scholarly journals Quantitative measurement of anti-SARS-CoV-2 antibodies: Analytical and clinical evaluation

2021 ◽  
Author(s):  
Victoria Higgins ◽  
Anselmo Fabros ◽  
Vathany Kulasingam
Keyword(s):  
Antibody Response ◽  
Clinical Evaluation ◽  
Predictive Value ◽  
Cross Reactivity ◽  
Analytical Sensitivity ◽  
Past Infection ◽  
Clinical Sensitivity ◽  
Limit Of Quantitation ◽  
Sensitivity Specificity ◽  
Roche Diagnostics

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of Coronavirus Disease 2019 (COVID-19). While molecular-based testing is used to diagnose COVID-19, serologic testing of antibodies specific to SARS-CoV-2 is used to detect past infection. While most serologic assays are qualitative, a quantitative serologic assay was recently developed that measures antibodies against the S protein, the target of vaccines. Quantitative antibody determination may help determine antibody titer, facilitate longitudinal monitoring of the antibody response, including antibody response to vaccines. We evaluated the quantitative Roche Elecsys® Anti-SARS-CoV-2 S assay. Specimens from 167 PCR-positive patients and 103 control specimens were analyzed using the Elecsys® Anti-SARS-CoV-2 S assay on the cobas e411 (Roche Diagnostics). Analytical evaluation included assessing linearity, imprecision, and analytical sensitivity. Clinical evaluation included assessing clinical sensitivity, specificity, cross-reactivity, positive predictive value (PPV), negative predictive value (NPV), and serial sampling from the same patient. The Elecsys® Anti-SARS-CoV-2 S assay exhibited highest sensitivity of 84.0% 15-30 days post-PCR positivity, no cross-reactivity, specificity and PPV of 100%, and NPV between 98.3%-99.8% ≥14 days post-PCR positivity, depending on the seroprevalence estimate. Imprecision was <2% at 9.06 U/mL across 6 days, the negative QC was consistently negative (<0.40 U/mL), the manufacturer’s claimed limit of quantitation of 0.40 U/mL was verified, and linearity across the analytical measuring range was observed, except at the low end (<20 U/mL). Lastly antibody response showed high inter-individual variation in level and time of peak antibody titre and trends over time.

A prospective nationwide observational study of agreement of antigen tests on oral pharyngeal swabs or less invasive testing with RT-qPCR, for detecting SARS-CoV-2 in adults: Protocol description (Preprint)

2021 ◽  
Author(s):  
Uffe Vest Schneider ◽  
Jenny Dahl Knudsen ◽  
Anders Koch ◽  
Nikolai Søren Kirkby ◽  
Jan Gorm Lisby
Keyword(s):  
Confidence Intervals ◽  
Predictive Value ◽  
Large Scale ◽  
Reference Method ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Sampling Location ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Antigen Tests

BACKGROUND The SARS-CoV-2 pandemic has resulted in an unprecedented level of world-wide testing for epidemiologic and diagnostic purposes, and due to the extreme need for tests, the gold standard reverse transcription polymerase chain reaction (RT-qPCR) testing capacity has been unable to meet the overall global testing demand. Consequently, although current literature has shown the sensitivity of rapid antigen tests (RATs) to be inferior to RT-qPCR, RATs have been implemented on a large scale without solid data on performance. OBJECTIVE This study will compare analytical and clinical sensitivities and specificities of 50 lateral flow or laboratory based RATs and three Strand Invasion Based Amplification (SIBA)-rt-PCR tests from 30 manufacturers to RT-qPCR on samples obtained from the deep oropharynx. In addition, the study will compare sensitivities and specificities of the included RATs as well as RT-qPCR on clinical samples obtained from the deep oropharynx, anterior nasal cavity, saliva, deep nasopharynx and expired air to RT-qPCR from deep oropharyngeal samples. METHODS In the prospective part of the study, 200 individuals found SARS-CoV-2 positive and 200 individuals found SARS-CoV-2 negative by routine RT-qPCR testing will be re-tested with each RAT applying RT-qPCR as the reference method. In the retrospective part of the study, 304 deep oropharyngeal cavity swabs divided into four groups based on RT-qPCR Cq levels will be tested by each RAT. RESULTS The results will be reported in several manuscripts with different aims. The first manuscript will report retrospective (analytical sensitivity, overall and stratified into different Cq range groups) and prospective (clinical sensitivity) data for RATs with RT-qPCR results as the reference method. The second manuscript will report results for RAT based on anatomical sampling location. The third manuscript will compare different anatomical sampling locations by RT-qPCR testing. The fourth manuscript will focus on RATs that rely on central laboratory testing. Test from four different manufactures will be compared for analytical performance data on retrospective deep oropharyngeal swab samples. The fifth manuscript will report the results of four RATs applied both as professional use and as self-test. The last manuscript will report the results from two breath tests participating in the study. Comparison of sensitivity and specificity between RATs will be done using McNemar for paired samples and chi-squared test for unpaired samples. Comparison of PPV and NPV between RATs will be done by bootstrap test. 95 % confidence intervals for sensitivity, specificity, positive predictive value and negative predictive value are calculated as bootstrap confidence intervals CONCLUSIONS The study will compare the sensitivities of a large number of RATs for SARS-CoV-2 compared to RT-qPCR and will address whether lateral flow based RATs test differ significantly from laboratory based RATS. The anatomical test location for both RAT and RT-qPCR will be compared. CLINICALTRIAL ClinicalTrials.gov NCT04913116

scholarly journals Development and clinical evaluation of a rapid antibody lateral flow assay for the diagnosis of SARS-CoV-2 infection

