Electrochemiluminescence Immunoassay Kit Research Articles

Impact of Covishield Vaccination in Terms of SARS CoV-2 Neutralizing Antibody Expression.

The vaccination efficacy can indirectly be assessed through the quantification of neutralizing antibodies. Very few data are available on Covishield efficacy in terms of neutralizing antibody expression upon vaccination. This study is focused on profiling of neutralizing antibody expression during and after the Covishield two shot vaccination and observing COVID-19 infection in vaccinated participants during the period. SARS CoV-2 neutralizing antibody concentrations in samples were estimated using electrochemiluminescence immunoassay kit for Lifotronics eCL8000. The sampling had been done sequentially at 45th, 85th day after 1st dose and 15th day after 2nd dose Covishield vaccination. Parallelly, in order to confirm the total SARS CoV-2 IgG response in COVID-19 infection, measured the IgG using SARS CoV-2 IgG lateral flow immunoassay test kit. The subjects previously infected with COVID-19 before 1st dose vaccination demonstrated high neutralizing antibody (> 10AU/ml). In COVID-19 uninfected subjects, there was a sudden incline in neutralizing antibody after the 2nd dose. Infection with SARS CoV-2 between 1st and 2nd dose of Covishield vaccination implicate that the level of neutralizing antibody in serum after 1st dose was not adequate to combat the virus and prevent infection. We observed COVID-19 infection in participants even after 2nd dose of vaccination. Interestingly, there was no protection against SARS CoV-2 even with a high neutralizing antibody expression of 188.5 AU/mL after the 2nd dose. Findings of Covishield efficacy in different cohort samples before and after 2 doses of Covishield vaccination provide impetus for improvement or development of next generation vaccines.

Development of chemiluminescence method based on serum type I collagen hydroxyl terminal peptide β special sequence (β-CTX)

The objective of this work is to develop and verify the analytical performance of a chemiluminescence immunoassay for the specific sequence β-carboxy-terminal cross-linking telopeptide of type I collagen (β-CTX) in human serum. Two specific monoclonal antibodies (mAb-8A03 and mAb-3D12) with high affinity for β-CTX were selected, and, under optimized conditions, a chemiluminescence immunoassay method (CLIA) for β-CTX was established. The CLIA of β-CTX detected β-CTX in a wide range of 2.0–6000 ng/L. The recovery rate in serum is 95–105%, the specificity is high, and the cross-reaction rate with common easily interfering substances is low (not more than 0.01%). The CLIA correlates well with Roche electrochemiluminescence immunoassay (ECLIA), with a correlation coefficient of 0.9551, which fully meets the requirements of clinical analysis. The developed β-CTX CLIA kit has high sensitivity and good stability. It has the same performance as the commercial Roche ECLIA kit and can be applied clinically.

A preliminary study for the establishment of a reference interval for vitamin B12 in China after performance verification of a second-generation ECLIA kit.

BackgroundThe second‐generation electrochemiluminescence immunoassay (ECLIA) kit of vitamin B12 is widely used in clinical laboratories, and the establishment of a reference interval (RI) is essential to provide the basis for clinical monitoring. The purpose of this study was to establish a laboratory RI for vitamin B12 in China and at the same time verify the method performance of the second‐generation kit.MethodsThe verification of the method performance was conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Based on these guidelines, a total of 580 serum samples were collected, and 391 serum samples were used for the establishment of the RI according to CLSI guidelines. The subjects were grouped by sex and age. The age groups were as follows: 21‐40, 41‐60, and 61‐80 years. The RI was defined by nonparametric 2.5th and 97.5th percentile intervals.ResultsThe performance of the second‐generation kit of vitamin B12 from the Roche Cobas E602 system was in compliance with laboratory requirements. Serum vitamin B12 levels conformed to a non‐Gaussian distribution. Harris‐Boyd's test did not indicate partitioning for different age and gender group. Besides, there was no significant difference between different age groups (P = .07) and gender groups (P = .2002). The RI for healthy Chinese adults (aged 21‐80 years) calculated by the nonparametric method was 250.8‐957.1 pg/mL.ConclusionsThe reference range of vitamin B12 was established, which provided a theoretical basis for the clinical application and monitoring of vitamin B12 detection.

