Detection of wheat gliadin contamination of gluten-free foods by a monoclonal antibody dot immunobinding assay

Unfractionated wheat gliadin was used to produce murine monoclonal antibodies to gliadin. A dot immunobinding assay, using these antibodies, was developed to detect possible gliadin contamination of nominally gluten-free flour, using dilute ethanol extracts spotted onto nitrocellulose membranes. The sensitivity of the assay was less than 10 micrograms/ml of unfractionated gliadin which permitted the detection of trace amounts of gliadin present in certain wheat starch based 'gluten-free' products. The assay detected not only wheat gliadin, but also prolamine extracts of rye, barley and oats; maize, soya and potato extracts as well as the control proteins casein and ovalbumin, gave negative results. The assay is of value as a simple and rapid method of screening foods for their suitability for consumption by patients with coeliac disease.     

The prolamin antibody reactivity against hordein polypeptides in sera from patients with coeliac disease

The antibody reactivity against the barley prolamin, hordein, was investigated by an immunoblotting technique, in sera from patients with untreated coeliac disease, patients with other gastrointestinal diseases and healthy controls. No characteristic hordein polypeptide antibody pattern could be connected to coeliac disease, as observed in a similar study using different fractions of the wheat prolamin, gliadin. Gliadin- and hordein-immunoadsorbent column experiments demonstrated that the prolamin reactivity originates from the same population of antibodies. It is speculated that distinct antigenic epitopes characteristic for untreated coeliac disease, might reside within a N-terminal repeat region of gliadin.     

A radioimmunoassay for alpha- and beta-gliadins

1. A rapid, sensitive specific radioimmunoassay for alpha- and beta-gliadin has been developed using an antiserum raised in rabbits to A-gliadin, a component of alpha-gliadin. 2. The antigen used in the assay was alpha-gliadin labelled with 125I; antigen-antibody complexes were collected after adsorption to Staphylococcus aureus in suspension. 3. The sensitivity of the assay, as judged by competitive binding with unlabelled antigen, was 1 ng of alpha- or beta-gliadin, which show complete cross-reaction with this antiserum. 4. Cross-reactivity to other wheat proteins was less than 1% and no cross-reactivity to extracts of rye, barley or oats was observed. 5. This radioimmunoassay for alpha- and beta-gliadin has been used to measure their amount in different varieties of wheat flour, several foods prepared from flour, e.g. bread, biscuits and products prepared as 'gluten free'. The possibility of assaying for alpha-gliadin in prepared foods is of special value since alpha-, beta-, gamma- and omega-gliadin have been shown to exacerbate coeliac disease.     

Groundnut Meal and Carrot Fortified Pasta: Optimization of Ingredients Level Using RSM

The present study was undertaken to optimize the level of groundnut meal, carrot juice and refined wheat flour for the development of pasta using response surface methodology. Different experimental combinations were designed using box-benken design of experiments considering 10–20 g groundnut meal, 14–30 mL carrot juice and 80–90 g refined wheat flour. Pasta samples with higher level of groundnut meal and carrot juice showed higher antioxidant activity and overall sensory acceptability. The samples with higher groundnut meal resulted in higher protein content. Pasta samples with higher amount of carrot juice showed higher rehydration ratio and lesser cooking time with low solid loss in cooking water. The food materials were optimized to obtain the best experimental combination for development of groundnut meal and carrot fortified pasta. Different levels of groundnut meal, carrot juice and wheat flour significantly affected the colour as well as cooking quality of pasta. The protein and antioxidant activity of pasta were increased with increasing level of groundnut meal and carrot juice in the sample. Optimized groundnut meal and carrot fortified pasta consisted of 17.64 % groundnut meal and 82.36 % wheat flour with 27.45 mL per 100 g formulation of carrot juice showing overall desirability as 0.849. This pasta sample required 3.49 min to cook with 4.71 % solid loss and rehydration ratio as 4.15 having 17.32 % protein content, 10.63 % antioxidant activity, 301.32 mg/100 g total phenols and overall sensory acceptability score as 8.7.     

