Exploring glycated sites in human serum albumin: impact of sample processing techniques on detection and analysis.

Glycation and the subsequent formation of advanced glycation end products (AGEs) disrupt and impair the physiological functions of proteins. This study presents a comprehensive glycation site mapping of human serum albumin (HSA) utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both in vitro glycation experiments and patient samples were investigated, exploring various enzymes, processing techniques, and their impacts on glycation site detection. A pilot study was conducted, analyzing sixteen serum samples, which spanned from healthy individuals to severe diabetic patients (with HbA1c values ranging from 5.7% to 18.1%). The aim was to comprehend the progression of glycation on various sites of HSA with increasing levels of glycation. Their glycated albumin levels (GA) spanned from 19.7% to 62.3%. Trypsin-mediated proteolytic digestion unveiled 12 glycation sites through direct in-solution digestion of whole serum. However, isolating albumin from serum enabled the identification of a higher number of glycation sites in each sample compared to direct serum digestion. Boronate affinity chromatography facilitated the segregation of less glycated albumin (LGA) from the more glycated albumin (MGA) fraction. Subsequent proteolytic digestion of both LGA and MGA samples revealed similar glycation sites. The MGA fraction exhibited a greater number of identified glycation sites, thereby elucidating which sites are particularly prone to glycation in highly glycated albumin samples. Changes in relative glycation levels were noted in the tryptic digests of albumin samples following the sample enrichment steps, as opposed to direct in-solution digestion of whole serum. Two enzymes, trypsin and Glu-C, were evaluated for efficacy in sequence coverage and glycation site analysis of HSA, with trypsin demonstrating superior efficiency over Glu-C.

Multicentric evaluation of a novel point of care electrochemical ELISA platform for SARS-CoV-2 specific IgG and IgM antibody assay.

New diagnostics technologies for the efficient detection and quantification of SARS-CoV-2 antibodies are very crucial to manage the COVID-19 pandemic, especially in the context of emerging vaccination paradigms. Herein, we report on a novel point-of-care Electrochemical ELISA platform with disposable screen printed electrodes functionalized with SARS-CoV-2 Spike Glycoprotein S1, to enable fast and accurate quantitative estimation of total antibody concentration (IgG and IgM) in clinical samples. The quantification is performed with a comparison of electrochemical redox current against the current produced by the spiked monoclonal antibodies with known concentration. The assay is validated through multicentric evaluation against 3 different FDA authorized Laboratory standard techniques, using both EDTA whole blood and serum samples. We demonstrate that the proposed assay has excellent sensitivity and specificity, making it a suitable candidate for epidemiological surveys and quantification of antibodies in COVID-19 vaccination programs.

Glycation of albumin and its implication in Diabetes: A comprehensive analysis using mass spectrometry.

AIM: To understand the mechanism of glycation of albumin and effects on cysteinylation and methionine oxidation. METHODS: The in vitro glycation of HSA and BSA was studied with varying concentrations of glucose. Clinical blood samples of diabetic subjects with varying HbA1c values, were analyzed to assess in vivo glycation. All samples and their tryptic digests were analyzed using liquid chromatography/mass spectrometry. Glycation sites were mapped on to the three-dimensional structure of the HSA and BSA. RESULTS: A total thirty-one sites for glycation and eight sites of Nε-carboxymethyl-lysine (CML) modification were identified on albumin. The site selectivity of glycation was correlated with the environment of the reactive residue in the three-dimensional structure. CONCLUSIONS: The maximum percentage glycation under extreme conditions was in the range of ~55 to 88% in four weeks. Two major glycation sites K-233 and K-525 were identified, which together accounted for 40-50% of total glycation. A correlation was observed between glycation and oxidation of methionine residues in samples glycated in vitro. The role of spatially proximate residues in facilitating the glycation process is evident. The tri- and tetra-glycated isoforms of albumin can serve as biomarkers for the severe uncontrolled diabetic state.

