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Biomedical subjects

A B Chen

Publications and source records attributed to A B Chen.

At least 19 recordsLinked to original sources
PubMed1992

An antibody capture bioassay (ACB) for DNase in human serum samples.

A novel assay for antibody captured bioactivity (ACB) has been developed to quantitate deoxyribonuclease I (DNase) in human serum samples. The procedure is simple, sensitive, reproducible and has a high throughput. Serum samples are diluted a minimum of 1/4 and assayed in 96-well microtiter plates coated with polyclonal antibodies specific to DNase. The serum is removed from the wells, the plates are washed and the antibody bound DNase is incubated at 37 degrees C with a DNA-methyl green substrate. The assay is sensitive to 0.8 ng/ml with a range to 10 +/- 2 ng/ml, depending upon the time of incubation (48 +/- 2 h). The recovery of rhDNase spiked into human serum samples averaged 84.4% +/- 6.7% in sera diluted 1/4 and 97.8% +/- 7.2% at a 1/8 serum dilution. Intra-assay precision ranged from 3.0 to 7.5% coefficient of variation (% CV) and interassay precision ranged from 5.0 to 10.2% CV for spiked serum controls. Endogenous DNase concentrations in 27 normal human sera were found to range from < 2.0 to 11.4 ng/ml. Endogenous DNase-like activity was found in Cynomolgus and Rhesus monkey sera; this activity diluted linearly and did not interfere with accurate quantitation of added rh DNase. No endogenous DNase-like activity could be detected in ten Sprague-Dawley rat sera. Bovine pancreatic DNase was found to have only very low cross-reactivity in this assay system. The ACB assay format can potentially be applied to the quantitation of other enzymes in serum and other biological samples.

Animals↗
PubMed1992

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

Amino Acid Sequence↗
PubMed1992

Quantitation of E. coli protein impurities in recombinant human interferon-gamma.

A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.

Antibodies, Monoclonal↗
PubMed1991

The measurement of staphylococcal protein A by ELISA in immunoglobulin preparations.

Chicken antibodies were used to develop an ELISA for the quantitation of parts-per-million levels of protein A in the purification of immunoglobulins or immunoglobulin-like molecules. Quantitation of protein A in the presence of excess human or murine immunoglobulins in this assay was compared with that obtained in ELISAs developed with rabbit antibodies specific either to protein A or to other molecules. Experiments demonstrate that protein A is bound to the immunoglobulins being purified and that this binding affects subsequent recognition by the antibodies used for the assay. Because of these effects and because fragments of protein A might not be detected in assays which rely on Fc binding of protein A, chicken antibodies that bind protein A specifically are an advantage for the quantitation of this protein by ELISA. In addition, comparison of the effect of different types of immunoglobulins on the protein A standard curve suggests that alternatives to including the immunoglobulin under purification with the standards can be utilized.

Animals↗
PubMed1990

Low incidence of antibodies to recombinant human tissue-type plasminogen activator in treated patients.

Sera from over 1,600 patients who received recombinant human tissue plasminogen activator (rt-PA) during clinical trials were assessed for the presence of antibodies to this therapeutic agent. The rt-PA was administered by a variety of dosage regimens for several different indications. Two different forms of rt-PA were used; one was predominantly two chain form, and the other was a predominantly one chain form. A sensitive radioimmunoprecipitation assay was used to measure antibodies to rt-PA in patients' serum before and after treatment. Of 932 patients tested with this assay, 929 were negative for antibodies to t-PA. Three patients developed low titers after treatment. Additional serum samples were obtained from these three patients within 2 years after rt-PA therapy and were negative for antibodies to t-PA. With the limited number of positive samples, no correlation could be found with dose or type of rt-PA, dosing regimen or clinical diagnosis. The virtual absence of antibody formation was confirmed in an additional 754 patients using a novel competitive two-site ELISA. It can be concluded that a single infusion of rt-PA was virtually unassociated with antibody formation, suggesting that repeat treatments could be given when necessary without the risk of immunologic complications as are seen with streptokinase or its derivatives.

Animals↗
PubMed1990

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.

1-Methyl-3-isobutylxanthine↗
PubMed1990

A sensitive radioimmunoprecipitation assay for the detection and quantitation of antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1).

A radioimmunoprecipitation (RIP) assay was developed to detect antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1). The assay, which utilized recombinant gp120 (rgp120), was quantitative, reproducible, and specific for antibodies to rgp120 or antibodies to native gp120 resulting from natural infection with HIV. Polyethylene glycol-8000 (PEG), used in the assay at a final concentration of 10% to precipitate immune complexes, was demonstrated to be effective in titering sera from different animal species. Provided samples were diluted at least 1:100, antibody titers could be determined either by the classical dilution method or by interpolation from a calibration curve prepared with a positive serum. The humoral response of animals immunized with rgp120 was monitored and a positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize. In addition, RIP titers of HIV-positive human sera correlated very well with reactivity obtained in a commercial HIV immunoblot assay. The assay has the advantage of quantitation, fast turnaround time, and versatility.

Animals↗
PubMed1989

Immunization procedures for E. coli proteins.

