Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies

Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision < or =20% CV and accuracy within +/-20% of the nominal values. Intra- and inter-assay coefficients of variation were within 9% and accuracies within +/-10% of the nominal values. The validated method was applied to the study of the cellular uptake of native and cationized anti-tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.     

An analysis of the fluid phase C1q binding assay. The effect of endogenous C1q on the precipitation and detection of an immune complex model

We examined the effect of endogenous C1q on the sensitivity of the fluid-phase C1q binding assay (C1qBA) in detecting an immune complex (IC) model, heat-aggregated IgG (HAIgG), at concentrations of 10-10,000 micrograms/ml sample. Results in normal human serum (NHS) or plasma (NHP) were compared with those in heat-inactivated NHS (NHS/56) in which most endogenous C1q was depleted by heat denaturation. Higher HAIgG concentrations were required in NHP and NHS to produce the same 125I-C1q precipitation seen in NHS/56. This decreased sensitivity varied from 70% at low HAIgG concentrations to 0% at high concentrations, as predicted for a large pool of endogenous C1q, in equilibrium with 125I-C1q, but in excess of that which could bind to all but the highest concentrations of IC model. In serum depleted of functional C1q on an immunoadsorbant of HAIgG, the precipitation of radiolabeled HAIgG under C1qBA conditions was concentration dependent and generated a saturation curve, showing that only a fraction of IC are usually precipitated in this assay. HAIgG precipitation was enhanced 1.4-fold in NHS/56 (8 micrograms C1q/ml) and three-fold in NHS (67 micrograms C1q/ml) suggesting that IC size is increased by endogenous C1q. In dual label experiments using 131I-HAIgG, the precipitation of 125I-C1q in NHS/56 was directly proportional to IC model precipitation, but markedly discordant in NHP, showing the measurement of IC in heat-inactivated sera superior to that in native serum. A comparison of the C1q:HAIgG ratio in PEG precipitates with that in samples, indicated that equilibrium was established between C1q and IC model. Thus the precipitation of 125I-C1q in the C1qBA represents (1) the fraction of total C1q bound to IC, and (2) the fraction of IC precipitated by PEG.     

Genotypic and phenotypic analysis of a case with inherited coagulation factor X deficiencyCSCD

Objective: To explore the correlation between F10 gene mutation and its phenotype in a Chinese pedigree affected with FX deficiency. Methods: Prothrombin time(PT), activated partial thromboplastin time(APTT), fibrinogen, F II activity(FII: C), FVII activity (FVII: C), FIX activity (FIX: C), FX activity(FX: C) were determined with a one-stage clotting assay. The FX antigen(FX: Ag) was detected with an enzyme linked immunosorbent assay(ELISA). The 8 exons, introns and 5' and 3' untranslated regions(UTR) of the F10 gene of the proband and her family members were subjected to PCR amplification and Sanger sequencing. Suspected mutation was confirmed by reverse sequencing. Polymorphisms were excluded by direct sequencing of 100 healthy individuals. Results The PT and APTT of the proband have prolonged to 16. 1 s and 49. 0 s, respectively. Her FX: C and FX: Ag were reduced by 27% and 56%, and her mother's PT, APTT, FX: C and FX :Ag were 14.8 s, 37.4 s, 44%, 34%, respectively. Her grandmother's PT, APTT, FX: C and FX: Ag were 15. 8 s, 42. 2 s, 31%, 45%, respectively. The results of her father and other family members were all within the normal range. Genetic analysis has revealed a heterozygous G>A mutation in the proband at position 28076 in exon 8 of the F10 gene, which resulted in a p. Gly363Ser substitution. The same mutation was also found in her mother and grandmother. No mutation of the F10 gene was found in her father. Gly363Ser may result in changes in the secondary structure of the F X protein and reduction of its activity. Conclusion The g. 28076G > A (p. Gly363Ser) mutation of the F10 gene probably underlies the F X deficiency in this pedigree. The mutation was discovered for the first time in Chinese patients. © 2018 MeDitorial Ltd. All rights reserved.     

