Enzyme-linked Immunoassay Method Research Articles

Competitive Enzyme-Linked Immunoassay for Sialoglycoprotein of Edible Bird’s Nest in Food and Cosmetics

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.

Sample Dilution in Omentin Measurement

In the studies, specific enzyme linked immunoassay (ELISA) method is generally used to determinate the plasma omentin level (1,2). There may be matrix effect interferences the omentin measurement. According to international union of pure and applied chemistry, matrix effect is the combined effect of all components of the sample other than the analyte on the measurement of the quantity. In standard laboratory procedures to determinate the plasma omentin by ELISA method, various proportions of dilution should be applied to the samples to reduce matrix interference effect. If the dilution operation is performed for any reason, the measured value must be multiplied by the dilution factor in good laboratory practice. The dilution factor will be 5, if 5fold dilution is applied to samples to reduce the matrix effect. If the sought parameter is measured 23 ng/mL in diluted sample, this value is multiplied by a dilution factor and 115 ng/mL should be reported which is the real value. In one of ours study, we used the BioVendor (BioVendorLaboratomi Medicina, Brno, Czech Republic) Human Omentin-1 ELISA reagent. In standard laboratory procedure, 40-fold dilution had been applied to the samples. In this case, the results had to be multiplied by 40. In this study, the average omentin values of the patients and control group were found 606.6±313.0 ng/mL and 357.5±147.4 ng/mL respectively (3). Although study of the Omentin-1 reference range not enough, manufacturer of the reagent we used in ours study has reported the omentin-1 level approximately 480±21 ng/mL in precision analysis. It has seen that the dilution factor was taking into account in some other similar studies, when the omentin levels were analyzed (4, 5).

Autoimmune thyroid diseases andHelicobacter pylori: The correlation is present only in Graves's disease

To investigate the correlation between autoimmune thyroid diseases (ATDs) and the prevalence of Cag-A positive strains of Helicobacter pylori (H. pylori) in stool samples. Authors investigated 112 consecutive Caucasian patients (48 females and 4 males with Graves' disease and 54 females and 6 males with Hashimoto's thyroiditis HT), at their first diagnosis of ATDs. Authors tested for H. pylori in stool samples using an amplified enzyme immunoassay and Cag-A in serum samples using an enzyme-linked immunoassay method (ELISA). The results were analyzed using the two-sided Fisher's exact test and the respective odds ratio (OR) was calculated. A marked correlation was found between the presence of H. pylori (P ≤ 0.0001, OR 6.3) and, in particular, Cag-A positive strains (P ≤ 0.005, OR 5.3) in Graves' disease, but not in Hashimoto's thyroiditis, where authors found only a correlation with Cag-A strains (P ≤ 0.005, OR 8.73) but not when H. pylori was present. The marked correlation between H. pylori and Cag-A, found in ATDs, could be dependent on the different expression of adhesion molecules in the gastric mucosa.

Evaluation of the Quantum Blue® rapid test for faecal calprotectin

Calprotectin is an acute-phase protein used extensively in the assessment of gastrointestinal inflammation. It can readily be measured by enzyme-linked immunoassay (ELISA) and recently by point-of-care testing (POCT). We evaluated the Quantum Blue(®) POCT in this study and compared it with our existing ELISA method. The method comparison study used faecal samples (n = 47) sent to the laboratory for routine calprotectin analysis. Linearity was assessed by serial dilution of extracted faeces (n = 4). Extraction efficiency was determined by repeat extraction of three different stools. The variation in results as a consequence of reading the POCT cartridges either side of the recommended 12 min was also assessed. The assay was linear across the range stated by the manufacturer. When multiple samples were taken from the same stool, results varied from -31.3% to +31.5%. For the clinical arm of our study, strictly applying the 50 μg/g cut-off recommended for both assays as positive for gastrointestinal inflammation, there were four patients where results fell a different side of the clinical cut-off; two patients had results higher by Quantum Blue(®) and two higher by ELISA. In our hands, the Quantum Blue(®) method was a suitable screening test for excluding inflammatory bowel disease. It may be of value to laboratories wishing to offer calprotectin but who do not have sufficient numbers to warrant ELISA methodology or in 'one stop' gastrointestinal clinics where an immediate result is required.