Multicentric Evaluation of a Novel Point of Care Electrochemical ELISA Platform for SARS-CoV-2 Specific IgG and IgM Antibody Assay

New diagnostics technologies for the efficient detection and quantification of SARS-CoV-2 Antibodies is very crucial to manage the COVID-19 pandemic, especially in the context of emerging vaccination paradigms. Herein, we report on a novel point-of-care Electrochemical ELISA platform with disposable screen printed electrodes functionalized with SARS-CoV-2 Spike Glycoprotein S1, to enable fast and accurate quantitative estimation of total antibody concentration (IgG and IgM) in clinical samples. The quantification is performed with a comparison of electrochemical redox current against the current produced by the spiked monoclonal antibodies with known concentration. The assay is validated through multicentric evaluation against 3 different FDA authorized Laboratory standard techniques, using both EDTA whole blood and serum samples. We demonstrate that the proposed assay has excellent sensitivity and specificity, making it a suitable candidate for epidemiological surveys and quantification of antibodies in COVID-19 vaccination programs.

Application of a Nanotechnology-Based, Point-of-Care Diagnostic Device in Diabetic Kidney Disease.

INTRODUCTION: Early detection of diabetes mellitus (DM) and diabetic kidney disease (DKD) is important for preventing end-stage renal failure and reducing cardiovascular complications. Availability of a validated point-of-care (PoC) device that can measure various DKD markers would be useful in this respect, especially in resource-poor parts of the world. METHODS: We validated a novel nanotechnology-based multianalyte PoC device (minimally invasive and does not require trained medical personnel) against laboratory gold standard tests for the detection of 5 biomarkers related to management of DM and DKD. The prospective study was funded by an International Society of Nephrology American Nephrologists of Indian Origin grant in 2 phases: (i) proof of concept: random samples were tested for the analytes with the PoC device and correlated with the laboratory gold standard; and (ii) clinical validation in a well-characterized cohort of patients. A nonenzymatic- and nonantibody-based electrochemical PoC device for quantitative measurement of markers-glycosylated hemoglobin (HbA1c), hemoglobin, serum albumin, microalbuminuria, urine creatinine, and albumin-to-creatinine ratio-was developed and used in this study. The disposable strips were interfaced with a multipotentiostat hand-held PoC device (3.7-V rechargeable lithium battery, 5-inch touch screen, Bluetooth enabled) working in amperometry mode, which provided the results in <1 minute. Data were analyzed using linearity plots and Bland-Altman difference plot analysis. RESULTS: A total of 4717 individuals were screened during the study (phase 1: 2576 and phase 2: 2141.) In phase 2, samples were tested in 529 subjects (346 females)-120 subjects with type 1 DM, 255 subjects with type 2 DM, 54 subjects without DM, 400 subjects with stage 2 chronic kidney disease, and 30 subjects with stage 3 chronic kidney disease. CONCLUSION: A nanotechnology-based PoC device for quantitative measurement of HbA1c, hemoglobin, serum albumin, microalbuminuria, and the urine albumin-to-creatinine ratio was developed for detection of early DKD and showed excellent correlation between the device and laboratory results. This device has the potential for early detection of DM and/or DKD, especially in remote communities in underserved areas of the world where prevalence of diabetes is rapidly increasing.

Aza-heterocyclic Receptors for Direct Electron Transfer Hemoglobin Biosensor.

Direct Electron Transfer biosensors, facilitating direct communication between the biomolecule of interest and electrode surface, are preferable compared to enzymatic and mediator based sensors. Although hemoglobin (Hb) contains four redox active iron centres, direct detection is not possible due to inaccessibility of iron centres and formation of dimers, blocking electron transfer. Through the coordination of iron with aza-heterocyclic receptors - pyridine and imidazole - we report a cost effective, highly sensitive and simple electrochemical Hb sensor using cyclic voltammetry and chronoamperometry. The receptor can be either in the form of liquid micro-droplet mixed with blood or dry chemistry embedded in paper membrane on top of screen printed carbon electrodes. We demonstrate excellent linearity and robustness against interference using clinical samples. A truly point of care technology is demonstrated by integrating disposable test strips with handheld reader, enabling finger prick to result in less than a minute.