Usefulness of the hepatitis C virus core antigen assay for screening of a population undergoing routine medical checkup

We studied the usefulness of the recently designed Trak-C assay for the detection and quantification of the hepatitis C virus (HCV) core antigen (Ag) for the screening of HCV infection in 4,201 subjects selected from 74,150 consecutive volunteers undergoing routine medical checkups. Subjects were selected for screening because they had risk factors (group II, n = 321) and/or elevated alanine transaminase activity (group I, n = 3847). Initially, the anti-HCV antibody assay and the Trak-C assay were performed on each patient. Subsequently, the Trak-C assay was performed only when the anti-HCV enzyme immune assay (EIA) was positive. Positive samples were further evaluated for anti-HCV antibodies by a third-generation strip immunoblot assay and for HCV RNA. Four samples (1.2%) from group II and 113 (2.9%) from group I were anti-HCV EIA positive. We also tested 33 subjects who previously tested positive for anti-HCV in our medical center. Among the 150 anti-HCV EIA-positive samples, the HCV core Ag result was in accord with the HCV RNA result in 146 cases (97.3%). When the EIA result was positive, the HCV core Ag concentration and the HCV RNA load were correlated (r(2) = 0.78; P < 0.001). Four samples with low viral loads were Trak-C negative but HCV RNA positive. Among the 2,395 anti-HCV EIA-negative serum samples collected during the first part of the study, 17 (0.7%) were found to contain very low levels of HCV core Ag (<8.5 pg/ml, the cutoff value being 1.5 pg/ml). All these samples were HCV RNA negative and considered to be false positives. This was confirmed by HCV core Ag neutralization analysis. The HCV core Ag assay is a useful method in the screening strategy of HCV infection and provides a reliable means of distinguishing between current and cleared HCV infections that is well correlated with HCV RNA testing.     

A sensitive immunoassay for von Willebrand factor

We have developed an ELISA specific for canine von Willebrand factor antigen (vWF:Ag) that also strongly reacts with the VWF:Ag of humans and many other vertebrates. This assay was designed to avoid the use of immunoreagents of human origin, however, commercially available antibodies to human vWF:Ag may also be used. von Willebrand factor (vWF) was quantitated using a modified double-sandwich ELISA with polyclonal antibodies specific for canine vWF:Ag. The assay was as sensitive for measuring canine vWF:Ag as previously published immuno-radiometric assays and the most sensitive ELISA for human vWF:Ag. Employing commercially available antibodies to human vWF:Ag in the same double-sandwich configuration, the lower limit of detection for human vWF:Ag was 4.8 x 10(-6) units/ml, lower by a factor of ten than previously reported ELISAs. In addition, a wide range of vWF:Ag levels can be determined with just a single plasma dilution. The assay readily distinguishes type III von Willebrand disease from other types of von Willebrand disease having very low levels of vWF. This vWF ELISA can be used to evaluate large numbers of plasma samples simultaneously and is therefore well-suited for large-scale screening programs.     

Direct analysis of artemisinin in plasma and saliva using coupled-column high-performance liquid chromatography with a restricted-access material pre-column

A previously established HPLC system with post-column derivatization for the analysis of artemisinin was coupled to an ADS (alkyl-diol silica) pre-column, allowing direct and repetitive injection of protein-rich fluids such as plasma. The limit of quantitation for 100 microl of plasma was 10 ng/ml (CV=10.5%) while concentrations down to 2 ng/ml could be quantified for 1.00 ml saliva samples (CV=11.1%). The system was linear in the tested range of 10-2000 ng/ml for plasma and 2-240 ng/ml for saliva samples, respectively. This paper introduces coupled column HPLC as a simplified method for the routine analysis of artemisinin in biological fluids.     

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 microl of plasma extracts onto a 100x3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y2). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and -0.2%, exhibiting precisions (C.V.) of +/-6.5 and +/-7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.     

