Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.     

Difference in iron metabolism may partly explain sex-related variability in the manifestation of Wilson’s disease

Background/aimWilson’s disease (WD) is a hereditary disorder characterized by abnormal metabolism of copper. For unknown reasons, the clinical picture of this disease appears to be sex-dependent. Because the metabolism of copper and iron is interrelated, we aimed to evaluate whether the variability in the clinical picture of WD could be explained by the sex difference in iron metabolism.MethodsA total of 138 WD patients were examined in this study: 39 newly diagnosed, treatment naive patients and 99 individuals already treated with decoppering drugs. The serum concentration of ceruloplasmin (Cp) and copper were measured using an enzymatic colorimetric assay and by atomic absorption spectroscopy, respectively. The parameters of iron metabolism were determined by using standard laboratory methods and enzyme immunoassays.ResultsIn the treatment naive group men had a higher median serum concentration of ferritin (290.5 vs. 81.0 ng/mL, p < 10⁻⁴), and hepcidin (Hepc) (55.4 vs. 22.8 ng/mL, p < 10^-3) compared to women, and tended to have higher concentration of iron, hemoglobin (HGB) and number of red blood cells (RBC). In the treated group men had higher median ferritin (122.0 vs. 46.0 ng/mL, p < 10⁻⁴), Hepc (23.5 vs. 10.8 ng/mL, p < 10⁻⁴), iron (102.5 vs. 68.0 μg/dL, p < 10⁻⁴), HGB (15.0 vs. 13.2 g/dL, p < 10⁻⁴), and RBC (5.0 vs. 4.5 M/L, p < 10⁻⁴) than women.ConclusionIron metabolism differs between men and women with WD, which may partly explain the sex difference noted in the disease manifestation.     

A sandwich enzyme-linked immunosorbent assay for adducts of polycyclic aromatic hydrocarbons with human serum albumin

Adducts of benzo[a]pyrene-diolepoxide (BPDE) with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive enzyme-linked immunosorbent assay (ELISA) that employs monoclonal antibody 8E11 to detect benzo[a]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we used 8E11 as the capture antibody in a sandwich ELISA to detect BPDE-HSA adducts directly in 1-mg samples of HSA or 20 μl of serum/plasma. The assay employs an anti-HSA antibody for detection, and this is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is approximately 10 times more sensitive than the competitive ELISA. To validate the assay, HSA samples were assayed from three populations with known high PAH exposures (coke oven workers), medium PAH exposures (steel factory control workers), and low PAH exposures (volunteer subjects) (n = 30). The respective geometric mean levels of BPDE-HSA adducts—67.8, 14.7, and 1.93 ng/mg HSA (1010, 220, and 28.9 fmol BPDE equiv/mg HSA)—were significantly different (P < 0.05). The sandwich ELISA will be useful for screening PAH exposures in large epidemiologic studies and can be extended to other adducts for which capture antibodies are available.     

A luminescent oxygen channeling immunoassay for the determination of insulin in human plasma

The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 microL. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R(2) = 0.98). This method is convenient and represents an alternative to ELISA.     

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay

Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme‐linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity‐purified anti‐C‐terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin‐linked horseradish peroxidase. Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2‐ to 5‐fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or β‐estradiol, in cultured adipocytes reduces resistin protein levels in a dose‐dependent manner. Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.     

Picogram detection levels of asialofetuin via the carbohydrate moieties using the light addressable potentiometric sensor

Fetal calf serum asialofetuin was assayed in the sandwich format using biotinylated and fluoresceinated ricin toxin (B-RCA and F-RCA). The sandwiched species was captured on a biotin-BSA coated nitrocellulose membrane with streptavidin. Anti-fluorescein antibody-urease conjugate was bound to the complex, and detected and quantitated under microvolume conditions using the light addressable potentiometric sensor. As little as 250 pg of asialofetuin was detectable whereas fetuin gave no response at conditions as high as 32 ng. Using a competitive inhibition assay, we established that the binding constant for the asialofetuin-ricin complex was 3.6×10⁸ m ^–1. This is in good agreement with data published using glycopeptides derived from asialofetuin, and RCA and the ricin agglutinin, RCA₁₂₀.     

