Advances of diagnosis and clinical assessments for pancreatic disorders. Remarks

In spite of the marked advances of laboratory medicine, diagnostic parameters or tools for the pancreatic disorders were now in obscure. However, advances of immunochemical procedures, especially advances of monoclonal antibodies and high performances of immunoassay techniques make it possible to reveal the chemico-pathological status of pancreatic disorders. Moreover, advances of ultrasonic echography and ultrasonically guided biopsy more than make up for the defects of biochemical and immunochemical information. In this symposium, these advances of diagnosis for pancreatic disorders will be studied and discussed.     

Dosage sérique des chaînes légères libres (CLL) d'immunoglobulines : de la biologie à la clinique

Detection and measurement of serum or urine immunoglobulin free-light-chains have been associated with a large variety of analytical problems (low amounts of immunoglobulin free-light-chains in biological fluids, interindividual variability of the structure and the immunochemical characteristics, no international standard…). Recently, a quantitative immunoassay has been available for immunoglobulin free-light-chains dosage in both serum and urine. This review aims at discussing advantages and potential pitfalls of this new immunoassay in daily laboratory practice, and at analysing the main clinical relevance of this test in clinical diagnosis.     

An immunoassay for distinguishing between crustacean tailmeat and white fish

An immunoassay has been developed which facilitates discrimination between white fish, such as coley, and crustacean tailmeat, such as scampi. Preliminary studies with this non‐competitive indirect ELISA clearly demonstrate the feasibility of using immunochemical tests for detecting adulteration of high value crustacean tailmeat products with lower value white fish.     

Antistreptolysin O]

Automated analyzers based on the quantitative immunochemical methods have been developed and can allow quantitative measurements of antistreptolysin O concentrations in the clinical laboratories. Automated ASO determinations improve in point of operation, time, effort, precision and accuracy, compared with the usual semiquantitative methods of tube dilution techniques based on the method of Rantz and Randall or some modification thereof, microtitration of the Edward method and agglutination test using latex or other particles. However, confusion and many kinds of problems have been brought about by the rapid development of automated ASO measurements in routine use. The principle quantitative immunochemical measurements of automated ASO determinations are immune agglutination assays, which are nephelometric immunoassay (NIA), latex agglutination photometric immunoassay (LAPIA) and turbidimetric immunoassay (TIA). Principles and methods of these automated immunoassay, reagents, ASO standard serum, automated analyzers, precision and accuracy, the present conditions and problems of automated analysis in the clinical laboratories were discussed in comparison with usual semiquantitative measurements. There was a good correlation between automated immune agglutination assay (NIA, LAPIA and TIA) and the usual semiquantitative assay. However, the quantitative accuracy of automated immune agglutination assay was decreased in the regions of low and high ASO values. Availability, precision and accuracy of ASO measurement were improved by the automated analysis. However, the methods of ASO measurements were increased and varied by the automated assay. Therefore, it is necessary to standardize the automated ASO measurements. The limitation and the clinical significance of the automated immune agglutination assay of ASO have to be studied for practical use in the future.     

Plasma determination of homocysteine on CPC Immulite 2000: comparison with determination on IMX Abbott

Plasma total homocysteine is a parameter frequently included in the biological investigation of arterious or venous thrombotic diseases. Different techniques, chromatographic, enzymatic, and immunochemical, are used in the measurement of homocysteine. Among immunochemical methods, some are conceived to function on multiparametric automates, leading to a greater accessibility of the test for most of the clinical laboratories. In this study, we have evaluated a new immunoassay proposed by the DPC Company for the Immulite 2000 analyzer (chemiluminescence) and compared its performance against the Abbott's immunoassay on IMx (fluorescence polarization). The results obtained show very good general performance of the DPC's technique. Linearity is also excellent. The within run CVs are 9.9, 7.0 and 5.4% and the between run CVs are 8.2, 3.9 and 4.3% respectively for homocysteine levels of 4.2, 13.9 and 27.7 pmol/L. We found a very good correlation between DPC's and Abbott's methods (regression analysis:y=0.948 x + 0.05; r=0.895). The mean of differences between both methods is -0.55 micromol/L. On the whole, the DPC technique appeared in our experience as easily exchangeable, from the analytical point of view, with Abbott's technique.     