One-step quantitative cortisol dipstick with proportional reading

Rapid quantitative immuno-detection of haptens by the lateral flow assay without "typical" competitive inhibition results is studied. In the present study, we describe an immuno-threshold-based assay for the quantification of cortisol. It gives a signal which is directly proportional to the cortisol concentration in plasma samples with a performance time of only 5 min. This technique provides a practical calibration curve with detection limit of 3.5 ng/ml. The precision of the assay is 6% (intra-assay coefficient of variation, CV) and 10% (inter-assay CV). Cross-reactivity with related steroids is acceptably low: corticosterone (3.38%), cortisone (2.08%), deoxycorticosterone (2.00%), 17alpha-hydroxyprogesterone (0.39%), and progesterone (0.05%). Furthermore, the test strips show the advantages of long storage time and high stability that allow mass production and preparation of large batches. A one-step cortisol whole blood test derived from this plasma lateral flow assay has then been performed. It is a rapid chromatographic immunoassay designed for quantitative determination of cortisol in whole blood samples. It requires no sample pretreatment and gives result within 15 min. In principle, with this rapid and sensitive immunoassay, the immuno-test strips can be employed for detecting all low-molecular-weight haptens. It may also be a useful and convenient dipstick format for drug detection.     

Absence of a bleeding tendency in severe acquired von Willebrand's disease. The role of platelet von Willebrand factor in maintaining normal hemostasis

A 67-year-old male with a prolonged activated partial thromboplastin time (APTT) of 43 seconds (normal, 25-40 seconds) was found to have laboratory features of von Willebrand's disease and IgA myeloma but had a normal bleeding time and no bleeding tendency. Plasma Factor VIII coagulant activity (F.VIII:C) was 80 U/L (0.08 U/mL), Factor VIII antigen (F.VIII:Ag) 70 U/L (0.07 U/mL), and von Willebrand's factor antigen (vWF:Ag) 50 U/L (0.05 U/mL) and ristocetin cofactor (vWF:RiCoF) 10 U/L (0.10 U/mL). The platelet vWF:Ag level was normal, whereas both platelet lysate and plasma vWF high molecular weight multimers were decreased. Patient plasma had no inhibitory effect on either F.VIII:C or vWF:RiCoF. However, when patient plasma was incubated with normal plasma, crossed immunoelectrophoresis for vWF:Ag demonstrated the presence of immune complexes. Infusion of 1-desamino-8-D-arginine vasopressin led to a transient correction of the plasma vWF:Ag multimer pattern. The survival of all components of vWF/F.VIII was decreased, as also occurred after cryoprecipitate. The levels of plasma F.VIII/vWF increased as the IgA values decreased after chemotherapy, whereas the platelet high molecular weight multimers remained decreased. The data suggest that the plasma vWF/F.VIII deficiency results from complexing of the IgA myeloma protein with vWF, resulting in premature clearance of the vWF/F.VIII complex. The absence of clinical bleeding likely results from the combination of a normal platelet vWF:Ag level and persistence of intermediate molecular weight vWF multimers.     

Seven human immunodeficiency virus (HIV) antigen-antibody combination assays: evaluation of HIV seroconversion sensitivity and subtype detection

In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability to detect p24 antigen from HIV-1 groups M and O, antibody-positive plasma samples from HIV-1 groups M and O, HIV-2, and 19 HIV seroconversion panels. Results indicate that although all combination assays can detect antibodies to HIV-1, group M, subtypes A to G, circulating recombinant form (CRF) A/E, and HIV-1 group O, their sensitivity varied considerably when tested using diluted HIV-1 group O and HIV-2 antibody-positive samples. Among combination assays, the AxSYM, Murex, and VIDAS HIV Duo Ultra assays exhibited the best antigen sensitivity (at approximately 25 pg of HIV Ag/ml) for detection of HIV-1 group M, subtypes A to G and CRF A/E, and HIV-1 group O isolates. However, the VIDAS HIV Duo Ultra assay had a lower sensitivity for HIV-1 group M and subtype C, and was unable to detect subtype C antigen even at 125 pg of HIV Ag/ml. The HIV antigen sensitivity of the VIDAS HIV Duo and Genscreen Plus combination assays was approximately 125 pg of HIV Ag/ml for detection of all HIV-1 group M isolates except HIV-1 group O while the sensitivity of Vironostika HIV Uniform II Ag-Ab and Enzygnost HIV Integral Ag-Ab assays for all the group M subtypes was >125 pg of HIV Ag/ml. Among the combination assays, the AxSYM assay had the best performance for detection of early seroconversion samples, followed by the Murex and VIDAS HIV Duo Ultra assays.     

Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry

A HPLC assay with tandem mass spectrometric detection in the positive-ion atmospheric pressure chemical ionisation (APCI) mode for the sensitive determination of retigabine [(I), D-23129] and its acetyl metabolite [(II), ADW 21-360] in plasma was developed, utilising the structural analogue (D-10328), (III), as internal standard. Automated on-line solid-phase extraction of diluted plasma samples, based on 200-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of retigabine and the acetyl metabolite down to 1 ng/ml. Injection of 500 microl of diluted plasma onto a C2 stationary phase-based column switching system in combination with a 75 mm x 4 mm reversed-phase analytical column at a flow-rate of 0.5 ml/min provided cycle times of 4 min per sample. The standard curves were linear from 1 to 1000 ng/ml using weighted linear regression analysis (1/x2). The method is accurate (mean accuracy < or = +/- 10%), precise (RSD < +/- 15%) and sensitive, providing lower limits of quantification in plasma of 1 ng/ml for retigabine (I), and 2.5 ng/ml for the metabolite (II) with limits of detection of 0.5 ng/ml for both analytes. Up to 200 unknowns may be analysed each 24 h per analyst.     

Quantification of human high molecular weight kininogen by immunoblotting with a monoclonal anti-light chain antibody

Activation of the Hageman factor-dependent pathways in human plasma leads to the cleavage of high molecular weight kininogen (HMWK) into a disulfide-linked two-chain (heavy and light chain) molecule and release of bradykinin, a vasoactive peptide. We have utilized murine monoclonal antibodies to the light chain of HMWK (Blood (1988) 71, 1344) and developed a very sensitive immunoblotting assay to detect and quantify the amount of cleaved or uncleaved HMWK in whole plasma. The total HMWK content of plasma from apparently healthy donors was 55 micrograms/ml by this method. Cleaved HMWK was detected when only 2% of the plasma had been activated and the method was sensitive down to 2 ng of HMWK. Because of the extreme lability of bradykinin in body fluids, quantification of cleaved HMWK provides an important adjunct which reflects contact activation and permits calculation of a theoretical upper limit of the potential kinin formed.     

Characterization of the coagulation system in healthy dolphins: the coagulation factors, natural anticoagulants, and fibrinolytic products

In dolphins, blood pooling and acidosis from lack of oxygenation with prolonged underwater stay are not associated with intravascular clotting as it would be in terrestrial mammals, while shed blood clots promptly, and intravascular clots form after death. This intriguing physiologic adaptation prompted further investigation of the coagulation system in the dolphin. We studied the plasma from 17 dolphins (Tursiops truncatus) for coagulation factors and from 12 of the 17 for natural anticoagulants and fibrinolytic products using reagents and equipment currently used for humans. We obtained the following results: prothrombin time, 13.3±0.5 s, activated partial hromboplastin time, no clot; fibrinogen, 343±74 mg/dl; functional assays of factors II, 0.4±0.07 U/ml; V, 2±0.5 U/ml; VII, 0.5±0.08 U/ml; VIII, 7.5±1 U/ml; IX, 2.4±0.5 U/ml; X, 0.9±0.1 U/ml; XI, 0.8±0.2 U/ml; and XII, not detected, antithrombin functional assay, 105.7±12.5%; antithrombin antigen, 96±8.3%; protein C activity 125.1±10.8%; C antigen, undetectable; protein S activity 15.6±6.8%; S antigen, undetectable. Fibrin degradation products and D-dimer were not increased above the normal human range. Our study suggests that the contact system in healthy dolphin may play a role in hindering intravascular clotting of stagnant blood. Low levels of functional protein S were not associated with any increased risk for thrombosis.     