Baker's asthma. Clinical and immunological studies

Seven bakers with respiratory symptoms were evaluated by skin tests, RAST assay for specific IgE antibodies to rye and wheat, inhalation challenge with methacholine for the determination of non-specific bronchial reactivity, and bronchoprovocation with rye and wheat extracts for the determination of antigen-specific bronchial reactivity. An immediate asthmatic response to antigen challenge was observed in four subjects and all of them had a high level of flour-specific IgE antibodies. The serum RAST values provided a more accurate predictive value than the degree of cutaneous sensitivity determined by skin testing with respect to the bronchial response to antigenic challenge. Among those who reacted positively to antigenic bronchoprovocation, a much lower antigen dose was required to elicit a positive reaction if the subject also had an increased degree of non-specific bronchial reactivity. An elevated RAST value was not found in thirty-eight asymptomatic bakers or in ten asthmatics who had no occupational exposure to flour. Thus, baker's asthma appears to be a form of allergic asthma to cereal flours mediated by specific IgE antibodies. Both the level of serum IgE antibodies and the degree of non-specific bronchial reactivity are important factors which may influence a baker's bronchial response upon inhalation of cereal flours.     

Gliadin-specific, HLA-DQ(alpha 1*0501,beta 1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients

Celiac disease (CD) is most probably an immunological disease, precipitated in susceptible individuals by ingestion of wheat gliadin and related proteins from other cereals. The disease shows a strong human HLA association predominantly to the cis or trans encoded HLA- DQ(alpha 1*0501,beta 1*0201) (DQ2) heterodimer. T cell recognition of gliadin presented by this DQ heterodimer may thus be of immunopathogenic importance in CD. We therefore challenged small intestinal biopsies from adult CD patients on a gluten-free diet in vitro with gluten (containing both gliadin and other wheat proteins), and isolated activated CD25+ T cells. Polyclonal T cell lines and a panel of T cell clones recognizing gluten were established. They recognized the gliadin moiety of gluten, but not proteins from other cereals. Inhibition studies with anti-HLA antibodies demonstrated predominant antigen presentation by HLA-DQ molecules. The main antigen- presenting molecule was established to be the CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer. The gluten-reactive T cell clones were CD4+, CD8-, and carried diverse combinations of T cell receptor (TCR) V alpha and V beta chains. The findings suggest preferential mucosal presentation of gluten-derived peptides by HLA-DQ(alpha 1*0501, beta 1*0201) in CD, which may explain the HLA association.     

Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease

BACKGROUND: Celiac disease (CD) is induced by wheat gliadins and related cereal proteins. Anti-gliadin antibodies (AGAs) are present in the serum of CD patients, but these antibodies have lower diagnostic specificity and sensitivity than autoantibodies [anti-endomysium antibodies (AEmAs) and anti-tissue transglutaminase antibodies (AtTGAs)]. Recently, AGAs from CD patients were found to recognize deamidated gliadin peptides, probably formed by the action of tissue transglutaminase. METHODS: We synthesized several gliadin peptides and their glutamine-glutamic acid-substituted counterparts on cellulose membranes and tested their recognition by IgA in sera of 52 AEmA-positive CD patients and 76 AEmA-negative controls in a luminescence assay. For comparison, we assayed IgA concentrations of AGAs, AtTGAs, and AEmAs. For measurement of AtTGAs, we used the human recombinant antigen. RESULTS: We identified several nonapeptides that were detected with high specificity by IgA in CD patients. Diagnostic accuracy of the peptide antibody assay was highest when peptide PLQPEQPFP was used in combination with peptide PEQLPQFEE within one assay. AGAs were above the cutoff in 14 of the controls, but only 5 of the controls were positive for peptide antibodies. For comparison, 82% and 94% of samples were correctly classified by AGAs and the combination nonapeptide assay, respectively (P = 0.007), and the AtTGAs correctly classified 98%. CONCLUSION: The peptide antibody assay has higher diagnostic accuracy than AGAs for distinguishing patients with CD from controls, and has diagnostic accuracy similar to that of AtTGAs.     