Methylated septin 9 gene for noninvasive diagnosis and therapy monitoring of breast cancer

Background: Blood-based biomarkers specific for breast cancer is urgently demanded to facilitate the screening and reduce breast cancer-related deaths. This study evaluated the potential of methylated septin 9 gene ( mSEPT9 ) as a biomarker for breast cancer. Methods: A total of 86 breast biopsy samples were collected from 59 patients with breast cancer and from 27 patients with benign breast tumor. Two hundred and seven plasma samples were collected from 80 patients with breast cancer, 27 patients with benign breast tumor patients, and 100 healthy volunteers. mSEPT9 was detected using the Epi proColon 2.0 CE test. Additionally, 40 tissue cases of the tumor samples were analysed by real time PCR for SEPT9_v2 mRNA expression and by immunohistochemical staining for SEPT9 protein expression. Serum CEA, CK19 and CA15-3 concentrations were analyzed using an Electrochemiluminescence Immunoassay Kit. Results: The sensitivity of plasma mSEPT9 for detecting breast cancer was 26.3% with a specificity of 97.6%. The plasma mSEPT9 -positive rate was higher in patients with later stage lymph node metastasis (P=0.018), intravascular tumor thrombus (P=0.035), ki67-positive (P=0.033) and p53-positive (P=0.044). Despite its limitation in sensitivity, conversion of plasma mSEPT9 to negative status in mSEPT9 -positive breast cancer patients following cancer treatment was correlated with favorable responses, while persistent positivity was associated with poor responses. mSEPT9 performed comparably with CK19 (P>0.05) and better than CEA (P=0.002) and CA15-3 (P mSEPT9 was detected in 83.1% (49/59) of the breast cancer tissues, whereas 8.0% (2/25) of the benign breast tumor tissues were mSEPT9 positive (P mSEPT9 in breast cancer tissue was associated with tumor size of >2 cm (P=0.026) and with invasion of adipose tissue (P=0.015). Conclusions: Our results indicate that mSEPT9 test has the potential to become a valuable tool in the diagnosis and treatment monitoring of breast cancer for Chinese patients.

Evaluation of serum ferritin and thyroid function in the second trimester of pregnancy.

Ferritin is a universal intracellular protein that acts as an iron carrier. Several studies have indicated that iron deficiency affects thyroid function in non-pregnant women. Our objective was to assess the relationship between serum ferritin levels and thyroid function in pregnant women during the second trimester. Pregnant women with sufficient iodine intake and normal antithyroid antibodies during the second trimester were recruited from the obstetric outpatient department of the Fifth People's Hospital of Fudan University. Serum ferritin (SF) levels, thyroid function, anti-thyroid antibodies and vitamin B12 were determined by electrochemiluminescence immunoassay kit. Maternal serum iron (Fe), unsaturated iron binding capacity (UIBC), hemoglobin (Hb), creatinine (Cr), fasting blood glucose (FBG), and alanine aminotransferase (ALT) were also evaluated. Stepwise regressions performed to evaluate the associations between SF and other maternal parameters. In the second trimester, 11.4% pregnant women had a SF concentration less than 12 μg/L, and 7.6% pregnant women were anemic. SF levels were negatively correlated with serum TSH levels (r = -0.219, p < 0.05), and positively correlated with FT4 levels (r = 0.203, p < 0.05). Linear regression analysis showed only SF, age, week of gestation were significant predictors of regression with TSH as the dependent variable (β: -0.007, -0.059, and 0.118 respectively; all p < 0.05). However consistent relation between the SF levels and FT4 was not observed in stepwise linear regression. Maternal iron status is a determinant of TSH concentrations during pregnancy in pregnant women during the second trimester.

Evaluation of a newly developed quantitative determination kit for tumor marker CA15-3 with chemiluminescent assay.