The subcutaneous administration of the vasopressin analogue 1-desamino-8-D-arginine vasopressin in patients with von Willebrand's disease and hemophilia

Summary Twenty-one patients suffering from mild von Willebrand's disease (vWd) and patients suffering from mild or moderate hemophilia A received 1-desamino-8-D-arginine vasopressin (DDAVP) (Minirin^®, Ferring AG) s.c. at a dose of 0.4 µg/kg body weight. Additionally, two hemophiliacs and 22 patients with vWd received DDAVP i.v. Within the observation period of 3 h Factor (F) VIII:C levels increased 2.4 × baseline levels in hemophiliacs, and the maximal effect was observed 3 h post DDAVP s.c. In patients with vWd post DDAVP s.c. (i.v.) a 2.7 (3.4), 2.1 (1.9) and 2.2 (2.8) fold increase for F VIII: C, F VIIIR:Ag and F VIII:Rcof was observed. In eight patients suffering from vWd with additional F XII deficiency a small and transitory but significant increase of F XII levels was detected post DDAVP s.c. No local or systemic side effects were observed. In five patients with vWd tooth extractions were performed without bleeding complications under DDAVP s.c. treatment. Two patients practiced self-treatment by injecting the drug s.c. at home. We thus conclude that s.c. DDAVP is an effective, reliable, and cost-reducing form of treatment that does not bring with it the risk of transmitting infectious diseases in patients with vWd and hemophilia and that can be administered at home.Abbreviations F VIII:C factor VIII procoagulant activity - F VIIIR:Ag factor VIII related antigen - F VIII:Rcof ristocetin cofactor - aPTT activated partial thromboplastin time     

Quantitation of protein A in human plasma is possible after heat inactivation of the samples

Quantitative determination of staphylococcal protein A in plasma is often hampered by the interaction between protein A and immunoglobulins. In human plasma, these interactions may not only involve the non-immune binding to the Fc or Fab regions of Ig but also antigen/antibody interaction by specific antibodies directed against protein A. In this paper we describe a method which can be used to quantitate nanogram amounts of protein A in the presence of human plasma. The ELISA used for the quantification of protein A is based on a double antibody solid-phase assay utilizing chicken anti-protein A as both capture and detector antibody. Protein A may be measured down to 5 ng/ml in plasma and 0.5 ng/ml in buffer. The plasma samples were heat-inactivated before analysis and this eliminated interference in the assay caused by interaction of protein A with an excess of immunoglobulins. This assay provides a reliable and convenient method for the detection and quantitation of soluble protein A in human plasma.     

Enzyme-linked immunosorbent assay for detection of Salmonella typhi protein antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was designed for the detection of Salmonella typhi protein antigen. The optimal concentration of antibody for coating the plate was found to be 50 micrograms/ml. The optimum conditions for antibody coating and antigen and conjugate incubation were 37 degrees C for 3 h, 37 degrees C for 2 h, and 4 degrees C overnight, respectively. The enzyme-substrate reaction was allowed to take place at 30 degrees C for 1 h. The established ELISA was found to be reproducible, with an inter-run coefficient of variation of less than 12% for the detection of an S. typhi protein antigen concentration of 0.5 to 50 micrograms/ml. The minimal detectable level of the antigen was 0.5 micrograms/ml. Cross-reactions were observed with the high level (50 micrograms/ml) of protein antigens obtained from Salmonella typhimurium, Escherichia coli, Salmonella paratyphi A, and Salmonella enteritidis. The ELISA established was used for the detection of S. typhi protein antigen in serum from 62 patients with typhoid, 30 patients with clinically diagnosed typhoid fever, 21 patients with paratyphoid, 17 patients with pyrexia caused by other bacteria, and 160 normal, healthy individuals. It was found that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of this assay were 83.87, 89.04, 87.93, 67.53, and 95.31%, respectively.     

