbent assays.

Microsticks are machine-tooled or molded pegs of plastic or stainless steel which were developed as solid-phase carriers for the enzyme-linked immunosorbent assay (ELISA). They consist of a stem, which can be coated with plastic to be used as the reactive surface, and a shaft designed for easy handling and labeling and for positioning the sticks in microtiter wells and transfer plates. Microsticks permit a wide selection of coating materials and afford the user greater control over quality and standardization of the solid-phase surface. Polycarbonate and nitrocellulose coatings were tested in ELISAs for antibodies to measles virus, toxoplasma, and human gamma globulin. The Microsticks were found to give reproducible, sensitive, and specific ELISA results and minimized problems due to lot-to-lot and batch-to-batch variations found with other plastic carriers.     

Antigen-specific cell adherence assay: a new method for separation of antigen-specific hybridoma cells

A new method for the detection and separation of antigen-specific antibody-producing cells on the basis of antibody-mediated recognition of solid-phase immobilized antigen molecules is described. Hybridoma cells are placed on microtiter plate wells coated with antigen molecules, and antigen-specific antibody-producing cells bind to the immobilized antigen molecules; antibody nonproducing or nonspecific antibody-producing cells can be easily separated from the bound cells by inverting the plate. Cells bound to solid-phase immobilized antigen molecules can readily be quantitated by counting under a light microscope, and the cells recovered can produce antibody in culture. Unspecific binding of cells in antigen-specific cell adherence assay (ASCAA) is optimally below 5%. Also, effect of drugs interfering with processes related to antibody production of antigen-specific cells can be detected and evaluated by ASCAA.     

Assessment of coating-efficiency in ELISA plates by direct protein determination

A commercially available protein determination method (Pierce BCA protein determination kit) was used to assess the efficiency of coating of microtiter plates. Since the method is insensitive to detergents it permits the determination of adhered protein after washing the microtiter plate with a detergent-containing solution. Thus only tightly bound protein is determined. Our results suggest that coating of microtiter plates with antibody is optimal at a certain concentration, above which coating is less efficient.     

IMPROVED ANTIBODY COATING PROTOCOL USING A SECOND ANTIBODY ANTISERUM. APPLICATION TO TOTAL THYROXIN IMMUNOASSAY

A complete antibody coating protocol for the preparation of dry antibody coated tubes is presented. This protocol is based on a recently described antibody immobilization principle. We modify this immobilization principle in order to improve and simplify the coating procedure. In addition, we propose a drying procedure that provides long-term storage stability of the antibody coated tubes. According to the modified protocol, polystyrene plastic tubes are first coated with rabbit γ-globulins. The tubes are incubated with a sheep anti-rabbit IgG antiserum dilution. After incubation, antigen-specific antibody antiserum raised in rabbits is added directly into the tubes containing the sheep anti-rabbit IgG antiserum solution (difference from the original protocol). Finally, the tubes are washed, blocked, and dried following the drying procedure developed.

The suitability of the modified protocol for the development of immunoassays requiring high loading of antibody was exemplified through the development of a RIA for total thyroxin. The estimated assay characteristics (detection limit 4 μg/L, dynamic range up to 210 μg/L, within-run CV 2.7– 5.7%, between-run CV 5.1–7.3%, recovery 84.4–112%, crossreactivity for T₃ 1.9%) were comparable with those provided by commercially available RIA kits for the determination of thyroxin.     

Highly sensitive enzyme immunoassay of human interferon-beta 1

A highly sensitive sandwich enzyme immunoassay for human interferon-beta 1 (HuIFN-beta 1) was developed. HuIFN-beta 1-containing samples and horseradish peroxidase (HRP)-labeled mouse anti-HuIFN-beta 1 monoclonal antibody (Fab') were incubated overnight at 2-10 degrees C in the wells of a 96-well microtiter plate, onto which affinity-purified rabbit anti-HuIFN-beta 1 polyclonal antibody was coated. The EIA was able to detect 0.5 IU/ml of HuIFN-beta 1, thus showing higher sensitivity than bioassay. The values obtained by the EIA closely paralleled those obtained by bioassay in the concentration which bioassay can detect. In order to detect the concentration below 0.5 IU/ml of HuIFN-beta 1, the avidin/biotin-amplified EIA was also developed. The use of biotinylated mouse anti-HuIFN-beta 1 monoclonal antibody (F(ab')2) and HRP-avidin in the EIA made it possible to detect 0.1 IU/ml of HuIFN-beta 1. These EIAs were applied for the studies such as process control of HuIFN-beta 1 production, pharmacokinetics of HuIFN-beta 1, and determination of serum level of HuIFN-beta 1 in healthy subjects.