Three immunization procedures were compared for the production of antibodies to the minor components of a complex E. coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins. An indirect ELISA showed that a maximum ELISA antibody titer was obtained with all the procedures 60 d after immunization. Analysis of these antisera by two-dimensional SDS-PAGE immunoblots, however, demonstrated that antibody reactivity to minor components in the mixture was not achieved until 112 d. This analysis also showed that a marked improvement in antibody response to minor components was obtained with the cascade immunization procedure. The mean titer and spectrum of antibody reactivity was similar for each group, and suggested that, although some individual variation was noted, the improvements observed were the result of the protocol used. Thus, for these ECPs, and multiple antigen mixtures in general, the preferred immunization protocol should employ at least three hosts and utilize the cascade immunization of Thalhamer and Freund. Characterization of the resulting antisera is best performed by use of silver stained two-dimensional SDS-PAGE and immunoblotting.

Animals↗
PubMed1989

Variability in the quantitation of circulating growth hormone using commercial immunoassays.

The quantitation of human GH in a serum sample is not consistent among various commercially available immunoassays. We measured serum GH concentrations with four RIAs [Cambridge, Kallestad, National Hormone and Pituitary Program, and Radioassay Systems Laboratories (RSL)] and two immunoradiometric assays (IRMAs; Hybritech and Nichols). Serum GH concentrations measured by the RIAs were between 1.9 and 2.8 times higher than those determined by the Hybritech IRMA, whereas the concentrations measured by the Nichols IRMA were approximately 3.0 times higher than the Hybritech values. We evaluated the effects of differences in standards, assay diluents, and antibody specificity on GH measurement in the various assays. When GH standards from each of the assays were measured in the Hybritech IRMA, only the RSL standard was less immunoreactive than the other assay standards. Different assay diluents also resulted in varying GH values. In the RIAs, GH diluted in serum was more immunoreactive than GH diluted in phosphate-buffered saline-0.5% BSA. This enhanced immunoreactivity appeared to be due to a nonspecific effect generated by serum. The Nichols and Hybritech IRMAs provide standards diluted in horse serum. In the Nichols assay, GH diluted in human serum was more immunoreactive than GH diluted in horse serum, whereas the immunoreactivity of GH diluted in either serum was equal in the Hybritech IRMA. These IRMAs also differ in that the Nichols assay detected the 20K variant of GH, whereas the Hybritech assay did not. Considering these discrepancies, comparison of data obtained using different assays should be made carefully.

Animals↗
PubMed1989

An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys.

A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.

Amino Acid Sequence↗
PubMed1988

Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins.

Two immunoassays have been developed for the quantitation of part-per-million levels of contaminants likely to co-purify with monoclonal antibodies produced in tissue culture and purified by protein A affinity chromatography. These contaminants are bovine IgG originating from the fetal bovine serum used in cell culture, and protein A. Mouse IgG was shown not to interfere in the bovine IgG assay, where contamination levels of 0.2-0.7% bovine IgG were measured in the lots of monoclonal antibody tested. The protein A ELISA was developed with monoclonal antibody included in the standard, and in the preparations of monoclonal antibody tested, 64 parts per million (ppm) or less of protein A were demonstrated. An additional immunoassay was developed to quantitate monoclonal antibody contamination of two recombinant proteins, rHBsAg and rgp 120 from HIV, purified by affinity chromatography with such antibodies. Possible interference of monoclonal antibody quantitation by the respective antigens was examined in this ELISA, and contamination levels of less than 56 ppm of antibody were determined in the purified recombinant proteins. The three immunoassays were shown to be specific for the major protein contaminants in either monoclonal antibodies or the recombinant proteins and were necessary in demonstrating their purity.

Animals↗
PubMed1988

Monitoring of hemostasis parameters during coronary thrombolysis with recombinant tissue-type plasminogen activator.

The monitoring of changes in the blood coagulation and fibrinolytic systems during thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) may be complicated by artifacts due to in vitro activation after blood collection and to interference of other agents (e.g., heparin) in the assays. In 106 patients with early acute myocardial infarction, infused with 150 mg of rt-PA (G11044) intravenously over 5 to 8 hours, blood samples were collected into liquid citrate supplemented with the plasmin inhibitor aprotinin (200 KIU/ml plasma) or on a lyophilized mixture of acidified citrate and the synthetic t-PA inhibitor D-Phe-Pro-Arg-CH2Cl (PPACK). A good correlation between precipitable (sulphite) and functional (clotting rate) fibrinogen levels was observed in plasma collected on citrate before therapy (r = 0.76) and in samples collected after 3 hours on either aprotinin (r = 0.87) or PPACK (r = 0.82). Precipitable fibrinogen levels were approximately 10% higher than functional level, in baseline samples collected on citrate alone and approximately 20% higher in 3 hour samples collected on either PPACK or aprotinin. Fibrinogen levels measured with both assays correlated well, but were somewhat higher in samples collected on PPACK than on aprotinin. rt-PA antigen levels assayed in plasma collected in either inhibitor correlated well (r = 0.90) but were 10-20% higher in PPACK containing samples. Addition of heparin up to 9 units/ml to plasma had no effect on the functional fibrinogen assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1987

Development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-alpha in rodents.

