Enzyme-linked immunoassay of somatostatin.

An enzyme-linked immunoassay for somatostatin using somatostatin-alkaline phosphatase conjugate as "labeled" antigen was developed. Minimal detectable dose at present was 40 pg per tube. Serial dilutions of rat hypothalamic extract gave a gradual change of antibody-bound alkaline phosphatase activity which was parallel to that with standard somatostatin. Precision and accuracy of the method were comparable to those in radioimmunoassay reported by others. This method will be a useful tool for the determination of somatostatin, especially in tissues.     

Enzyme-linked immunoassay of ursodeoxycholic acid in serum.

A sensitive and specific enzyme-linked immunoassay for the measurement of ursodeoxycholic acid in human serum was developed. Ursodeoxycholic acid conjugated to alkaline phosphatase (from calf intestine) was used as a tracer. An antiserum to ursodeoxycholic acid serum was raised in rabbits using ursodeoxycholic acid--bovine serum albumin conjugate as an antigen. The binding required 1 hr at 42 degrees C; separation of the bound tracer was achieved by addition of a second antibody, and alkaline phosphatase activity of this bound complex was measured colorimetrically. The ratio of bound to total enzyme activity decreased linearly with a logarithmic increase in ursodeoxycholic acid concentration from 20 to 900 pmol. The specificity and sensitivity of this enzyme-linked immunoassay were similar to those of a radioimmunoassay reported previously. The serum ursodeoxycholic acid levels measured by this method correlated well with those determined by gas--liquid chromatography and radioimmunoassay. Based on these findings, this enzyme-linked immunoassay of bile acid might be useful as a tool for the routine clinical analysis of serum bile acids.     

Enzyme immunoassay of ampicillin in milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Enzyme-linked immunoassay. II. A simple method for synthesis of the rabbit antibody-beta-D-galactosidase complex and its general applicability.

Rabbit anti-human IgG antibody was mildly reduced in the presence of 10 mM 2-mercaptoethylamine and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide. Human IgG and the anti-human IgG antibody-beta-D-galactosidase complex were successively adsorbed on Sepharose 4B binding rabbit anti-human IgG antibody. In this way amounts of human IgG as small as 5X10(-15) moles could be measured by determining the activity of beta-D-galactosidase bound to the Sepharose.     

Enzyme-linked immunosorbent analysis for aflatoxin B1.

An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B2alpha, and least specific for aflatoxin G1.     

Enzyme-linked immunosorbent analysis for aflatoxin B1.

An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B2alpha, and least specific for aflatoxin G1.     

Comparison of recombinant human haptocorrin expressed in human embryonic kidney cells and native haptocorrin

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies.     

Miniaturised hybrid immunoassay for high sensitivity analysis of aflatoxin M1 in milk

An ultra-sensitive sandwich ELISA was developed for detection of AFM1 in milk. The assay involved the immobilization of rat monoclonal antibody of AFM1 in 384 microtiter plate to capture AFM1 antigen. This was detected by tracer secondary rabbit poly-clonal antibody labelled with horseradish peroxidase upon addition of a luminol-based substrate. Milk samples with different fat percentage were analyzed after pre-treatment. Linear range of AFM1 detection 250-6.25 pg/mL was achieved in 3% fat milk. The miniaturised assay (10 μL) enabled ultra trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005 pg/mL. A sensitive magnetic nanoparticles (MNPs) based ELISA was also developed and coupled with micro plate ELISA for analysis in milk. The hybrid-assay, by coupling the 1°Ab immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5 pg/mL) and high AFM1 contamination (200 pg/mL). The most promising feature of this MNPs-ELISA is the small column size, high capture efficiency and lower cost over other reported materials. The proposed assay can be deployed for simultaneous analysis and monitoring of AFM1 in milk.     

Comparative analysis of cobalamin binding kinetics and ligand protection for intrinsic factor, transcobalamin, and haptocorrin.

