Enzyme-Linked Immunosorbent Assay of Fungal NADP-Glutamate Dehydrogenase

A sensitive and reliable method has been developed for the quantitation of NADP⁺-glutamate dehydrogenase from the phytopathogenic Ascomycete Sphaerostilbe repens using a two-step competitive enzyme-linked immunosorbent assay. Purified enzyme was adsorbed noncovalently to polystyrene wells and rabbit immunserum was allowed to bind to antigensensitized wells. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently linked to alkaline phosphatase using paranitrophenylphosphate as the substrate. Increasing amounts of purified enzyme or crude fungal extracts were quantitated by their ability to inhibit specific antibody adsorption to antigen-coated polystyrene wells. This system proves to be useful in the range of 10 to 80 nanograms of enzyme level. Using this assay, identical amounts of NADP⁺-glutamate dehydrogenase were found in mycelia grown on nitrate and ammonia sources.     

Staphylococcal Enterotoxin B: Solid-Phase Radioimmunoassay

An immunoassay employing (125)I-labeled enterotoxin B and polystyrene tubes coated with specific antibody was used for assaying purified and crude enterotoxin. Antibody was adsorbed to untreated polystyrene tubes. Unlabeled enterotoxin competed with (125)I-labeled enterotoxin for antibody-combining sites. The uptake of (125)I-labeled toxin reflected the concentration of unlabeled toxin present. The test is sensitive to 1 to 5 ng of purified and crude enterotoxin B per ml, and cross-reactions with heterologous enterotoxins did not interfere with the specificity. This test possesses the combination of sensitivity and objectivity absent in current methods for assaying enterotoxin and provides a model for investigating other enterotoxin serotypes.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

Two-Site Immunoassay for Acetylcholinesterase in Brain, Nerve, and Muscle

Two-site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4 degrees C. Bound enzyme was detected with an 125I-labeled antibody against a different AChE epitope. The assay signal was quasi-linearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two-site methods with a different pair of species-selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54 degrees C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anticholinesterases or trypsin. A biotinylated second antibody detected by alkaline-phosphatase-conjugated avidin was used to develop an AChE enzyme-linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radiometric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two-site method showed closely parallel variations in immunoreactivity and enzyme activity.     

Secreted forms of human neutrophil collagenase

Collagenase in human neutrophils is found within intracellular granules which can be stimulated to be secreted with phorbol myristic acetate. This extracellular secreted form of neutrophil collagenase was isolated by immunoaffinity chromatography using a monoclonal antibody previously shown to specifically recognize neutrophil collagenase. The enzyme efficiently bound to this column and was eluted with NaSCN as three major species of 75, 57, and 22 kDa, respectively. These proteins were closely related immunologically since, after radiolabeling and separation by gel filtration, each of the three proteins was precipitated by the monoclonal antibody. Also, the 75- and 57-kDa proteins exhibited collagenase activity after elution from polyacrylamide gels run under nondenaturing conditions. Further, the 57-kDa protein autodegraded into a 22-kDa protein with time. Polyclonal antibody, prepared to the 57-kDa enzyme, also recognized the 75- and 22-kDa proteins using an immunoblot technique. When crude neutrophil supernatants containing latent collagenase were immunoblotted, both the 75- and the 57-kDa enzymes were present. Our immunoaffinity purified active enzymes, although activated during the course of purification, resemble the latent enzymes in crude neutrophil supernatants. The multiple forms of secreted collagenase from degranulated leukocytes may resemble more closely that seen in inflammation.     

Staphylococcal Enterotoxin C: Solid-Phase Radioimmunoassay

A solid-phase radioimmunoassay test employing 125I-labeled enterotoxin C and polystyrene tubes coated with specific antibody was used for the detection and quantitation of entertoxin C in condensed milk, cheddar cheese, custard, and ham salad. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 16% or less.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Two immunologically and catalytically distinct arachidonate 12-lipoxygenases of bovine platelets and leukocytes

12-Lipoxygenases were found in the cytosol fraction of bovine leukocytes and platelets. The bovine leukocyte enzyme was immunoprecipitable by a monoclonal antibody directed to 12-lipoxygenase of porcine leukocytes, but not by a monoclonal antibody against the human platelet enzyme. In contrast, the bovine platelet enzyme cross-reacted only with antibody against the human platelet enzyme. The leukocyte and platelet enzymes were partially purified to final specific enzyme activities of 1.1 and 0.3 mumol/min/mg protein, respectively, by immunoaffinity chromatography using each cross-reacting antibody as a ligand. The leukocyte enzyme reacted with various octadecapolyenoic acids as well as eicosapolyenoic and docosapolyenoic acids, whereas the platelet enzyme was almost inactive with octadecapolyenoic acids. Moreover, the two enzymes showed different heat-instabilities and reaction time courses. Thus, the 12-lipoxygenases of bovine leukocytes and platelets were immunologically and catalytically distinct enzymes.     

Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay for Cellobiohydrolase I

A double-antibody sandwich enzyme-linked immunosorbent assay was developed for quantifying cellobiohydrolase I (CBH I) in crude preparations of the cellulase complex from Trichoderma reesei. The other enzymes (endoglucanase and β-glucosidase) in this complex and other ingredients in culture broth did not interfere with this assay. The antibody configuration that resulted in the highest specificity for the assay of CBH I employed a monoclonal antibody to coat wells in polystyrene plates and peroxidase-labeled polyclonal antibody to detect cellobiohydrolase bound to the immobilized monoclonal antibody. Previously, procedures have not been available for the direct assay of CBH I activity in the presence of the other enzymes in the complex, and current indirect procedures are cumbersome and inaccurate. The direct procedure described here is highly specific for CBH I and useful for quantifying this enzyme in the range of 0.1 to 0.8 μg/ml.     

Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.     

Affinity of pertussis toxin produced by Bordetella pertussis for human haptoglobin: application to the in vitro assay of the toxin

Pertussis toxin formed a stable complex with human (Hp). Hemagglutinating activity of the toxin was inhibited in the presence of Hp, but leukocytosis activity was not.An enzyme linked immunosorbent assay for the toxin, Hp-ELIS, was developed on the basis of its specific affinity for Hp. A polystyrene microplate coated with Hp was incubated with samples containing the toxin. The bound was measured by sequential reaction with antipertussis toxin goat IgG, alkaline-labelled anti-goat IgG and p-nitrophenylphosphate.The Hp-ELISA method showed high specificity, high sensitivity and good correlation with leukocytes promoting activity in vivo. One ng of pertussis toxin could be detected within 3 h.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Development of quantitative immunoenzyme and radioimmunologic analysis of lambda-free immunoglobulin light chains

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light lambda-chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.     

Development of Quantitative Enzyme Immunoassay and Radioimmunoassay of λ-Free Light Chains of Immunoglobulins

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light -chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B₂ (TXB₂). As a specific antigen, the bovine serum albumin conjugate of TXB₂ was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB₂ per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB₂ per sample. The suitability of the assay was checked with TXB₂-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r=0.960 and r=0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r=0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B2 (TXB2). As a specific antigen, the bovine serum albumin conjugate of TXB2 was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB2 per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB2 per sample. The suitability of the assay was checked with TXB2-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r = 0.960 and r = 0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r = 0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

Measuring Myeloperoxidase Activity in Biological Samples

Background

Enzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.

Principal findings

We found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.

Conclusion

We validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.     

Development and optimization of an enzyme-linked immunosorbent assay employing two murine monoclonal antibodies for absolute quantitation of human beta-glucuronidase.

We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) for absolute quantitation of human beta-glucuronidase. This is a double antibody sandwich system employing two murine monoclonal antibodies specific for human beta-glucuronidase developed in our laboratories. The method involves (a) coating of the high binding polystyrene microtitration plate with the first antibody (7B6 IgG), (b) blocking of remaining active sites with 3% bovine serum albumin in phosphate-buffered saline, (c) application of samples, (d) addition of the biotinylated second antibody (6D2 IgG), (e) addition of streptavidin-horseradish peroxidase, and (f) development of color with o-phenylenediamine dihydrochloride-H2O2 and reading in a microplate reader at a wavelength of 490 nm. The method is highly sensitive with an optimal range of 10 to 100 ng/ml of the enzyme and is reproducible with intraday and interday precisions of 3.2 and 4.1%, respectively. The enzyme contents of 20 urine and 20 bile samples quantitated by this ELISA method were, respectively, 148 +/- 101 and 6380 +/- 3780 ng/ml (means +/- SD) which correlated well with their enzyme activities. Such a method for absolute quantitation of human beta-glucuronidase is essential for studying its pathophysiologic roles in cholelithiasis and carcinogenesis and can also be used clinically as an indicator for tissue damage or malignancy.     

Polyclonal antibody based immunopurification of an acid stable alpha-amylase produced byTalaromyces emersonii

Summary A polyclonal antibody preparation specific for an alpha-amylase produced by the thermophilci fungusTalaromyces emersonii has been raised in rabbits. Using an enzyme linked immunoadsorbant assay (ELISA) it has been demonstrated that this antibody preparation is highly titred and has the ability to detect less than 20ng of purified alpha-amylase. Specificity of the antibody has been demonstrated using Western immunoblotting analysis following resolution of crude preparations on polyacrylamide gradient gels. A purified IgG fraction of this serum has been used to construct an immunoaffinity column and this was used to develop a rapid and simple purification procedure for this potentially biomedically important acid stable alpha-amylase.     

USE OF POLYMYXIN B IN A CAPTURE ENZYME IMMUNOASSAY FOR DETECTION OF SALMONELLAE SPP. LIPOPOLYSACCHARIDE

A capture enzyme immunoassay for detection of salmonellae sp. lipopolysaccharide was developed. The assay made use of polymyxin B sulfate, passively attached to a polystyrene matrix, to capture lipopolysaccharide. Bound lipopolysaccharride was then detected with a monoclonal antibody, specific for salmonellae spp. followed by goat antimouse antibody conjugated with horseradish peroxidase.
The analytical sensitivity of the assay was approximately 1 ng/ml of lipopolysaccharide. The results are comparable to those obtained with a competitive enzyme immunoassay previously developed. The sensitivity of the polymyxin B assay decreased to 4–5 ng/ml when the salmonellae spp. lipopolysaccharide was mixed with 1–100 μg/ml of Escherichia coli lipopolysaccharide, while this level of heterogeneous lipopolysaccharide, did not decrease the sensitivity of the competitive enzyme immunoassay.
The polymyxin B capture assay was advantageous in that polymyxin B is a standardized reagent that is relatively inexpensive and does not require extensive preparation or containment facilities. The assay is robust; however, because of the light sensitivity of polymyxin B, its stickiness to other reagents and interference by other lipopolysaccharides, this assay requires careful attention to detail and may therefore be an unsuitable assay for field use.