Hepcidin: clinical utility as a diagnostic tool and therapeutic target

Iron is essential for life, yet excessive iron can damage tissues and organs. To prevent iron deficiency and overload, iron balance is regulated by the hormone hepcidin. Hepcidin levels increase in response to iron sufficiency, decreasing intestinal iron absorption and inhibiting release of iron from stores and macrophages. Iron deficiency lowers hepcidin, leading to enhanced iron absorption and mobilization of iron from stores. Hepcidin is also increased by inflammation, and has a major role in the anemia of chronic disease. Chronic kidney disease (CKD) is associated with increased hepcidin levels, and this likely contributes to the incidence and severity of anemia, and resistance to erythropoiesis-stimulating agents (ESAs). Elevated hepcidin contributes to the dysregulation of iron homeostasis in CKD. In patients with CKD, although parenteral iron in CKD can bypass some of the iron-blocking effects of hepcidin, free iron and iron stores increase, anemia is only partially corrected, and ESA dose requirements remain significantly higher than physiological replacement. Agents that lower hepcidin or inhibit its actions may be effective strategies to restore normal iron homeostasis, and overcome anemia of chronic kidney disease. We review the regulation of hepcidin, its role in CKD-related anemia, and discuss the potential for hepcidin as a clinical marker, and several investigational methods to lower hepcidin for treatment of anemia in CKD.

A realtime and continuous assessment of cortisol in ISF using electrochemical impedance spectroscopy

This study describes the functioning of a novel sensor to measure cortisol concentration in the interstitial fluid (ISF) of a human subject. ISF is extracted by means of vacuum pressure from micropores created on the stratum corneum layer of the skin. The pores are produced by focusing a near infrared laser on a layer of black dye material attached to the skin. The pores are viable for approximately three days after skin poration. Cortisol measurements are based on electrochemical impedance (EIS) technique. Gold microelectrode arrays functionalized with Dithiobis (succinimidyl propionate) self-assembled monolayer (SAM) have been used to fabricate an ultrasensitive, disposable, electrochemical cortisol immunosensor. The biosensor was successfully used for in vitro measurement of cortisol in ISF. Tests in a laboratory setup show that the sensor exhibits a linear response to cortisol concentrations in the range 1pM to 100nM. A small pilot clinical study showed that in vitro immunosensor readings, when compared with commercial evaluation using enzyme-linked immunoassay (ELISA) method, correlated well with cortisol levels in saliva and ISF. Further, circadian rhythm could be established between the subject's ISF and the saliva samples collected over 24h time-period. Cortisol levels in ISF were found reliably higher than in saliva. This research establishes the feasibility of using impedance based biosensor architecture for a disposable, wearable cortisol detector. The projected commercial in vivo real-time cortisol sensor device, besides being minimally invasive, will allow continuous ISF harvesting and cortisol monitoring over 24h even when the subject is asleep. Forthcoming, this sensor could be interfaced to a wireless health monitoring system that could transfer sensor data over existing wide-area networks such as the internet and a cellular phone network to enable real-time remote monitoring of subjects.

Detection of Listeria monocytogenes in pork and beef using the VIDAS® LMO2 automated enzyme linked immunoassay method

Listeria (L.) monocytogenes, a foodborne pathogen, is known to be a possible contaminant of foods during production and processing. Samples (n=985) of raw meat and by-products obtained from beef and pork were first screened by the VIDAS system for the presence of Listeria spp., followed by testing for the presence of L. monocytogenes. Positive L. monocytogenes results were confirmed by plating on selective agars: 14% of the samples were positive for Listeria and 4% tested positive for L. monocytogenes, of which 3% were confirmed on selective agars. In by-products (17%) the contamination with listeriae was higher than in meat cuts (10%). Only samples strongly positive for Listeria spp. by VIDAS were positive for L. monocytogenes. Overall, the prevalence of L. monocytogenes in beef and pork samples was rather low in comparison to most previous studies. The VIDAS system was shown to be a suitable method for screening out Listeria-negative samples; the main advantage being a markedly reduced assay time.