Quality of timothy pollen (Phleum pratense) from different pollen seasons and different suppliers

We investigated seven batches of timothy (Phleum pratense) pollen from four different pollen seasons and three different manufacturers in the USA and Europe by several biochemical and immunochemical methods. By measuring the contents of hexoses, proteins and the total allergenic activity using RAST inhibition we could not find any significant differences in quality between pollen of different seasons, different geographical origin and different manufacturers. There were only slight differences in the staining of some protein bands and in peak height of some antigens in isoelectric focusing (IEF), SDS-PAGE and crossed immunoelectrophoresis. These differences in IEF and SDS-PAGE patterns seem to be specific for the pollen samples from the USA and Europe, respectively. We also observed minor differences in IEF immunoprint patterns, mainly for some basic proteins. The allergen patterns in crossed radioimmunoelectrophoresis were very similar, with only minor differences in concentration of the basic allergens. It appears that timothy pollen from different years can be produced in different geographical areas by different manufacturers with fairly constant allergenic activity.     

An atomic force microscopy study in the detection of hepatitis B surface antigen by enzyme-linked immunosorbent assay

Atomic force microscopy (AFM) was employed to study certain enzyme immunoassay steps for the detection of hepatitis B surface antigen (HBsAg). Physical adsorption of monoclonal antibodies (mAb), blocking of surface active sites free of antibodies by neutral proteins, and capture of HBsAg particles by sensitized surfaces were visualized successively in microplate wells of standard immunological plates from various manufacturers. The previously undescribed details such as "etching holes" up to 20?nm in depth were observed on the surface of plates some companies. The quantitative relationships between the optical density (OD) values obtained by enzyme immunoassay (EIA) and the number of antigen-antibody complexes in AFM were calculated.     

Immunoblotting and dot immunobinding. Emerging techniques in protein immunochemistry

Immunoblotting (also known as Western blot) and dot immunobinding (also known as dot blot) immunoassays, used extensively in immunochemical research, have great potential significance for diagnostic testing in the clinical laboratory. Immunoblotting has distinct versatility in immunochemical test applications. Immunoblotting combines the power of gel electrophoresis for resolving the electrophoretic variants of immunologically cross-reacting proteins with the ease and sensitivity of immunodetection on a solid-phase immunoassay. The potential for diagnostic applications includes: assay of antigens from pathologic serum samples, body fluids, and tissue; assay of patient serum samples for antibody against known antigen; and separation and assay of patient immunoglobulin. The usefulness of these applications is enhanced by the possibility of simultaneous use of nonantibody ligand, reversible protein staining, or, in the case of enzyme proteins, the use of substrate to detect activity. Dot immunobinding, in a similar fashion, permits assays of multiple specimens simultaneously on single strips of blotting media using sample sizes as small as 0.1 microL. Also, both immunoblotting and dot immunobinding techniques permit sequential probing of antigens with different antisera, with subsequent elution and recovery of specific antibody probes.     

Immunochemical differences between oral and nonoral strains of Bacteroides asaccharolyticus.

We undertook a morphological and immunochemical comparison of purified outer membrane antigens from oral and nonoral isolates of Bacteroides asaccharolyticus. Electron micrographs of thin sections of whole bacteria revealed a compact, electron-dense capsule external to the outer membrane of oral strains. A loose, web-like material was noted on the surface of several nonoral strains that was distinct from the dense capsule seen on oral strains. Polyacrylamide gel electrophoresis showed distinct differences in the protein band pattern between oral and nonoral isolates; sugar composition was similar with a few exceptions. An indirect fluorescent-antibody test utilizing antiserum to a purified capsular antigen from a single oral strain cross-reacted with all of numerous oral and nonoral strains of B. asaccharolyticus, thereby demonstrating a shared antigen that is species specific for B. asaccharolyticus. However, antibodies to an oral strain-derived capsular antigen were detectable by enzyme immunoassay only in serum from rabbits immunized with oral strains. Thus, definite morphological and immunochemical differences were found between oral and nonoral isolates of B.asaccharolyticus.     

Measurement of C9 concentrations using an immunochemiluminometric assay

A 2-site immunochemiluminometric assay is described for the measurement of complement component C9 concentrations in biological fluids as an aid in the diagnosis and management of immune-based diseases. The assay utilises 2 monoclonal antibodies one of which is labelled with a chemiluminescent acridinium ester and one of which is covalently coupled to reprecipitated aminoaryl cellulose. The incubation time is 1 h with simultaneous reagent addition. The working range of the assay is 10-2500 micrograms/1 at CVs less than or equal to 10% and the results are in excellent agreement with those of an immunoradiometric assay. The assay exhibits superior performance to other immunochemical and immunoassay techniques and has the advantage of using stable, non-radioactive reagents.     