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kesheng Li ◽  
Chongxiang Tong ◽  
Xiaoqin Ha ◽  
Chaoning Zeng ◽  
Xia Chen ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
False Negative ◽  
Antibody Test ◽  
Cross Reactivity ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
N Protein ◽  
Test Kit ◽  
Positive Rate ◽  
Rapid Antibody

Abstract Background The novel coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has quickly spread worldwide since its outbreak in December 2019. One of the primary measures for controlling the spread of SARS-CoV-2 infection is an accurate assay for its diagnosis. SARS-CoV-2 real-time PCR kits suffer from some limitations, including false-negative results in the clinic. Therefore, there is an urgent need for the development of a rapid antibody test kit for COVID-19 diagnosis. Methods The nuclear capsid protein (N) and spike protein 1 (S1) fragments of SARS-CoV-2 were expressed in Escherichia coli, and rapid antibody-based tests for the diagnosis of SARS-CoV-2 infection were developed. To evaluate their clinical applications, the serum from COVID-19 patients, suspected COVID-19 patients, recovering COVID-19 patients, patients with general fever or pulmonary infection, doctors and nurses who worked at the fever clinic, and health professionals was analyzed by the rapid antibody test kits. The serum from patients infected with Mycoplasma pneumoniae and patients with respiratory tract infection was further analyzed to test its cross-reactivity with other respiratory pathogens. Results A 47 kDa N protein and 67 kDa S1 fragment of SARS-CoV-2 were successfully expressed, purified, and renatured. The rapid antibody test with recombinant N protein showed higher positive rate than the rapid IgM antibody test with recombinant S1 protein. Clinical evaluation showed that the rapid antibody test kit with recombinant N protein had 88.56 % analytical sensitivity and 97.42 % specificity for COVID-19 patients, 53.48 % positive rate for suspected COVID-19 patients, 57.14 % positive rate for recovering COVID-19 patients, and 0.5−0.8 % cross-reactivity with other respiratory pathogens. The analytical sensitivity of the kit did not significantly differ in COVID-19 patients with different disease courses (p < 0.01). Conclusions The rapid antibody test kit with recombinant N protein has high specificity and analytical sensitivity, and can be used for the diagnosis of SARS-CoV-2 infection combined with RT-PCR.

scholarly journals Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245164
Author(s):  
Yee Ling Lau ◽  
Ilyiana binti Ismail ◽  
Nur Izati binti Mustapa ◽  
Meng Yee Lai ◽  
Tuan Suhaila Tuan Soh ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Cross Reactivity ◽  
Recombinase Polymerase Amplification ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Primary Means ◽  
Amplification Assay ◽  
Recombinase Polymerase Amplification Assay

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

scholarly journals Development and Clinical Evaluation of a Recombinant-Antigen-Based Cytomegalovirus Immunoglobulin M Automated Immunoassay Using the Abbott AxSYM Analyzer

2000 ◽  
Vol 38 (4) ◽  
pp. 1476-1481 ◽  
Author(s):  
G. T. Maine ◽  
R. Stricker ◽  
M. Schuler ◽  
J. Spesard ◽  
S. Brojanac ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Clinical Evaluation ◽  
Measles Virus ◽  
Parvovirus B19 ◽  
Relative Sensitivity ◽  
Cross Reactivity ◽  
Immunoglobulin M ◽  
Cmv Infection ◽  
Varicella Zoster ◽  
Sensitivity Specificity

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

A step forward in identifying the right human chorionic gonadotropin assay for testicular cancer

2020 ◽  
Vol 58 (3) ◽  
pp. 357-360 ◽  
Author(s):  
Simona Ferraro ◽  
Mauro Panteghini
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Residual Disease ◽  
Germ Cell Tumors ◽  
Cross Reactivity ◽  
Analytical Sensitivity ◽  
Early Tumor ◽  
Definition Of ◽  
The Right ◽  
Roche Diagnostics