Low-Level Laser Therapy Associated toN-Acetylcysteine Lowers Macrophage Inflammatory Protein-2 (MIP-2) mRNA Expression and Generation of Intracellular Reactive Oxygen Species in Alveolar Macrophages

The aim of this work was to investigate the low-level laser therapy (LLLT) effect on alveolar macrophages (AM) activated by oxidative stress and lipopolysaccharide (LPS). LLLT has been reported to actuate positively relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased MIP-2 mRNA expression and intracellular reactive oxygen species (ROS) generation observed in acute lung inflammation (ALI) can be influenced by LLLT. Rat AM cell line (AMJ2-C11) was cultured with LPS or H(2)O(2) and laser irradiated. MIP-2 mRNA and ROS production in the AM were evaluated by Real Time-PCR and the 2',7'-dichlorofluorescin diacetate (DCFH-DA) respectively. The NF-κB protein in the AM was measured by the enzyme linked immunoassay method. To investigate the antioxidant effect of laser, the AM were prebathed with N-acetylcysteine (NAC) and then irradiated with laser. LLLT was also studied in the presence of an inhibitor of NF-κB (BMS 205820). In addition, the effect of LLLT on NF-κB protein was investigated. LLLT attenuated the MIP-2 mRNA expression and intracellular ROS generation after LPS or H(2)O(2). When the AM were pretreated with NAC, the laser effect was potentiated. BMS 205820 suppresses the effect of LLLT on MIP-2 mRNA expression and ROS generation, stimulated by LPS or H(2)O(2). On NF-κB transcription factor, both the LLLT and NAC reduced this protein in the AM exposed to LPS or H(2)O(2). The synergistic effect between LLLT and NAC on the reduction the NF-κB was also evidenced. Results indicate that there is a synergistic action of LLLT with NAC on MIP-2 mRNA expression from LPS- or H(2)O(2)-stimulated AM, and that both ROS intracellular generation and NF-kB signaling seem to be involved.

Interrelationship of growth hormone, glucose and lipid metabolism

The relationship between Growth hormone (GH) and the metabolism of glucose and lipid is not completely understood. The present study is to obtain further information that will clarify the relationships between growth hormone and the metabolism of glucose and lipid. The subjects were randomly selected 25 male (11) and female (14) healthy individuals aged 35.96 +/- 8.05 years. After an overnight fast (10-12 hours), blood was taken from the subjects into heparinised tubes, centrifuged at 5,000 rpm for 5 minutes and the plasma separated. Fasting plasma glucose (FBS) was determined by glucose oxidase method,, total cholesterol, LDL, HDL and, Triglyceride were determined by enzymatic methods. Hormone sensitive lipase was determined by, using dilaural-glycero-glutaric acid methyresoruffin as substrate and Cobas Integra 800 Auto-analyser. Growth Hormone was determined by Enzyme linked immunoassay method by using monoclonal antibodies and Access 2 Immunoassay system. All reagents were supplied by Roche Company. The results showedpositive correlations between GH vs age and GH vs BMI. On the contrary, negative correlations were shown between GH vs the fasting levels of glucose,GH vs lipid and GH vs HSL. ). GH caused the reduction of the blood levels of glucose and, lipid using HSL as mediator, by inhibiting gluconeogenesis and stimulating lipolysis, respectively.

Development of an ELISA for quantification of tumstatin in serum samples and tissue extracts of patients with lung carcinoma

Background Tumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples. Methods Recombinant tumstatin as an immunogen was expressed by E. coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies. Results The detection limit of the ELISA was 1.4 ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7–115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases ( P = 0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues ( P < 0.001). Conclusions The development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.