BackgroundTumor marker carbohydrate antigen 15‐3 (CA15‐3) is used as a biomarker to aid to diagnose and monitor the prognosis of breast cancer patients. A new quantitative determination kit for CA15‐3 with chemiluminescent assay was developed by Xiamen InnoDx Biotech Co., Ltd, China. Therefore, we conducted the report to evaluate the performance of the kit.MethodsAccording to the “Guiding principles on performance analysis of diagnostic reagents in vitro”, the calibration curve, limit of detection, reportable range, accuracy, precision, anti‐interference capability, cross‐reaction and comparison by measuring EDTA plasma and serum were carried out. In addition, the kit was performed in parallel to electrochemiluminescence immunoassay kit (Roche) to analyze the correlation between the two kits.ResultsRegression equation of calibration curve of the kit was Y=0.7914X+4.1032 (R2=.990). Limit of detection was 0.0347 U/mL. The reportable range was 0.5‐2400 U/mL. Recovery ratio was 100.0%‐104.8%. Coefficient of variations (CVs) of within‐run and between‐run were 4.8%‐7.6% and 5.8%‐7.4% respectively. No remarkable interferences (all Bias% were less than ±10%) were detected when samples contained hemoglobin ≤183.8 μmol/L, bilirubin ≤340 μmol/L, triglyceride ≤18.1 mmol/L, or rheumatoid factor ≤400 U/mL. No cross‐reaction was present in the kit. Moreover, compared with the results from electrochemiluminescence immunoassay kit (Roche) in 345 serum samples, there was a satisfied correlation coefficient of 0.977 (P<.01), and the kit was simultaneously fit for the detection of EDTA plasma and serum samples.ConclusionThe new kit validated satisfactorily, and it can be used for detecting CA15‐3 in clinical practice.

Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA)

A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and “sandwiched” by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90–110% for serum samples. A value of 0.88pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer.

The Establishment of an HE4-CLIA Method and the Combined Analysis of HE4 and CA125 in Ovarian Cancer.

The human epididymal secretory protein 4 (HE4) is a novel, verified biomarker for the early diagnosis of ovarian cancer. Magnetic beads were coated with capture antibodies and were used with acridinium ester labeled detection antibodies in a sandwich-type immunoassay. The patient's HE4 serum levels were measured simultaneously with the chemiluminescence immunoassay (CLIA) kit we developed and electrochemiluminescence immunoassay (ECLIA) kit from Roche (Mannheim, Germany). CA125 was also detected by time-resolved fluoroimmunoassay. The diagnostic value was analyzed. The assay demonstrated a linear range from 2.5 to 2,000 pmol/l, with an analytical sensitivity of 2.5 pmol/l. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. Compared with the ECLIA kit from Roche in 124 serum samples (40 patients with ovarian cancer, 35 patients with benign gynecological diseases, and 49 health controls), there is a satisfied correlation coefficient of 0.875. The area under the receiver-operating curve (ROC-AUC) was 0.903 (95% CI was 0.839-0.966, P < 0.001) for HE4, 0.787 (95% CI was 0.694-0.879, P < 0.001) for CA125, and 0.914 (95% CI was 0.866-0.962, P < 0.001) for combined analysis of HE4 and CA125. A quantitative method (HE4-CLIA) for detecting HE4 in serum was successfully established. Preliminary clinical sample analysis showed HE4-CLIA has a certain clinical value in the screening and diagnosis of ovarian cancer. Moreover, in distinguishing benign from malignant ovarian lesions, HE4 has higher demonstrated accuracy than CA125.

Serum Neuron-specific Enolase Levels in Preterm and Term Newborns and in Infants 1–3 Months of Age

Elevated serum levels of neuron-specific enolase (NSE) was initially assumed to be specific to neuronal tumors (particularly neuroblastoma), but is now known to accompany nontumoral conditions and tumors other than neuroblastomas. There is a need to establish normal ranges for NSE, especially in early infancy. The aims of this study were to determine reference values for NSE in newborns and young infants and to assess whether NSE levels in early infancy (i.e., preterm infants and term infants) differ from the adult reference range for this enzyme. We enrolled 140 healthy babies, which included 40 preterm newborns (3-15 days old and born at 28-42 weeks gestation), 40 term newborns (< 1 month old and born at term), and 60 young infants 1-3 months old (n=20 per subgroup of 1-, 2-, and 3-month-old infants). The determination of NSE levels was performed by the electrochemiluminescence immunoassay (ECLIA) method using the Elecysys 2010 device (Roche Diagnostics, Mannheim, Germany). The mean serum NSE levels for the preterm newborns was 21.83±15.06ng/mL [95% confidence interval (95%CI), 16.95-26.71ng/mL]; term newborns, 18.06±12.83ng/mL (95%CI, 13.94-22.19ng/mL); and young infants, 9.09±4.38ng/mL (95%CI, 7.96-10.23ng/mL). The mean serum NSE level for infants 1-3 months old was within the ECLIA kit's normal range (4.7-18ng/mL for adults), whereas the corresponding means for the preterm and term newborns were higher (p<0.001, for both). Our findings suggest that adult reference values should not be applied to the preterm and term age groups.