Characterization of a hemoglobin variant: HbQ-India / IVS 1-1 [G>T]-ß-thalassemia.

Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in heterozygous state and presents normal hematological blood picture. Here we report a rare case of HbQ-India with a thalassemic phenotype that has been analyzed using a combination of mass spectrometry, gene sequencing and PCR analysis. This combined analyses revealed the HbQ variant to be associated with a beta chain mutation, IVS 1-1 [G>T]. Though HbQ has earlier been reported with thalassemic trait using different techniques, this is the first report of a compound α and ß chain Hb heterozygous mutant involving HbQ and IVS1-1 being validated using Mass Spectrometry and Reverse dot blot hybridization.

Paraoxonase activity and antibodies to oxidized-LDL in chronic renal failure patients on renal replacement therapy.

Serum paraoxonase (PON1) and antibodies to oxidized-LDL (anti ox-LDL) were measured in chronic renal failure subjects on renal replacement therapy such as hemodialysis (HD) peritoneal dialysis (PD) and transplantation (Txp). Paraoxonase activity was significantly lower in HD and PD group (P<0.001) than in control subjects. In transplant patients, paraoxonase activity was not significantly different from that of controls. Antibodies to ox-LDL was significantly higher in HD, PD and Transplant patients (P<0.0001) compared to control subjects. High titers of antibodies were observed in the HD group compared to the PD and Transplant subjects. A decrease in paraoxonase activity and high titers of Antibodies to ox-LDL in the dialysis group suggest a decreased cardio protective effect of HDL and enhanced risk of premature cardiovascular complications. Whereas in case of transplant subjects, there seems to be restoration of PON1 activity, but elevated levels of anti-oxLDL could still be a potential atherogenic factor. Hence, we propose that estimation of these two parameters can be used as a useful index to measure the cardiac risk in the above patient category.

Ninety-minute accelerated critical pathway for chest pain evaluation.

Rapid, efficient, and accurate evaluation of chest pain patients in the emergency department optimizes patient care from public health, economic, and liability perspectives. To evaluate the performance of an accelerated critical pathway for patients with suspected coronary ischemia that utilizes clinical history, electrocardiographic findings, and triple cardiac marker testing (cardiac troponin I [cTnI], myoglobin, and creatine kinase-MB [CK-MB]), we performed an observational study of a chest pain critical pathway in the setting of a large Emergency Department at the Veterans Affairs Medical Center in 1,285 consecutive patients with signs and symptoms of cardiac ischemia. The accelerated critical pathway for chest pain evaluation was analyzed for: (1) accuracy in triaging of patients within 90 minutes of presentation, (2) sensitivity, specificity, positive predictive value, and negative predictive value of cTnI, myoglobin, and CK-MB in diagnosing acute myocardial infarction (MI) within 90 minutes, and (3) impact on Coronary Care Unit (CCU) admissions. All MIs were diagnosed within 90 minutes of presentation (sensitivity 100%, specificity 94%, positive predictive value 47%, negative predictive value 100%). CCU admissions decreased by 40%. Ninety percent of patients with negative cardiac markers and a negative electrocardiogram at 90 minutes were discharged home with 1 patient returning with an MI (0.2%) within the next 30 days. Thus, a simple, inexpensive, yet aggressive critical pathway that utilizes high-risk features from clinical history, electrocardiographic changes, and rapid point-of-care testing of 3 cardiac markers allows for accurate triaging of chest pain patients within 90 minutes of presenting to the emergency department.

Utility of B-natriuretic peptide levels in identifying patients with left ventricular systolic or diastolic dysfunction.