Chemiluminescent optical fiber immunosensor detection of Brucella cells presenting smooth-A antigen

Brucellosis is a worldwide distributed zoonotic re-emerging disease, causing abortions in domesticated animals and Malta fever in humans. Currently, the gold standard for confirmation of the existence of the causative agent genus Brucella is the isolation of the bacteria from body or liquid samples, whereas standard serological tests are used for diagnosis. The need for a rapid point of care identification of Brucella organisms has led us to develop a novel chemiluminescent optical fiber immunosensor. The immunosensor based on a conventional enzyme linked immunoassay (ELISA) technique was implemented on optical fibers and its performance was compared to the standard ELISA method. We show that depending on the detecting antibodies used specificity of the assay is achieved. Brucella cells presenting smooth-A O-chain determinant were detected at a minimal protein concentration of 1.098 ng/ml, correlating to 305 cfu/ml, while smooth-M O-chain cells, rough cells and two gram-negative bacteria other than Brucella sp. produced negative results, confirming the high specificity of the technique.

Residues determination of carbofuran in vegetables based on sensitive time-resolved fluorescence immunoassay

Using direct competitive time-resolved fluorescence immunoassay (TRFIA), a rapid, highly selective and sensitive method was developed for the determination of carbofuran residues in lettuce and carrot. The method was based on a direct competitive immunoassay using europium-labelled anti-carbofuran monoclonal antibody and carbofuran-ovalbumin as coated antigen. The sensitivity, estimated as the I 50 value, was 34.54 ng/mL, with a detection limit (I 10) of 1.94 ng/mL and a practical working range between 1.33 and 341.6 ng/mL. The average recoveries of carbofuran from lettuce and carrot were 81.26–108.0% and 113.9–124.8%, respectively. Eventually, confirmation test between TRFIA and enzyme-linked immunoassay (ELISA) was performed. It confirmed that the results given by the TRFIA method were in agreement with those of the ELISA method.

Combinatorial coating of adhesive polypeptide and anti-CD34 antibody for improved endothelial cell adhesion and proliferation

Improved attachment, adhesion and proliferation of the surrounding mature endothelial cells (ECs) and circulating endothelial progenitor cells (EPCs) is of primary importance to realize the in situ rapid re-endothelialization of cardiovascular stents. To achieve this, a combinatorial coating of synthesized mussel adhesive polypeptide mimics as well as anti-CD34 antibody was constructed onto the devices through a novel adsorption method in this study. To immobilize the polypeptide and target antibody effectively, polycaprolactone (PCL) was first spin-coated onto the substrate as intermediate. The immobilization of polypeptide and antibody was confirmed by the changes of water contact angles and the attachment, growth of ECs and EPCs on the substrates, respectively. The results showed that after adhesive polypeptide or/and antibody immobilization, the hydrophilicity of coated PCL substrate (PCLS) was obviously improved. The amount of the immobilized antibody, determined by enzymelinked immunoassay (ELISA) method, was enhanced with the increase of antibody concentrations in the range from 5 to 25 mug/ml. The coatings after BSA blocking prevented the unspecific protein adsorption as monitored by fluorescent microscopy. The results of in vitro cell culture showed that compared with the PCLS, polypeptide/anti-CD34 antibody coating could effectively enhance the attachment, growth and adhesion of ECs and EPCs, in particular EPCs. A platelet adhesion experiment revealed that the blood compatibility of the PCLS after polypeptide/anti-CD34 antibody coating was also obviously improved. The results showed that the surface modification with adhesive polypeptide and anti-CD34 antibody will be a promising coating technique for the surface modification of the intravascular prostheses for rapid re-endothelialization.

Reply Re: A Prospective Evaluation of a Quantitative D-dimer Assay in the Evaluation of Acute Pulmonary Embolism

Reply Re: A Prospective Evaluation of a Quantitative D-dimer Assay in the Evaluation of Acute Pulmonary Embolism

Development of a magnetic particle immunoassay for polybrominated diphenyl ethers and application to environmental and food matrices