Enzymatic detoxification of gluten by germinating wheat proteases: Implications for new treatment of celiac disease

Introduction. Currently the only treatment for celiac disease is a lifelong gluten-free diet. The diet is, however, often burdensome, and thus new treatment options are warranted. We isolated proteases from germinating wheat grain naturally meant for total digestion of wheat storage proteins and investigated whether these enzymes can diminish toxic effects of gluten in vitro and ex vivo.

Methods. Pepsin and trypsin digested (PT) gliadin was pretreated with proteases from germinating wheat, whereafter the degradation was analyzed by HPLC-MS (high-performance liquid chromatography and mass spectroscopy) and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The toxicity of cleaved PT-gliadin products was assessed in Caco-2 epithelial cells, celiac patient-derived T cells, and in human small intestinal mucosal organ culture biopsies.

Results. Proteases from germinating wheat degraded gliadin into small peptide fragments, which, unlike unprocessed PT-gliadin, did not increase epithelial permeability, induce cytoskeletal rearrangement or changes in ZO-1 expression in Caco-2 cells. Pretreated gliadin did not stimulate T cell proliferation in vitro or enhance the production of autoantibodies to culture supernatants and the activation of CD25+ lymphocytes in the organ culture to the same extent as unprocessed PT-gliadin.

Discussion. Germinating wheat enzymes reduce the toxicity of wheat gliadin in vitro and ex vivo. Further studies are justified to develop an alternative therapy for celiac disease.     

Sub-class of IgG in allergic disease. I. IgG sub-class antibodies in immediate and non-immediate food allergy

Previous studies have suggested that, apart from IgE-mediated reactions, some of the symptoms of food allergy may be caused by IgG antibodies to food proteins. This study was carried out to see if there were any distinctive features of the IgG sub-class antibody response to dietary antigens which occurs in food allergic patients. IgG sub-class antibodies were measured using a quantitative enzyme-linked immuno-sorbent assay (ELISA) to wheat gliadin, ovalbumin and bovine casein in twenty patients who had coeliac disease and in twenty-eight egg allergic patients. These were compared with twenty-one atopic dermatitis patients who did not have food allergy and twenty-six healthy control subjects. Coeliac disease patients tended to have raised IgG antibody levels (especially IgG1) to all three antigens but these overlapped considerably with that seen in egg allergic and atopic dermatitis patients. Coeliacs who avoided gluten had anti-gliadin antibody levels which did not differ from those seen in healthy subjects but nevertheless had raised anti-ovalbumin and casein-specific antibodies. The IgG antibody was largely restricted to IgG1 and IgG4 sub-class although the relative amount of each varied with the antigen. Although gliadin-specific antibodies were mainly IgG1, ovalbumin-specific antibodies were mainly IgG4. The increased antibody levels to all three antigens in coeliacs were caused by a raised IgG1 response, IgG4 antibodies were usually normal. Egg allergic patients also had raised IgG1 but not IgG4 antibodies to ovalbumin. These data show that the response to different dietary antigens can vary with the antigen. The fact that IgG1 and not IgG4 antibodies were raised to all three antigens in patients with coeliac disease suggests that they are a secondary consequence of the disease, perhaps reflecting increased transport of antigens across a damaged gut mucosa rather than a specific immunopathological reaction. However, the observation that antibodies to gliadin, and not ovalbumin or casein, fell following gluten avoidance shows that the response to gliadin, at least, is dependent upon continued exposure to antigen.     

Isotype distribution and serial levels of antibodies reactive with dietary protein antigens in dermatitis herpetiformis

Using a sensitive enzyme-linked immunoassay (ELISA), a significantly increased prevalence (p less than 0.001) of serum antibodies reactive with wheat gliadin, bovine milk or ovalbumin has been demonstrated in 75% (33/44) of adult patients with dermatitis herpetiformis (DH), compared with healthy adults. There was no significant difference in the prevalence of antibodies (79%) in patients on a gluten-free diet or not on a gluten-free diet (72%). These serum antibodies reactive with gliadin, milk and ovalbumin were of the IgG isotype. However, IgA anti-gliadin antibodies were also detected in DH patients, but only in patients who were not on a gluten-free diet. In contrast, IgA anti-milk antibodies were also detected in DH patients irrespective of whether the patient was on a gluten-free diet. In DH patients, antibodies reactive with ovalbumin were often restricted to the IgG4 subclass and antibodies reactive with bovine milk antigens (notably casein) were distributed predominantly in both IgG2 and IgG4 subclasses, a similar IgG isotype distribution to that observed in healthy individuals. However, anti-gliadin antibodies in DH patients showed no predominant IgG4 subclass restriction. IgG4 anti-ovalbumin antibodies and IgG4 and/or IgG2 anti-casein antibodies persisted for up to 4 yr without fluctuation, irrespective of whether DH patients were on a gluten-free diet.     

Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions.

The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.     

EXPERIMENTAL RICKETS IN RATS : V. THE EFFECT OF VARYING THE ORGANIC CONSTITUENTS OF A RICKETS-PRODUCING DIET.

1. Casein phosphorus does not completely prevent the development of rickets when substituted in Diet 84 in amount equivalent to a protective dose of basic potassium phosphate. 2. The protection given by lecithin is equivalent to its phosphorus content. 3. The protection given by yeast is at least proportional to its phosphorus content. An amount carrying sufficient vitamine B to promote growth, but insufficient to provide adequate phosphorus, does not prevent rickets. 4. Vitamine A, in the form of butter or butter fat to the amount of 10 per cent of the diet, neither prevents nor cures rickets. 5. The substitution of 10 per cent of egg albumin in Diet 84 improves the nutrition, but does not prevent rickets. 6. The addition of meat to Diet 84, thereby supplying an abundance of phosphorus, promotes normal growth and normal bone formation. A diet consisting solely of meat and flour is inadequate for proper growth, and leads to changes in the bones comparable with those observed on a diet low in calcium, but rich in phosphorus. 7. A diet has been found which contains the necessary food elements for approximately normal growth, and in which the only known deficiency is phosphorus. This leads regularly to the production of rickets.     

A beta-turn rich oats peptide as an antigen in an ELISA method for the screening of coeliac disease in a paediatric population

BACKGROUND: ELISA methods for the measurement of IgA antigliadin antibodies (AGA), both home-made and commercial systems, routinely employ wheat gliadin fractions as coating antigens. We investigate the sensitivity and specificity for CD diagnosis of a new ELISA method using a highly immunoreactive beta-turn rich gamma3-avenin peptide as an alternative coating antigen. METHODS: The assay was standardized with antihuman IgA peroxidase-conjugated as the second antibody. Alternatively, an ELISA based on the use of protein A-peroxidase was assayed to measure both IgG plus IgA antibodies. Sixty-three sera from healthy controls were analyzed to establish the system's cut-off point. Sera from 103 coeliac and from 65 noncoeliac children were tested; for diagnosis purposes, a small intestinal biopsy had been performed in all of them. RESULTS: For the IgA class antibodies assay a high sensitivity and specificity of 90.3% and 98.5%, respectively, was obtained, comparable to those achieved for IgA antiendomysium antibodies (EmA) with the same sera. CONCLUSIONS: In view of the high sensitivity and specificity obtained together with water solubility of the peptide and easiness for large-scale reproducible synthesis, the new AGA IgA avenin peptide ELISA represents a significant improvement in CD diagnosis in comparison with conventional established AGA IgA ELISA using crude gliadins as coating antigens.     

Antibodies in human sera to oncorna virus-like proteins from normal or leukemia marrow cell cultures

Some human marrows in culture release particles with oncornavirus-like properties. This study was designed to examine the immunological properties of similar particles in human marrow culture supernates. Leukemic and nonleukemic marrows were cultured for 5-7 days in the presence of [14C]uridine and [3H]leucine or [3H]glucosamine. Labeled supernatant components banding in sucrose gradient densities of 1.20-1.24 g/ml were used as antigen in a double antibody immunoprecipitation assay. The assay was validated by end point titrations and competition with unlabeled antigen; purified myeloma proteins were used as negative controls. Cross-reactivity with mammalian oncornaviruses, as judged by competitive inhibition of precipitation by these viruses, was slight and at the border of the sensitivity of the method. Precipitated antigens analyzed by SDS polyacrylamide gel electrophoresis contained three distinct polypeptides of about 70,000, 45,000 and 30,000 mol wt; these comigrated with the gp 70, pg 45, and p 30 of a murine leukemia virus. Similar polypeptides were obtained from both leukemic and nonleukemic marrow culture supernates. As determined by the radioimmunoprecipitation assay, 32 of 45 leukemic sera (71%), 36 of 45 normal sera (80%), 15 of 19 sera from family contacts of leukemic patients (79%), 14 of 21 cord blood specimens (67%), and 21 of 23 sera (91%) from patients with systemic lupus erythematosus had detectable antibody activity.     