Variations in Copper Concentration and Ceruloplasmin Activity of Dairy Cows in Relation to Lactation Stages with Regard to Ceruloplasmin to Copper Ratios

Variations of copper (Cu) concentration and ceruloplasmin (Cp) activity in serum and plasma of dairy cows at different stages of lactation were assessed in 240 Holstein dairy cows. Furthermore, ceruloplasmin to copper ratios were also investigated. The cows were classified according to their lactation stages into four different groups as close-up stage (3–1 week(s) antepartum), fresh lactation stage (0–1 week postpartum), early lactation stage (3–5 weeks postpartum), and mid-lactation stage (15–18 weeks postpartum). Each group consisted of 60 multiparous cows. Serum and heparinised plasma samples were obtained from each cow. Concentrations of serum copper (sCu) and plasma copper (pCu) as well as activities of plasma Cp (pCp) were higher in the group of fresh lactation stage than other groups (P < 0.05). Serum Cp (sCp) exhibited no significant difference between fresh lactation and early lactation groups (P > 0.05). Low correlations were obtained between sCp and pCu, sCp and sCu, and sCu and pCu. Plasma copper concentration and plasma ceruloplasmin activity were higher than serum copper concentration and serum ceruloplasmin activity, respectively (P < 0.001). The ratios of Cp activity to Cu concentration (Cp/Cu) were not significantly changed in the different lactation stages of dairy cows (P > 0.05). Use of sCp/pCu and sCp/sCu rather than pCp/pCu will reduce the calculated value of Cp/Cu. Furthermore, for evaluation of copper status, use of sCp/sCu or sCp/pCu identified more animals as ‘low’ and ‘marginal’ than using pCp/pCu (P < 0.001). It can be concluded that ceruloplasmin and copper undergo a physiological increase just after calving; thus, their values should be interpreted with caution during assessment of copper status. Plasma measurements should be used for calculation of Cp/Cu, and further research is required to refine diagnosis criteria for use of such ratio in determining copper status in dairy cows.     

A comparison of the inhibition of deacetylase in primary cultures of rat and human hepatocytes effecting metabolism and DNA-binding of 2-acetylaminofluorene

The metabolism of 2-acetylaminofluorene (AAF) in primary cultures of rat and human hepatocytes was investigated to determine if the activation of this well-studied chemical carcinogen proceeds via similar routes of metabolism between species. The total level of AAF metabolite(s) bound to hepatocellular DNA was determined in the presence of deacetylase inhibitors, diethyl(p-nitrophenyl) phosphate (paraoxon) or bis(p-nitrophenyl) phosphate (BPNPP). These compounds are known to inhibit deacetylase and to decrease the mutagenicity of AAF. Experiments with rat and human hepatocytes demonstrated inhibition in the deacetylation of AAF (5×10⁻⁴ M) with paraoxon or BPNPP. The BPNPP (5×10⁻⁴ M inhibited 99% of the AF formation in the human hepatocytes and 88% inhibition in the rat hepatocytes. Paraoxon at 10⁻⁴ M demonstrated a 98% inhibition of deacetylation with humans and a 92% inhibition with rats. The rat hepatocytes also showed a 53% decrease in DNA binding in the presence of paraoxon. In contrast with human hepatocytes, while paraoxon decreased the AF metabolite by > 97%, there was no change in total DNA binding.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Follitropin receptors in rat testis characterization with enzymatically 125I-labeled human follitropin

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The ¹²⁵I-labeled human follitropin exhibited high binding activity, with specific binding of up to 17% in the presence of an excess of testis homogenate.Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (K_a) determined at 24°C were not significantly different in immature and adult rat testis, and the mean value for K_a was 3.9 · 10⁹ M^?1. At 37°C, the K_a value obtained using immature rat testis was 1.3 · 10¹⁰ M^?1. The association of ¹²⁵I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30–60% of the preformed hormone · receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnant mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .     