A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about 1 h after sample cleanup. The assay has a limit of detection (LOD) below 0.1 ppb towards the following brominated diphenyl ether (BDE) congeners: BDE-47, BDE-99, BDE-28, BDE-100, and BDE-153, with the LOD approximately the same as GC–NCI–MS. The congeners most readily recognized in the ELISA were BDE-47 and BDE-99 with the cross-reactivitys of BDE-28, BDE-100, and BDE-153 being less than 15% relative to BDE-47. As anticipated, the sensitivities are proportional to the similarities between the hapten structure and the BDE congener structure. Some oxygenated congeners with structural similarity to the hapten showed high to moderate cross-reactivities. Very low cross-reactivity was observed for other PBDEs or chlorinated environmental contaminants. The assay gave good recoveries of PBDEs from spiked water samples and a very small within and between day variance. Comparison with GC–NCI–MS demonstrated the ELISA method showed equivalent precision and sensitivity, with better recovery. The lower recovery of the GC–NCI–MS method could be caused by the use of an internal standard other than an isotopically substituted material that could not be used because of the fragmentation pattern observed by this method. The cleanup methods prior to ELISA were matrix dependent, no pretreatment was needed for environmental water samples, while fish, milk, and soil samples required various degrees of cleanup. Analysis of this wide variety of environmental samples by both ELISA and GC–MS demonstrated ELISA provides a timely and cost-effective method to screen for PBDEs in a variety of samples.

The Antibody Response to Helicobacter pylori in the Sera from a Rural Population in the Central Anatolia Region of Turkey

Helicobacter pylori (H. pylori )i nfection is common worldwide. Although the seropositivity of H. pylori rates has been unclear in the Turkish population. In this study, anti-H. pylori IgG seroprevalence and anti-cytotoxin-associated gene A (CagA) IgG positivity were evaluated. The sera of 880 people without gastrointestinal symptoms (384 males, 496 females) were tested for anti-H. pylori IgG and anti-CagA IgG antibodies by enzyme linked immunoassay method. Anti-H. pylori IgG antibodies wer ep ositiv ei n263 sera (41%) and their rates increased with age. The seroprevalence of anti-H. pylori IgG was higher in females (43.8%) tha ni n males(38%). Of the anti-H. pylori IgG positiv es era, 194 (53%) were also positive for anti-CagA IgG. The anti-CagA IgG positivity did not significantly differ with age. However, the lowest rate (46.6%) was determined among individuals 20‐ 29 years of age and the highest rate (62.5%) among individuals over 60 years age .A nti-CagAIgG positivity rateswere higher in males (87.5%) than in females (37.5%).

Dramatic Changes in the Prevalence of Helicobacter pylori Infection During Childhood: A 10‐year Follow‐up Study in Russia

The prevalence and rate of acquisition of Helicobacter pylori infection in children from developing countries is higher than in developed countries. This phenomenon has been related to differences in socioeconomic status, sanitation, and household hygiene. Russia is in the process of transforming from an underdeveloped to a developed country. To examine the effect of recent improvements in standards of living on the prevalence of H pylori in Russian children. We conducted 2 cross-sectional studies among children in St Petersburg, Russia. The first study was conducted in 1995 and the second was conducted a decade later. H pylori status was evaluated by the same enzyme-linked immunoassay method for anti-H pylori immunoglobulin G. Demographic data were obtained from each individual, and socioeconomic class was assessed by the education level of the mother and family income. In 1995 the overall prevalence of H pylori infection was 44%; 10 years later it had decreased to 13%. In both studies, the prevalence increased with age. In 1995 the prevalence was 30% among children younger than 5 years. A decade later the prevalence in the same age group was 2% (P = 0.001). The age-specific prevalence of H pylori infection increased significantly with age in both study periods. During 1995 the prevalence of the infection increased from 30% at age <5 years to 48% in the age group 15 to 19 years (P = 0.001). In the 2005 study the prevalence of the infection increased from 2% at age <5 years to 25% in the age group 15 to 19 years (P = 0.001). The crude and the age-adjusted odds ratio risk of infection in children showed an inverse correlation between the mothers' and fathers' educational levels and H pylori seropositivity (OR 1.8, 95% CI 1-3.2; P = 0.06). No associations were found between the prevalence of H pylori and any factor tested, including sex, type of dwelling, income, or the number of people living in the home. Improvements in standards of living in Russia have resulted in a marked reduction in H pylori transmission. Different rates of acquisition of H pylori form the basis for the differences in prevalence of infection between and among populations. The change in the prevalence of H pylori within 10 years among the Russian population is an example of how sensitive H pylori acquisition is to improvement in standards of living. Increased use of anti-H pylori eradication therapy played an important role in the reduction of the prevalence of H pylori infection.