Treatment of healthy rats and mice with a single intravenous injection of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) caused a dose-dependent gastrointestinal inflammation. Within 30 min gastric emptying was blocked and tissue edema occurred in the small and large intestine. In the cecum hemorrhage occurred after 4 h at doses greater than or equal to 250 micrograms/kg. The cecum exhibited an acute inflammatory response following rHuTNF-alpha treatment similar to that seen in tumor necrosis at the same dose. The vascular endothelium became swollen, increased numbers of neutrophils and other leukocytes attached to and penetrated the endothelium, and finally hemorrhage occurred. Treatment of rats with daily injections of rHuTNF-alpha (250 micrograms/kg per d) for 3 wk failed to produce cachexia. Within 24-48 h rats became resistant to the hemorrhagic effect of rHuTNF-alpha, however, the cytokine still caused a transitory block of gastric emptying after 10 d of treatment. Treatment at 5- or 10-d intervals produced results similar to the initial injection. These results suggest that maximum hemorrhagic response will occur when rHuTNF-alpha is administered at intervals of 5-10 d rather than daily.

Animals↗
PubMed1986

D-Phe-Pro-Arg-chloromethylketone: its potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy.

In vitro artifacts due to proteolysis may occur in blood samples containing recombinant tissue-type plasminogen activator (rt-PA) due to continued activation of plasminogen to plasmin by rt-PA. The aim of this study was to identify a rapid inhibitor of rt-PA that would not interfere in assays designed to monitor thrombolytic events. When rt-PA was added at 5 micrograms/ml to whole blood and incubated at 25 degrees C, fibrinogen decreased 50 percent, plasminogen levels decreased 90 percent and alpha 2-antiplasmin decreased below detectable levels. If D-Phe-Pro-Arg-chloromethylketone (PPACK) or aprotinin were added before the addition of rt-PA there was no significant loss of fibrinogen. Only PPACK completely inhibited changes in fibrin degradation products, plasminogen and alpha 2-antiplasmin. PPACK was also found to inhibit the binding of rt-PA to plasma protease inhibitors in vitro. Rhesus monkeys were infused with rt-PA and blood samples were taken with either PPACK or aprotinin in the collection syringe. There was a significant increase in the recovery of immunoreactive rt-PA and consistent measures of fibrinogen, FDPs, plasminogen, and alpha 2-antiplasmin in the PPACK group as compared to the aprotinin group which indicates that PPACK will prevent the in vitro formation of artifacts due to the presence of active rt-PA.

Amino Acid Chloromethyl Ketones↗
PubMed1986

Immunoassay for the detection of E. coli proteins in recombinant DNA derived human growth hormone.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of part-per-million levels of the most probable E. coli polypeptide (ECP) contaminants of E. coli produced biosynthetic human growth hormone (hGH). The antibody preparation, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference ECPs (a collection of the most probable protein contaminants) by both affinity chromatography and immunoblot analysis. Affinity purification of this antibody preparation using immobilized reference ECPs resulted in an assay with a higher signal-to-noise ratio and also 'normalized' the antibody population to approach stoichiometric equivalence with the immobilized ECPs. Reference ECPs, size fractionated by gel filtration, were quantitated in agreement with their absorbance at 280 nm. The assay was demonstrated to be specific for ECPs obtained from the hGH purification process. Since the purification of each recombinant DNA derived protein from E. coli requires its own unique process, this means that no generic ECP assay can be developed. It is felt that the criteria established for this assay provide a comprehensive approach to the development of quantitative multiple antigen immunoassays.

Antibody Specificity↗
PubMed1986

A sensitive radioimmunoprecipitation assay for the detection of antibody to recombinant human gamma-interferon: comparison to a bioassay neutralization test.

This report describes a specific radioimmunoprecipitation (RIP) assay for the detection of antibodies to recombinant DNA (rDNA) derived human gamma-interferon (rHuIFN-gamma). The assay was shown not to detect antibodies to rHuIFN-alpha, rHuIFN-beta, human lymphotoxin, or E. coli proteins and was reproducible with intraassay and interassay coefficients of variation of 1.6 and 3%, respectively, for the log titer of a high positive control. Comparison of this assay with a standard bioassay for detection of neutralizing antibody (abrogation of the inhibitory effect of rHuIFN-gamma on EMC virus replication in A549 cells) demonstrated that the RIP assay was more sensitive for detection of HuIFN-gamma neutralizing monoclonal antibody. Nonneutralizing monoclonal antibody was detectable in the RIP assay but not in the bioassay neutralization test. Examination of polyclonal antisera (rabbit and monkey) that contained neutralizing antibodies also demonstrated the RIP system to be a more sensitive indicator of the presence of antibodies than the bioassay neutralization test. In preliminary studies of human samples (86 patients) from clinical trials using an assay precipitation system capable of detecting antibody of the IgG, IgM, IgA, and IgE classes, no antibody to rHuIFN-gamma was observed. These patients were also found negative for neutralizing antibody to rHuIFN-gamma.

Antibodies↗