PURPOSE: Although echocardiography is important for making the diagnosis of left ventricular dysfunction, its cost and lack of availability limit its use as a routine screening test. B-Natriuretic peptide levels accurately reflect ventricular pressure, and preliminary studies with a rapid assay have found that levels are sensitive and specific for diagnosing heart failure in patients with dyspnea. We hypothesized that B-natriuretic peptide levels obtained through the use of a rapid assay should correlate with echocardiographic abnormalities of ventricular function. SUBJECTS AND METHODS: We studied 400 patients who were referred for echocardiography at the San Diego Veteran's Healthcare System between June and August 2000 to evaluate ventricular function. B-natriuretic peptide levels were measured by a point-of-care immunoassay; cardiologists assessing left ventricular function were blinded to the assay results. Patients were grouped into those with normal ventricular function, systolic dysfunction only, diastolic dysfunction only, and both systolic and diastolic dysfunction. RESULTS: Mean (+/- SD) B-natriuretic peptide concentration was 416 +/- 413 pg/mL in the 253 patients diagnosed with abnormal left ventricular function, compared with 30 +/- 36 pg/mL in the 147 patients with normal left ventricular function. Patients with both systolic and diastolic dysfunction had the highest levels (675 +/- 423 pg/mL). The area under the receiver operating characteristic (ROC) curve for B-natriuretic peptide levels to detect any abnormal echocardiographic finding was 0.95 (91% confidence interval: 0.93 to 0.97). B-Natriuretic peptide levels were unable to differentiate systolic vs. diastolic dysfunction. In patients with symptoms of heart failure and normal systolic function, B-natriuretic peptide levels >57 pg/mL had a positive predictive value of 100% for diastolic abnormalities. CONCLUSIONS: A simple, rapid test for B-natriuretic peptide levels can reliably predict the presence or absence of left ventricular dysfunction on echocardiogram. For some patients, a normal level may preclude the need for echocardiography.

Mitigation of the clinical significance of spurious elevations of cardiac troponin I in settings of coronary ischemia using serial testing of multiple cardiac markers.

The ability to differentiate between true positives, false positives, and sporadically elevated cardiac troponin levels has grown in importance as cardiac troponins assume an increasingly dominant role in the diagnosis of coronary syndromes. In a population sample of 1,000 patients who presented consecutively to a large urban hospital emergency room, 50 of 112 patients who had elevated troponin levels (> 0.6 ng/ml) during evaluation for myocardial injury were subsequently found to have had an isolated, spurious elevation of cardiac troponin, and not a diagnosed myocardial infarction. Logistic regression analysis shows that by hierarchically analyzing electrocardiographic changes with concurrent creating kinase-MB and myoglobin levels at the time of the troponin elevation, one may predict with 91% accuracy whether the troponin elevation is actually indicative of a myocardial infarction in a patient. Spurious troponin elevations may be a common occurrence, and if not detected, may result in an increased number of falsely diagnosed myocardial infarctions.

A rapid test for B-type natriuretic peptide correlates with falling wedge pressures in patients treated for decompensated heart failure: a pilot study.

OBJECTIVES: To determine if changes in B-type natriuretic peptide (BNP) levels can accurately reflect acute changes in pulmonary capillary wedge pressure during treatment of decompensated heart failure. BACKGROUND: Tailored therapy of decompensated congestive heart failure with hemodynamic monitoring is controversial. Other than the expense and complications of Swan-Ganz catheters, its use in titration of drug therapy has no conclusive end point. Because BNP reflects both elevated left ventricular pressure and neurohormonal modulation and has a short half-life, we hypothesized that levels of BNP would decline in association with falling wedge pressures. Final BNP levels would perhaps signify a new set point of neuromodulation. METHODS AND RESULTS: Twenty patients with decompensated New York Heart Association (NYHA) class III-IV congestive heart failure (CHF) undergoing tailored therapy were studied. BNP levels were drawn every 2 to 4 hours for the first 24 hours (active treatment phase) and then every 4 hours for the next 24 to 48 hours (stabilization period). Hemodynamic data was recorded simultaneously. In 15 patients whose wedge pressure responded to treatment in the first 24 hours, there was a significant drop in BNP levels (55%) versus nonresponders (8%). There was a significant correlation between percent change in wedge pressure from baseline per hour and the percent change of BNP from baseline per hour (r = 0.79, P <.05). When the wedge pressure was kept at a stable, low level during the stabilization phase, BNP levels continued to fall another 37% (937 +/- 140 pg/mL at 24 hours to 605 +/- 128 pg/mL). Patients who died (n = 4) had higher final BNP levels (1,078 +/- 123 pg/mL v 701 +/- 107 pg/mL). CONCLUSIONS: The data suggest that rapid testing of BNP may be an effective way to improve the in-hospital management of patients admitted with decompensated CHF. Although BNP levels will not obviate the need for invasive hemodynamic monitoring, it may be a useful adjunct in tailoring therapy to these patients.