Use of Electroacupuncture at ST36 to Inhibit Anaphylactic and Inflammatory Reaction in Mice

Objective: Electroacupuncture (EA) has been used to treat myalgia, allergy and gastroenteropathy in Korea. To determine whether EA can treat anaphylactic and inflammatory reactions, the effect of EA was investigated in a murine model. Methods: EA stimulation of the ST36 acupoint was performed for 10 min. Using a passive cutaneous anaphylaxis (PCA) model, the antianaphylactic effects of EA were examined. Interleukin-6 and tumor necrosis factor-α were measured using the ELISA method. The level of nuclear factor (NF)-ĸB/RelA protein and NF-ĸB DNA-binding activity was determined using the Western blot analysis and the transcription factor enzyme-linked immunoassay method. Results: EA inhibits PCA and β-hexosaminidase release, IL-6 secretion on the PCA, and in addition, EA reduces NF-ĸB DNA-binding activity. Conclusion: These results indicate that EA may possess antianaphylactic and antiinflammatory properties.

Correlation between the Serum Values of Soluble Intercellular Adhesion Molecule-1 and Total Sialic Acid Levels in Patients with Breast Cancer

Background: In this study, we aimed to investigate serum total sialic acid (TSA) and soluble intracellular adhesion molecule-1 (sICAM-1) levels in breast cancer patients to find a correlation with the cancer stage. Methods: The parameters from sera of 61 patients with breast cancer were measured. The concentrations of serum sICAM-1 and TSA were measured in serum samples from 61 patients with breast cancer and 25 healthy control subjects using enzyme-linked immunoassay and thiobarbituric acid method. Results: Mean serum sICAM-1 and TSA levels were significantly higher in the total patient group than in the control group (p < 0.001). Thus, the correlation between TSA and sICAM-1 became more significant in metastatic breast cancer. There were significant positive correlations between TSA and sICAM-1 in stage I+II (r = 0.59, p < 0.05), stage III (r = 0.47, p < 0.05), and stage IV (r = 0.89, p < 0.01), and total patient group (r = 0.56, p < 0.001). Conclusion: SerumsICAM-1 and TSA levels were higher in patients with breast cancer, than that of the control group, and also in the metastatic breast cancer group. Significant correlations between serum sICAM-1 and TSA may reflect the similar function of these molecules as adhesion molecules, and their roles in the carcinogenesis of breast cancer as well as metastasis.

Laboratory Comparison of the VisuLize IX Antigen Assay with the Asserachrome IX Antigen Assay

Factor IX antigen levels are used primarily to assess factor IX levels in patients with liver insufficiency or who are receiving oral vitamin K antagonist therapy. We evaluated the test performance of a new factor IX antigen enzyme-linked immunoassay kit and compared it to our currently used enzyme-linked immunoassay method. Samples from normal donors and patients with known hemophilia B of varying severity were tested by using a predicate factor IX antigen method (Asserachachrome IX:Ag) and a new factor IX antigen method (VisuLize IX Antigen). The 2 methods were evaluated for performance, correlation, differences, and bias. Test samples included 46 normal donors, 14 commercial products, and 16, 18, and 15 severe, moderate, and mildly deficient factor IX deficient plasmas, respectively. The 2 methods correlated well using regression analysis. However, there were significant differences using the paired t test with bias plots indicating higher absolute values for the VisuLize IX antigen method in the severe and moderate deficiency range, but not the normal range. Testing commercially prepared survey material, the Asserachrome method demonstrated a consistently lower result than expected. The VisuLize factor IX is a new method for determining antigenic levels in human plasma. This method has a much shorter incubation time and demonstrates good correlation with current factor IX antigenic methods.