Could the new HIV combined p24 antigen and antibody assays replace p24 antigen specific assays?

The performance of twelve HIV combined p24 antigen and antibody assays available in Europe were compared. The assays were examined with a total of 1983 samples that included 1005 unselected HIV negative samples, 7 HIV-1 p24 Ag reference samples with HIV-1 Ag, 10 samples of a HIV antigen sensitivity commercial panel, 124 samples of 31 p24 antigen panels of different HIV-1 subtypes, 168 members of 24 HIV-1 seroconversion panels, 559 HIV-1 (groups M and O) antibody positive samples and 110 HIV-2 antibody positive samples. The specificity ranged from 99.4 to 100%. Ten of the 12 assays detected all anti-HIV positive samples irrespective of genotype while two assays missed one sample each (one subtype F and one subtype C). The combined assays could be classified into three groups. The first includes two assays (Enzygnost HIV Integral and Vironostika Ag/Ab) that have a clinical sensitivity similar to the two antibody only assays. The second includes the seven assays that detected infection after the p24 antigen only assay and show a delay from 3.3 to 5.17 days after HIV-1 RNA. The third group detected the infection before the p24 antigen assay and less than 3 days after nucleic acid testing (NAT). The improved ability to detect p24 Ag, at levels similar to specific HIV Ag assays, suggests that these new HIV combined Ag/Ab assays could replace p24 antigen only assays in situations for blood or organ screening when NAT is not feasible or not affordable.     

Simultaneous detection of hepatitis C virus core antigen and antibodies in Saudi drug users using a novel assay

Drug users and particularly, injecting drug users, are at increased risk for infection with hepatitis C virus (HCV). The aims of the study were to simultaneously detect HCV core antigen and specific antibodies in sera from Saudi drug users using the new HCV combination assay and to compare this data with HCV core antigen, anti-HCV antibodies and HCV RNA data from the same patients. A total of 297 patients who are followed up or admitted to a drug rehabilitation hospital over a period of 3 years were included in this study. Samples were analyzed using the new HCV Ag/Ab combination assay (Meurex), HCV core Ag assay, HCV antibodies and with the HCV RNA assay. Out of the 297 samples from Saudi drug users, 111 samples (37.4%) have detectable HCV core Ag, 112 samples (37.7%) have detectable HCV antibodies, 118 have detectable HCV RNA, and 116 samples were positive by the HCV Ag/Ab combination assay (39.1%). Out of the 116 samples, HCV core Ag was detected in 110 samples (94.8%), HCV antibodies were detected in 111 (95.7%) samples and HCV RNA was detected in 114 samples (98.3%). In the control group (n = 305), only 2 (0.66%) blood donor were positive by HCV antibodies assay, HCV RNA assay as well as HCV Ag/Ab combination assay. The new HCV Ag/Ab combination assay may well improve the overall quality of diagnosis of HCV infection especially in high risk population such as drug users that necessitates rigorous testing.     

Artificial factor VIII deficient plasma: preparation using monoclonal antibodies and its use in one stage coagulation assays.

Monoclonal antibodies to factor VIII antigen (VIII:Ag) and von Willebrand factor (vWf:Ag) were immobilised on Sephacryl S-1000 and tested for their ability to deplete normal human citrated plasma of factor VIII. A combination of two antibodies to VIII:Ag and one antibody to vWf:Ag was required to produce plasma containing less than 0.01 IU/ml. Its performance in the one stage coagulation assay of VIII:C was equivalent to that of congenital VIII deficient plasma for the assay of normal and haemophilic plasma and factor VIII concentrates. Storage of freeze dried aliquots of this product at -20 degrees C, +4 degrees C, and 37 degrees C showed that it could be used as a substrate for at least six months when stored at temperatures +4 degrees C and below.