A sol-particle immunoassay for determination of anti-rubella antibodies; development and clinical validation

A sol-particle immunoassay (SPIA) was developed for determination of anti-rubella antibodies. The test is based on antibody-coated gold sol particles, which may agglutinate in the presence of rubella haemagglutinin antigen. The agglutination which gives a decrease in optical density can be inhibited by specific antibodies present in sera incubated with the immunochemical reagents. The test is performed in microtitration plates and results can be read in a plate-reader. Clinical validation studies were performed comparing the anti-rubella SPIA with the haemagglutination inhibition test and with the latex agglutination test. The sensitivity of the SPIA was calculated to be 99%; the specificity of the test was at least 99.5%. The anti-rubella SPIA is highly suitable for large-scale screening to determine the rubella immune-status.     

Neurotrophic factors and autoantibodies to them as molecular predictors of cerebral dysfunctions

A complex of clinical and immunochemical studies was made in patients with chronic brain ischemia and ischemic stroke and in neonatal infants with CNS dysfunctions and retarded intrauterine development. Enzyme immunoassay was used to measure the levels of brain proteins with trophic properties--S100b, the major protein myelin, lectins CSL, R1, and the levels of primary and antiidiotypic antibodies to these proteins in the biological fluids of the patients. The findings suggest that the study brain proteins and autoimmune processes against these factors are involved in the mechanisms of the pathogenesis of the diseases in question and they enable changes and variations in the levels of neurotropic factors and their autoantibodies to be considered as predictors of brain ischemia and perinatal cerebral lesions.     

Evaluation of a multiplex flow cytometric immunoassay to detect PR3- and MPO-ANCA in active and treated vasculitis, and in inflammatory bowel disease (IBD)

This study compared the performance of a flow cytometric immunoassay for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO), with indirect immunofluorescence (IIF) and ELISAs from 12 different manufacturers. Sera were from patients with active (n=55) or treated (n=68) small vessel vasculitis, or inflammatory bowel disease (IBD, n=22). The immunoassay specificity was 88% compared with 96% for IIF and 94% (median, range 91-96%) for both ELISAs. Its sensitivity in treated disease was 82% compared with 84% for IIF and 69% (median, range 57-82%) for the ELISAs. The immunoassay's specificity was 88% which was the same as the median for both ELISAs (range 84-95%). The PR3- and MPO-ANCA immunoassay was almost as sensitive as IIF, and more sensitive than, but just as specific as, most ELISAs, in detecting ANCA in active and treated vasculitis. A major advantage of this assay is its ability to be further modified to simultaneously screen for a panel of autoantibodies relevant to vasculitis.     

Immunochemical detection of human blood in feces.

Current methods for testing stool samples for hemoglobin utilize peroxidase oxidation of chemical indicators such as guaiac or benzidine. These tests have frequent false-positive and false-negative results, complicating random screening for occult gastrointestinal bleeding. The authors have developed an immunochemical test for human blood in feces using goat antibodies to hemoglobin. When employed in radial immunoassay the test is uncomplicated by cross-reaction with common human foods or other nonhemorrhagic fecal fecal constitutents. The lower limit of sensitivity for hemoglobin in stool samples is 10 mg/dl, compared with a commonly reported threshold of 100 mg/dl for peroxidase tests. The test accurately detects hemoglobin in mixtures of human blood and feces. Immunochemical identification of human blood in stool offers improved detection of lower gastrointestinal bleeding.     

Development of Immunochemical Techniques for Detecting Karnal Bunt in Wheat

The protein profile of Tilletia indica, the causal agent of Karnal bunt, was analyzed by SDS-PAGE and compared to protein profiles of T. barclayana, T. tritici, T. controversa and T. laevis. Based on these results, antibodies were developed against a 64 kDa T. indica protein and used to construct both a microwell sandwich ELISA and a dipstick immunoassay. Both of these immunoassays can detect 1.25 ng/well of purified T. indica protein or 40 ng/well of crude spore extract, distinguishing Karnal bunt from all wheat smuts and, to some degree, the rice smut, T. barclayana. To enhance specificity, an innovative approach to species-specific antibody production was utilized to develop antibodies that discern Karnal bunt from all other fungal species. Using these new antibodies, a Karnal bunt specific immunoassay was developed that has all the attributes of the original Karnal bunt immunoassays and detects Karnal bunt in the presence of wheat. This is the first report of immunochemical diagnostic tools that will aid in controlling the spread of this wheat pathogen by accurately and efficiently detecting Karnal bunt teliospores in wheat.     