Utility of B-natriuretic peptide as a rapid, point-of-care test for screening patients undergoing echocardiography to determine left ventricular dysfunction.

BACKGROUND: Although echocardiography is an important tool for making the diagnosis of left ventricular (LV) dysfunction, the cost of this procedure limits its use as a routine screening tool for this purpose. Brain natriuretic peptide (BNP) accurately reflects ventricular pressure, and preliminary studies have found it to be highly sensitive and highly specific in diagnosing congestive heart failure in the emergency department. We hypothesized that BNP might therefore be useful as a screening tool before echocardiography in patients with suspected LV dysfunction. METHODS: Subjects included patients referred for echocardiography to evaluate the presence or absence of LV dysfunction. Patients with known LV dysfunction were excluded from analysis. BNP was measured by a point-of-care immunoassay (Biosite Diagnostics, San Diego, Calif). The results of BNP levels were blinded from cardiologists making the assessment of LV function. Patients were divided into those with normal ventricular function, abnormal systolic ventricular function, abnormal diastolic function, and evidence of both systolic and diastolic dysfunction. RESULTS: Two hundred patients in whom LV function was unknown were studied. In the 105 patients (53%) whose ventricular function was subsequently determined to be normal by echocardiography, BNP levels averaged 37 +/- 6 pg/mL. This was significantly less than in those patients with either ultimate diastolic dysfunction (BNP 391 +/- 89 pg/mL (P <.001) or systolic dysfunction (BNP 572 +/- 115 pg/mL (P <.001). A receiver-operator characteristic curve showing the sensitivity and specificity of BNP against the echocardiography diagnosis revealed the area under the curve (accuracy) was 0.95. At a BNP level of 75 pg/mL was 98% specific for detecting the presence or absence of LV dysfunction by echocardiography. CONCLUSIONS: A simple, rapid test for BNP, which can be performed at the bedside or in the clinic, can reliably predict the presence or absence of LV dysfunction on echocardiogram. The data indicate that BNP may be an excellent screening tool for LV dysfunction and may, in fact, preclude the need for echocardiography in many patients.

Usefulness of cardiac troponin I in patients undergoing open heart surgery.

BACKGROUND: Significant myocardial injury during cardiac surgery is associated with a 10-fold increase in 2-year complication rates, yet there remains no clinical gold standard for diagnosis. Troponin I has complete cardiospecificity and is clinically used for diagnosis of myocardial infarction in other settings. METHODS AND RESULTS: One hundred consecutive patients undergoing open heart surgery (71 coronary artery bypass grafts and 29 aortic valve replacements) were enrolled and blood samples were drawn preoperatively, at 5 AM and 5 PM on days 1 and 2 after surgery, and at 5 AM for 3 more days. Twelve-lead electrocardiograms were performed daily and echocardiographic studies were performed on patients with either; electrocardiographic changes signifying likely myocardial damage, intraoperative complications, or elevated creatine kinase subfraction MB or troponin values. Seventeen patients had either new wall motion abnormalities or new Q waves all with peak cardiac troponin I >40 ng/mL. Stratification of patients by peak troponin values <40 and >60 ng/mL was highly predictive (P <.001) of days in intensive care unit, days on ventilator, development of new arrhythmia, and especially cardiac events. These postoperative variables also showed a stronger correlation with peak cardiac troponin I than did peak creatine kinase subfraction MB. CONCLUSION: Peak troponin I values detect myocardial infarction the day after heart surgery and predicts patient outcome.

Utility of B-type natriuretic peptide in the diagnosis of congestive heart failure in an urgent-care setting.