Bread wheat gliadin cytotoxicity: a new three-dimensional cell model

BACKGROUND: In an attempt to clarify the role of gliadin toxicity in the pathogenesis of gluten intolerance (celiac disease), previous in vitro studies have been based on two-dimensional human cell cultures. However, the specific morphological and biochemical properties of in vivo tissue are better maintained in three-dimensional cell cultures (multicellular spheroids, MCS). The aim of this study was to develop a three-dimensional in vitro model to investigate the effects of gliadin on epithelial cells and broaden our understanding of the early tissue damage occurring in celiac disease. METHODS: The three-dimensionally growing Lovo cell line was exposed to increasing concentrations of peptic-tryptic-digested bread wheat gliadin (from 125 to 1000 microg/mL) for 7 days in order to evaluate cell viability (colony-forming assay), and at the standard concentration of 500 microg/mL for 7 days in order to evaluate MCS diameters, volumes and cell morphology using light and electron microscopy. RESULTS: In comparison with the controls, the cell viability of the gliadin-treated MCS was significantly reduced (20-80%), but there was no difference in size. Various degrees of cell damage (autophagic vacuoles and intra-cytoplasmic lipid-like droplets) were detected by both light and electron microscopy. CONCLUSION: This is the first study investigating the effects of gliadin on MCS. Lovo MCS seem to be responsive to gliadin exposure, thus confirming previous results obtained using two-dimensional cell cultures. The data suggest that three-dimensional cell cultures may be useful in broadening our understanding of some of the early effects of gliadin peptides on epithelial cells.     

Plasma amino acid response to single test meals in humans

The plasma amino acid response to single test meals in young adults was used for human biological evaluation of the supplementary effect of dried skim milk powder (DSM) and synthetic L-methionine on a vegetable protein mixture. The protein sources in the vegetable mixture were wheat flour, defatted soya bean flour and pea flour. The plasma amino acid responses were evaluated both as PAA ratios (a modification of the Longenecker and Hause method), and as delta MR% (percentage change in the postprandial essential amino acid molar ratios according to Graham and Placko). Both evaluation methods indicated that there was an adequate supply of all the essential amino acids in the basic vegetable mixture, except for a small deficit of methionine. Supplementation (5% and 10%) with DSM did not significantly improve the low plasma amino acid response of methionine. The addition of synthetic L-methionine proved to be very effective in this respect. 1 g of L-methionine per 100 g of proteins from the vegetable mixture gave a plasma methionine response similar to that of the other essential amino acids. This implies that the deficit of methionine in the basic wheat/soya bean/pea mixture was about 30%.     

Measurement of faecal immunoglobulin a levels in young children

A nephelometric immunoassay was developed to quantify immunoglobulin A (IgA) in children's stools. This method enables IgA in faecal protein extracts to be measured over a large range of concentrations (1.61-51.50 mg/L) with good accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation (CVs) of 1-6%). An excellent recovery (105%) was obtained in stool samples overloaded by purified colostral IgA, demonstrating that the method used for faecal IgA extraction is adapted, not to induce significant IgA degradation, and probably allow a complete extraction of IgA. The amount of faecal IgA, as determined in stool samples from 125 children (6-24 months old), was an average of 14 mg per 100 g of stools (about 10% of the total protein stool content), with large individual variation (3-30 mg per 100 g of stools). No correlation was observed between faecal IgA amounts and the children's age or sex. Such an immunoassay could enable exhaustive noninvasive investigations of the maturation of the intestinal immune system, as well as accurate studies of the effect of oral dietary supplementation on IgA regulation.     