Diagnostic value of S100B protein in the differential diagnosis of acute vertigo in the emergency department.

Vertigo is a common presenting complaint resulting from central or peripheral etiologies. Because central causes may be life-threatening, ascertaining the nature of the vertigo is crucial in the emergency department (ED). With a broad range of potential etiologies, distinguishing central causes from benign peripheral causes is a diagnostic challenge. Cranial magnetic resonance imaging (MRI) is the recommended neuroimaging method when clinical findings are ambiguous. However, MRI scanning for every patient with an uncertain diagnosis may not be efficient or possible. Therefore, to improve ED resource utilization for patients with vertigo, there is a need to identify the subset most likely to have MRI abnormalities. It has previously been shown that S100B protein provides a useful serum marker of stroke, subarachnoid hemorrhage, and traumatic brain injury. This study evaluated whether S100B levels could predict central causes of vertigo as identified by cranial MRI in the ED. This prospective, observational study was conducted with adult patients with acute-onset vertigo (within 6 hours) in the ED of a teaching hospital in Kocaeli, Turkey. Patients with nausea or dizziness complaints without previously known vertigo or cranial pathology, and who agreed to participate in the study, were included. Patients with trauma or with neurologic findings that developed concurrent with their symptoms were excluded. Serum levels of S100B were measured with an electrochemiluminescence immunoassay kit. All subjects underwent cranial MRI. The predictors of positive MRI results were evaluated using logistic regression analysis. Sensitivity and specificity of S100B​ levels for identifying subjects with central causes of vertigo on MRI were calculated with receiver operating characteristic (ROC) curve. Of the 82 subjects included in the study, 48 (58.5%) were female, and the mean (±SD) age was 51 (±16) years. Thirty-one (37.8%) subjects had positive MRI results. Median (with interquartile range [IQR]) serum S100B levels were significantly different between MRI-negative and MRI-positive groups (median = 27.00 pg/mL, IQR = 10.00 to 44.60 vs. median = 60.94 pg/mL, IQR = 38.25 to 77.95, respectively; p = 0.04). In logistic regression analysis, subjective "he or she is spinning" (p = 0.030, odds ratio [OR] = 1.63, 95% confidence interval [CI] = 1.38 to 2.49), systolic blood pressure (sBP; p = 0.045, OR = 1.044, 95% CI = 1.021 to 1.080), and serum S100B level (p = 0.042, OR = 1.22, 95% CI = 1.018 to 1.445) were found to be independent predictors of MRI abnormalities. In the ROC analysis, S100B > 30 pg/mL predicted the clinical outcome with 83.9% sensitivity (95% CI = 66.3% to 94.5%) and 51.0% specificity (95% CI = 36.6% to 65.2%). The area under the ROC curve was 0.774 (95% CI = 0.666 to 0.881). To the best of our knowledge this is the first study assessing the utility of serum S100B levels for diagnosis of acute-onset vertigo. Serum S100B levels are associated with the presence of central causes of vertigo on cranial MRI. However, serum S100B levels are not sufficiently sensitive to exclude candidates from cranial MRI.

Evaluation of α-fetoprotein (AFP) in human serum by chemiluminescence enzyme immunoassay with magnetic particles and coated tubes as solid phases

In this work, the monoclonal antibodies (McAbs) to α-fetoprotein (AFP) were immobilized on two different solid phases, i.e., magnetic particles (MP) and coated tubes (CT). Based on this, a MP based chemiluminescence enzyme immunoassay (MP-CLEIA) and a CT based CLEIA (CT-CLEIA) were proposed for the evaluation of AFP in human serum and their analytical merits were studied and compared. By detailed discussion of several performance variants, including the concentration of immobilized McAb, dilution ratio of horseradish peroxidase (HRP) labeled McAb (HRP-McAb), total assay time, substrate volume, chemiluminescent kinetics, and hook effect concentration, the advantages of MP-CLEIA became conspicuously apparent. Moreover, in the presence of MP, the catalytic activity of labeled enzyme was kept to high extent and the stability of immunoreagents was satisfied. Finally, 59 human serum samples were detected by the MP-CLEIA and a good correlation was obtained when comparing the results with that from a commercial electrochemiluminescence immunoassay kit.