OBJECTIVES: The goal of this study was to evaluate the utility of a rapid "bedside" technique for measurement of B-type natriuretic peptide (BNP) in the diagnosis of congestive heart failure (CHF) in an urgent-care setting. BACKGROUND: B-type natriuretic peptide is a protein secreted from the cardiac ventricles in response to pressure overload. One potential application of measurements of BNP in blood is distinguishing dyspnea due to CHF from other causes. METHODS: B-type natriuretic peptide concentrations were measured in a convenience sample of 250 predominantly male (94%) patients presenting to urgent-care and emergency departments of an academic Veteran's Affairs hospital with dyspnea. Results were withheld from clinicians. Two cardiologists retrospectively reviewed clinical data (blinded to BNP measurements) and reached a consensus opinion on the cause of the patient's symptoms. This gold standard was used to evaluate the diagnostic performance of the BNP test. RESULTS: The mean BNP concentration in the blood of patients with CHF (n = 97) was higher than it was in patients without (1,076 +/- 138 pg/ml vs. 38 +/- 4 pg/ml, p < 0.001). At a blood concentration of 80 pg/ml, BNP was an accurate predictor of the presence of CHF (95%); measurements less than this had a high negative predictive value (98%). The overall C-statistic was 0.97. In multivariate analysis, BNP measurements added significant, independent explanatory power to other clinical variables in models predicting which patients had CHF. The availability of BNP measurements could have potentially corrected 29 of the 30 diagnoses missed by urgent-care physicians. CONCLUSIONS: B-type natriuretic peptide blood concentration measurement appears to be a sensitive and specific test to diagnose CHF in urgent-care settings.

A rapid bedside test for B-type peptide predicts treatment outcomes in patients admitted for decompensated heart failure: a pilot study.

OBJECTIVES: The goal of this study was to determine if B-type natriuretic peptide (BNP) levels predict outcomes of patients admitted with decompensated heart failure. BACKGROUND: Treatment of decompensated congestive heart failure (CHF) has often been based on titration of drugs to relieve patient's symptoms, a case that could be made for attempting to also treat neurohormonal abnormalities. Because BNP reflects both elevated left ventricular pressure as well as neurohormonal modulation, we hypothesized that BNP might be useful in assessing outcomes in patients admitted with decompensated CHF. METHODS: We followed 72 patients admitted with decompensated New York Heart Association class III to IV CHF, measuring daily BNP levels. We then determined the association between initial BNP measurement and the predischarge or premoribund BNP measurement and subsequent adverse outcomes (death and 30-day readmission). RESULTS: Of the 72 patients admitted with decompensated CHF, 22 end points occurred (death: n = 13, readmission: n = 9). In these patients, BNP levels increased during hospitalization (mean increase, 233 pg/ml, p < 0.001). In patients without end points, BNP decreased (mean decrease 215 pg/ml). Univariate analysis revealed that the last measured BNP was strongly associated with the combined end point. In patients surviving hospitalization, BNP discharge concentrations were strong predictors of subsequent readmission (area under the receiver operator curve of 0.73). CONCLUSIONS: In patients admitted with decompensated CHF, changes in BNP levels during treatment are strong predictors for mortality and early readmission. The results suggest that BNP levels might be used successfully to guide treatment of patients admitted for decompensated CHF.

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

Inhibition of Fe(II) catalyzed linoleic acid oxidation and DNA damage by phosvitin.

The oxidation of linoleic acid catalyzed by Fe(II) is strongly inhibited by phosvitin, while chelation with EDTA, NTA or deferoxamine produced only partial inhibition. Interestingly, the DNA degradation catalyzed by Fe(II) in the presence of H2O2 is also inhibited by phosvitin or deferoxamine. In contrast, chelation of the metal ion with EDTA or NTA enhanced the DNA degradation. The results suggest that the nature of interaction between the metal ion and the complexing agent may be an important factor in the generation of active oxygen intermediates.

Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis.

An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II. The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose-N-acetylgalactosamine binding lectin. This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography. The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis. Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA. The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001). Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.