Diagnostic efficacy of the ELISA test for the detection of deamidated anti‐gliadin peptide antibodies in the diagnosis and monitoring of celiac disease

Background and Aim: We evaluated the diagnostic performance of an ELISA test for anti‐gliadin IgA and IgG antibodies, which uses synthetic deamidated gliadin peptides (anti‐gliadin antibodies, AGAs) as coating; the results were compared with a test that uses extracted gliadin (AGAe). Methods: The study was conducted on the sera of 144 patients suffering from celiac disease (CD), including 20 patients with IgA deficiency and 9 who were following a gluten‐free diet (GFD), and 129 controls. Results: In the 115 CD patients (without IgA deficiency), the sensitivity of AGAe IgA and IgG was 32.2 and 60.9%, whereas that of AGAs IgA and IgG was 59.1 and 72.2%. The specificity for AGAe IgA and IgG, and AGAs IgA and IgG was 93.8 and 89.9%, and 96.9% and 99.2%, respectively. Of the 20 patients with CD and IgA deficiency, 7 tested positive for AGAe IgG and 14 for AGAs IgG. The test using deamidated gliadin peptides performed better in terms of sensitivity and specificity than the AGA tests with extracted antigen. Conclusions: The very high specificity of the AGAs IgG test (99.2%) also suggests that patients who test positive with this assay require a thorough followup, even if the anti‐tissue transglutaminase antibodies (anti‐tTG) and anti‐endomysial autoantibodies (EMA) assays are negative. J. Clin. Lab. Anal. 23:165–171, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of fibrinolytic capacity by a combined assay system for tissue-type plasminogen activator antigen and function using monoclonal anti-tissue-type plasminogen activator antibodies

An assay system has been developed that allows consecutive quantification of tissue-type plasminogen activator (t-PA) activity and t-PA antigen in the same plasma sample. In the first step t-PA is bound to an immobilized IgM monoclonal anti-t-PA antibody and functional activity of bound t-PA is quantified by its plasminogen-activating activity. In the second step the amount of bound t-PA antigen is determined by using a different peroxidase-labeled monoclonal anti-t-PA antibody. In this combined assay system t-PA functional activity was found to depend not only on the amount of t-PA antigen but also on the amount of plasminogen activator inhibitor (PAI), whereas in the t-PA antigen assay PAI did not affect the results. In plasma samples obtained from normal controls t-PA activity was detected only in post-venous occlusion plasma (3.7 +/- 2.5 IU/ml), whereas 2.7 +/- 0.5 ng/ml t-PA antigen was found before and 12.6 +/- 4.4 ng/ml after venous occlusion. Using this combined assay system to study plasma samples from patients who did not respond to venous occlusion with shortening of the euglobulin clot lysis time (ECLT), it was possible not only to confirm that in none of these patients could t-PA activity be detected in the postocclusion plasma samples but also to subdivide that group of patients into a group of about 39% not reacting with normal t-PA antigen release to venous occlusion and into a second group of about 61% that reacted with normal t-PA antigen release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay of an antitumor protein, neocarzinostatin, and its antibody by fluorescence polarization.

I evaluated use of the fluorescence polarization technique to measure neocarzinostatin, a proteinaceous antitumor antibiotic, and its antibody, in serum. The antigen (neocarzinostatin), labeled with fluorescein isothiocyanate, was allowed to interact with its antibody in a cuvet, in the instrument, yielding an increase in the fluorescence polarization value. Antibody content was determined in the presence of a definite amount of the labeled antigen, fluorescence polarization values increasing in parallel with each addition of antibody. Antigen content was determined with a known amount of antibody, which reacted at first with an unknown amount of antigen in samples, followed by addition of a definite amount of the labeled antigen (competition). I used the method to determine a pharmacokinetic parameter, the apparent volume of distribution for neocarzinostatin in rabbits, using drug-injected rabbit sera. I evaluated precision, accuracy, and reproducibility, using various samples or possible interfering substances such as bilirubin and hemoglobin, and also compared results for antigen with those by single radial immunodiffusion assay. The present assay is fast (less than 2 min), sensitive (less than 10 nmol/liter can be detected), and simple (there is no separation step before readout of the results).