Sandwich Enzyme Immunoassay for Rat Retinol-Binding Protein Using Antibody against Recombinant Antigen and Its Application

The development of a sandwich enzyme immunoassay for rat retinol-binding protein using molecular biological techniques was described. Rat retinol-binding protein gene cloned by the PCR method was expressed by a fusion vector pEZZI8 in Escherichia coli strain HB101. A recombinant retinol-binding protein fused with IgG-binding domain ZZ of protein A was purified with IgG-Sepharose. Antibody against the recombinant protein was found to be specific to rat retinol-binding protein in plasma by immunoblot analysis. Affinity-purified anti-recombinant protein IgG was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range is 1.9-60 ng/ml and the coefficients of variation within and between the assay series (assay range: 4-30 ng/ml) are 4.30 ± 4.33 and 5.32 ± 1.45%, respectively. Cross-reactivity of the immunoassay was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse serum. In an in vitro experiment, retinol-binding protein produced by rat hepatocytes could be measured by the sandwich enzyme immunoassay.     

Participation of high-density lipoprotein in vitellogenesis in Japanese eel hepatocytes

To investigate effect of estradiol-17β (E₂) treatment in vivo on binding of eel hepatocytes to HDL, we developed hepatocytes binding assay. When hepatocytes were incubated with 200 times excess of eel HDL, the binding of hepatocytes to HDL precoated on wells was inhibited competitively. This indicates that eel hepatocytes bound specifically to HDL. E₂ treatment in vivo induced vitellogenin (VTG) synthesis. Hepatocytes prepared from the same E₂ treatment eel showed 3-fold higher ability of binding to HDL compared to hepatocytes prepared from ells without E₂ treatment. We also examined effects of E₂ and HDL on VTG induction in cultured hepatocytes. VTG, determined by enzyme-linked immunosorbent assay (ELISA), induction was about two-times higher in the presence of both 10⁻⁵ M of E₂ and 400 μg of HDL than in the presence of 10⁻⁵ M E₂ alone. At concentrations below 10⁻⁶ M E₂, VTG was not induced in the presence or absence of HDL. By SDS–PAGE and immunoblotting, VTG was detected only in the presence of both 10⁻⁵ M of E₂ and HDL. Our findings strongly support the idea that HDL correlates with vitellogenesis in eel liver.     

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed inEscherichia coli as periplasmic inclusion bodies. Their variable heavy (V_H) and light (V_L) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II ofDrosophila melanogaster and rat MAb Yol1/34, specific for pig brain α-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv′)₂ was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv′)₂ have binding constants quite close to those of the parental MAbs and fourfold higher than scFv′ monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.     

Immunological detection of the type V collagen propeptide fragment,PVCP-1230, in connective tissue remodeling associated with liver fibrosis

Aim: Liver fibrosis involves excessive remodeling and deposition of fibrillar extracellular matrix (ECM) components, which leads to malfunction of the organ, causing significant morbidity and mortality. The aim of this study was to assess whether levels of a type V collagen fragment, the propeptide CO5-1230, indicate the amount of collagen deposited during liver fibrosis.

Methods: A specific competitive enzyme-linked immunosorbent assay (ELISA) was developed to measure CO5-1230 levels. The sequence TAALGDIMGH located at the start of the C-terminal propeptide between amino acid position 1230′ and 1239′ (CO5-1230) of the α2 chain was selected as the immunogen. Monoclonal antibodies were raised against this fragment. An assay developed using the biotin–streptavidin system was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl₄)-treated rats, for up to 20 weeks.

Results: The ELISA was capable of measuring CO5-1230 in serum specifically, with an intra-assay variation of 3.46% and inter-assay variation of 5.09%. Mean CO5-1230 levels were significantly elevated in CCl₄ rats compared with controls [8 weeks: 57.4?ng/mL, controls 45.5?ng/mL (P?=?0.0020); 12 weeks: 81.3?ng/mL, controls 50.2?ng/mL (P?=?0.0020); 16 weeks: 85.1?ng/mL, controls 51?ng/mL (P?=?0055); 20 weeks: 92?ng/mL, controls 47.8?ng/mL (P?=?0.0033)]. CO5-1230 levels correlated with the total amount of collagen in sections from the injured livers, quantified from Sirius red stains (Spearman, R²?=?0.5580). In BDL rats, serum levels of CO5-1230 were also elevated compared with controls [2 weeks: 160.1?ng/mL, controls 78.9?ng/mL (P?=?0.0007); 4 weeks: 111.3?ng/mL, controls 62.2?ng/mL, (P?=?0.0068)] and showed a linear correlation to the total collagen content (Spearman, R²?=?0.3305).