Changes in the absorbance spectrum of aquo-cobalamin (Cbl x OH(2)) revealed that its binding to transcobalamin (TC) is followed by slow conformational reorganization of the protein-ligand complex (Fedosov, S. N., Fedosova, N. U., Nex?, E., and Petersen, T. E. (2000) J. Biol. Chem. 275, 11791-11798). Two phases were also observed for TC when interacting with a Cbl-analogue cobinamide (Cbi), but not with other cobalamins. The slow phase had no relation to the ligand recognition, since both Cbl and Cbi bound rapidly and in one step to intrinsic factor (IF) and haptocorrin (HC), namely the proteins with different Cbl specificity. Spectral transformations observed for TC in the slow phase were similar to those upon histidine complexation with Cbl x OH(2) and Cbi. In contrast to a closed structure of TC x Cbl x OH(2), the analogous IF and HC complexes revealed accessibility of Cbl's upper face to the external reagents. The binders decreased sensitivity of adenosyl-Cbl (Cbl x Ado) to light in the range: free ligand, IF x, HC x, TC x Cbl x Ado. The spectrum of TC x Cbl small middle dotAdo differed from those of IF and HC and mimicked Cbl x Ado participating in catalysis. The above data suggest presence of a histidine-containing cap shielding the Cbl-binding site in TC. The cap coordinates to certain corrinoids and, possibly, produces an incapsulated Ado-radical when Cbl small middle dotAdo is bound.     

Degree of structural homology of lactoferrins from milk and neutrophilic granulocytes

Some physico-chemical properties of human and pig lactoferrins from milk and neutrophilic granulocytes were compared. It was shown that the lactoferrins from different cell and tissue sources of the same species (humans or pigs) are identical in terms of electrophoretic mobility, molecular weight, iron-binding capacity, absorbance spectra, amino acid and sugar compositions and peptide maps. Human and pig lactoferrins show a high degree of structural homology (approximately 50%), but are immunochemically different.     

Purification and characterization of rabbit haptocorrin

Rabbit haptocorrin (R-binder) has been purified from serum by affinity chromatography on cobalamin-Sepharose and Blue Sepharose. It proved to be a protein with a relative molecular mass of 60 000 and an amino acid content very similar to that of other haptocorrins (Nexø, E. and Olesen, H. (1981) in B₁₂ (Dolphin, D., ed.), Wiley Interscience, New York/London, in the press). The pH optimum (pH 6–9) for binding of cyanocobalamin and the affinity to dicyanocobinamide were like those of human and hog haptocorrins. In spectral studies, the extinction coefficient of cyanocobalamin at 363 nm (γ₁-band) increased by about 16% on binding to rabbit haptocorrin. Binding of azidocobalamin gave spectra changes similar to those for binding to rabbit transcobalamin.     

Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.     

Progesterone in milk: a simple experiment illustrating the estrous cycle and enzyme immunoassay

Experiments designed for students in reproductive physiology are rare. Here, we describe a simple experiment concerning a physiological aspect of the reproductive system. Milk samples are obtained from cows in estrus, in midcycle, 21 days after insemination, and in gestation. With these samples, the gestation or estrous stage is determined according to the progesterone level in milk that is measured using enzyme immunoassay. This experiment can therefore be used to demonstrate assay techniques and to illustrate the variations in progesterone concentrations during an estrous cycle and gestation. This exercise should be given after the reproduction section of the animal physiology course so that students can apply their knowledge concerning hormonal profiles during an estrous cycle.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

Quantitative immunochromatographic analysis: theory and application to theophylline immunoassay

The development of an immunochromatographic technique suitable for rapid analysis of biological fluids is described. Quasi-one-dimensional antibody lattices specific for theophylline were constructed by packing Sepharose beads conjugated with specific antibody into specially designed narrow capillary tubes. The design of these capillary columns was such that they would subtract a preset threshold quantity of antigen (label and analyte) from the total amount presented. Labeled antigen, which appeared in the flowthrough, could then be used to precisely quantitate the analyte present. The ideal format would permit very precise subtraction of 100% of the available antigen up to the threshold amount and none of the remainder. The microcolumn described here comes close to this ideal behavior through the attainment of very high ratios of bound/free antigen. The elevated bound/free ratio could be explained by theoretical analysis of the effect on equilibria of the high antibody concentration in this quasi-one-dimensional system. Lattices containing anti-theophylline antibodies were used to develop a competitive enzyme immunoassay for theophylline which demonstrated a dose-response that was closely similar to that predicted by theoretical treatment. The entire assay procedure was performed in less than 30 min and demonstrated a sensitivity limit of approximately 20 ng/ml. Preliminary studies on clinical serum samples suggest that this assay has potential for the routine analysis of biological fluids.     

Use of soybean peroxidase for the enzyme immunoassay of sulfamethoxipyridazine in milk

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3-63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 microg/l) by several orders of magnitude.     

Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk

As a result of immunization of rabbits with neomycin B (NM) conjugated to sodium periodate-oxidized (SP) transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5–10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/106), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the MRL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.     

Use of soybean peroxidase for the enzyme immunoassay of sulfamethoxipyridazine in milk

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3–63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 μg/l) by several orders of magnitude.