Mediator release assays based on human or murine immunoglobulin E in allergen standardization

BACKGROUND: The biological potency of allergens can be measured by provoking mediator release from effector cells. As established immunochemical methods in allergen standardization only determine inhibition potency or major allergen content, routine tests for biological potency may enhance standardization and batch control of allergen products. OBJECTIVE: The general performance and application potential of biological in vitro assays in batch control and standardization of allergens and as a tool for verifying activity and stability of allergen standards were analysed. METHODS: Allergen extracts of five clinically relevant allergens from three to five different manufacturers were investigated. A CAP-IgE-inhibition assay was compared with mediator release assay (MRA)s based on murine or human basophils. Rat basophilic leukaemia (RBL) cells were passively sensitized with pooled murine allergen-specific IgE-containing sera. Humanized RBL cells and human-stripped basophils were sensitized with pooled patient's sera, which were also used for the CAP-IgE-inhibition assay. Allergen specificity of the sera was determined by immunoblotting. RESULTS: A good batch-to-batch consistency was found with each assay among all manufacturers and allergens tested. Between different manufacturers, the products showed differences in activity and the various assays indicated an almost identical ranking. However, the biological assays revealed qualitative differences of biological activity or composition of allergen preparations undetectable by IgE-inhibition assay. CONCLUSIONS: MRAs provide refined information on allergen activity, either confirming the results of IgE-inhibition assay, or indicating differences requiring further investigation, and represent a highly sensitive novel tool in allergen standardization. By using permanently cultivated cell lines, repeated venepuncture to obtain human basophils is avoided. As in the RBL assay, the coefficient of variation for the release values were below 15% and for the ED50 below 25%, the assay is suitable to determine differences that are relevant for batch control purposes.     

Synthesis and immunochemical properties of the recombinant major surface glycoprotein E2 of the classical swine fever virus

Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.     

In vitro modulation of keratinocyte-derived interleukin-1 alpha (IL-1 alpha) and peripheral blood mononuclear cell-derived IL-1 beta release in response to cutaneous commensal microorganisms.

The ability of a range of skin commensal microorganisms to modulate interleukin-1 (IL-1) release by cultured human keratinocytes and peripheral blood mononuclear cells (PBMCs) was investigated by a combination of enzyme-linked immunosorbent assays and bioassays. Three fractions (formaldehyde-treated whole cells, culture supernatants, and cellular fractions) were prepared from Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, and Malassezia furfur serovar B. The levels of immunochemical IL-1 alpha released by cultured keratinocytes during coincubations with these microbial fractions ranged from 0 to 136 pg/ml and were maximal after 72 h. No microbial fraction consistently upregulated immunochemical IL-1 alpha release by freshly isolated keratinocytes from two donors and a transformed cell line, all of which produced the cytokine constitutively to various extents. Bioassays revealed that most of the IL-1 released was biologically inactive. In contrast, whole cells of formaldehyde-treated P. granulosum and S. epidermidis significantly stimulated release of IL-1 beta by PBMCs from three donors compared with the negative control (culture medium). Release was maximal at 24 h. Coincubation with intact cells of the yeast M. furfur significantly decreased levels of IL-1 beta below the values for the negative control by PBMCs from all three donors. There was good correlation between bioassay data and immunoassay data for IL-1 beta, and the depressive effect of M. furfur cells on cytokine production by all three cultures of PBMCs was mirrored in the levels of bioactive cytokine. This reduction in IL-1 beta release by PBMCs by M. furfur may provide an explanation why dermatoses thought to be caused by this yeast are essentially noninflammatory or only mildly inflammatory.     

A two-site monoclonal enzyme immunoassay for pregnancy-associated alpha 2-glycoprotein

A two-site monoclonal enzyme immunoassay (MEIA) was developed for the determination of pregnancy-associated alpha 2-glycoprotein (PA alpha 2G) in serum. The minimum detectable level was 35 ng/ml. No immunochemical interaction with the closely related alpha 2-macroglobulin was found. Serum levels in 145 normal healthy males and non-pregnant females were 1.8 +/- 2.8 micrograms/ml (mean +/- SD), respectively, both significantly lower than previously reported. The distribution in the normal population was characteristically different when males and females were compared. No increase with age was found. During pregnancy, a significant increase in serum concentration was observed with average levels of 320 +/- 200 micrograms/ml at term (mean +/- SD), a 50-fold increase in concentration. As a tumor marker for breast and ovarian cancer, PA alpha 2G was found to be of no value. The present study emphasizes that a reevaluation of PA alpha 2G levels must be undertaken in order to assess the clinical and biological significance of this protein.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.