Conclusions: Increased serum levels of CO5-1230 were associated with the extent of collagen deposition in two different models of fibrotic processes in the liver. The data indicate that formation of type V collagen may be of value as a disease-specific diagnostic biomarker that reflects the total burden of disease. The amino acid sequence selected is located in the first 10 amino acids of the C-terminal propeptide section, which is a formation-specific region.     

The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay

A radioreceptor assay using [³H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1–0.2 picomoles/ml and is linear from 4 × 10^?11 to 4 × 10^?9 M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [³H]nitrendipine binding (24 percent inhibition per 20 μ1 serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 – 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.     

Competitive immunoassay for analysis of vitamin B(12)

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B₁₂ by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B₁₂ and purified using affinity chromatography. Checkerboard assay was performed with vitamin B₁₂ antibody and vitamin B₁₂–alkaline phosphatase conjugate followed by its conjugate characterization using ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (K_a) of the vitamin B₁₂ antibody was found to be 4.23 × 10⁸ L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B₁₂ that showed 12% to 35%. The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2% and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity, specificity, precision, and recovery of vitamin B₁₂ with spiked multivitamin injections, tablets, capsules, and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000 to 10,000 ng/ml. After extraction of vitamin B₁₂ using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.     

MMP mediated degradation of type VI collagen is highly associated with liver fibrosis--identification and validation of a novel biochemical marker assay

Background and Aims

During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis.

Methods

Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats.

Results

Intra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl₄-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R² = 0.58, p<0.0001) in CCl₄- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003).

Conclusions

This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.     

Maintenance of liver function in long term culture of hepatocytes following In Vitro or In Vivo Ha-ras EJ transfection

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-ras^EJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 10⁶ cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 10⁶ cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 10⁶ cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 10⁶ cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-ras ^EJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.Abbreviations BWE basal Williams' medium E - FBS fetal bovine serum - F10 or F12 basal Ham's F10 or F12 medium - Ha-ras ^EJ EJ allele of the human cellular ras oncogen of Harvey - hGH human growth hormone - hsp heat shock protein gene - LE-p liposome encapsulated plasmid - N-OH-2-AFAF N-hydroxy-2-acetylaminofluorene - RLECC rat liver epithelial cell - SF serum-free - SS serum-supplemented - UGG serum substitute UGltroser G® - 1-OH-, 3-OH-2-AFAFF 1-hydroxy-, 3-hydroxy-2-acetylaminofluorene - 2-AFAF 2-acetylaminofluorene - 2-AFF 2-aminofluorene     

Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to support these pharmacokinetic studies. For this immunoassay, we raised anti-idiotype antibodies in rabbits. After purification of the rabbit material, the anti-idiotype antibodies are used as capturing antibodies on the ELISA plate. After trastuzumab has bound to the catcher antibody, a sandwich ELISA procedure is followed whereby biotinylated anti-idiotype antibodies can bind to trastuzumab. Detection is performed by streptavidin-polyHRP (poly-horseradish peroxidase) conjugate and (3,5,3′,5′)-tetramethylbenzidine (TMB) substrate. The reaction is stopped using sulfuric acid, and the absorbance is measured at 450 nm. The calibration range of the assay is 0.039 to 5 ng/ml in well. Because samples are analyzed in multiple dilutions, the validated range corresponds to 1.6 to 1600 ng/ml in undiluted serum. Samples above the upper limit of quantification (ULOQ) can be diluted before transfer to the assay plates. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum and plasma. The assay is now used to support pharmacokinetic studies with trastuzumab in human serum and plasma.