AbstractClinical practice guidelines for the management of germ cell tumors recommend the measurements of human chorionic gonadotropin (hCG) and/or free hCGβ subunit for earlier diagnosis/recognition of the residual disease, for the prognostic evaluation and for the post-chemotherapy surveillance. However, the marketed hCG assays are validated and approved only for pregnancy purposes, with the sole exception of the Elecsys ‘hCG+β’ assay (Roche Diagnostics), cleared in Europe for oncological application. Theoretically, the hCG assay design for oncological purposes should fulfil the recommendations of the International Society of Oncology and Biomarkers requiring the use of antibodies displaying an equimolar recognition of both intact hCG and hCGβ monomer. Further analytical requirements should also be considered, such as optimal analytical sensitivity to allow an early tumor detection and low cross-reactivity for luteinizing hormone (LH). For the Elecsys assay, the detection limit (0.2 U/L) and the reported cross-reactivity for LH (0.12%) may be considered adequate if compared with the recommended requirements. Another issue is the definition of decision limits for oncologic purposes. After 3 years of clinical experience using the Elecsys assay in the oncology setting, we were able to define limits partitioned by sex and age as follows: males <50 years, 0.3 U/L; males >50 years, 2.3 U/L; female <50 years, 2.1 U/L; female >50 years, 5.6 U/L. There is an urgent need to disseminate appropriate educational information and to boost the clinical use of selective, highly sensitive and precise assays, specifically manufactured for cancer application.

scholarly journals Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak

2020 ◽  
Vol 14 (05) ◽  
pp. 494-501
Author(s):  
Carolina Garciglia Mercado ◽  
Ramon Gaxiola Robles ◽  
Felipe Ascencio ◽  
Jesus Silva-Sanchez ◽  
Maria Teresa Estrada-Garcia ◽  
...  
Keyword(s):  
Predictive Value ◽  
Lamp Assay ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Clinical Sensitivity ◽  
Hospital Facility ◽  
Hospital Outbreak ◽  
Carbapenem Resistant ◽  
Sensitivity Specificity

Introduction: Carbapenem-resistant A. baumannii (CRAB) represents a public health threat increasing worldwide. We assess the suitability of a loop-mediated isothermal amplification (LAMP) method for on-site screening of CRAB in a hospital facility. Methodology: A set of six primers were designed for recognizing eight distinct sequences on six targets: blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, and blaVIM. A LAMP method was developed, optimized and evaluated for the identification of CRAB in thirty-three environmental samples from an outbreak in an Intensive Care Unit (ICU) facility. Results: The sensitivity of the LAMP assay for the detection of A. baumannii was ten-fold higher than the PCR assay (1.0 ng.µL-1). The LAMP assays showed a higher detection rate for CRAB samples and robust diagnosis performance in comparison to a conventional PCR, with clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100% for blaOXA-23-like, blaOXA-51-like and blaVIM. Conclusions: The developed LAMP assays are powerful tools that can be useful in on-site screening of CRAB causing local outbreaks in clinics and hospitals facilities where costs and equipment restraints are imperative.

scholarly journals Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™ - Antigen-detecting point-of-care device for SARS-CoV-2

2021 ◽  
Author(s):  
Lisa Johanna Krüger ◽  
Julian A.F. Klein ◽  
Frank Tobian ◽  
Mary Gaeddert ◽  
Federica Lainati ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Scale Up ◽  
Cross Reactivity ◽  
Ease Of Use ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Clinical Sensitivity ◽  
Lateral Flow Assays

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

scholarly journals Antibody response patterns in COVID-19 patients with different levels of disease severity—Japan

2020 ◽  
Author(s):  
Kazuo Imai ◽  
Yutaro Kitagawa ◽  
Sakiko Tabata ◽  
Katsumi Kubota ◽  
Mayu Nagura-Ikeda ◽  
...  
Keyword(s):  
Antibody Response ◽  
Disease Severity ◽  
Cross Reactivity ◽  
Response Patterns ◽  
Igg Antibodies ◽  
Past Infection ◽  
Infection Pattern ◽  
Human Coronaviruses ◽  
Novel Coronavirus ◽  
Different Levels

AbstractBackgroundWe analyzed antibody response patterns according to level of disease severity in patients with novel coronavirus disease 2019 (COVID-19) in Japan.MethodsWe analyzed 611 serum specimens from 231 patients with COVID-19 (mild, 170; severe, 31; critical, 30). IgM and IgG antibodies against nucleocapsid protein (N) and spike 1 protein (S1) were detected by enzyme-linked immunosorbent assays.FindingsThe peaks of fitting curves for the OD values of IgM and IgG antibodies against N appeared simultaneously, while those against S1 were delayed compared with N. The OD values of IgM against N and IgG against both N and S1 were significantly higher in the severe and critical cases than in the mild cases at 11 days after symptom onset. The seroconversion rates of IgG were higher than those of IgM against both N and S1 during the clinical course based on the optimal cut-off values defined in this study. The seroconversion rates of IgG and IgM against N and S1 were higher in the severe and critical cases than in the mild cases.ConclusionOur findings show that a stronger antibody response occurred in COVID-19 patients with greater disease severity and there were low seroconversion rates of antibodies against N and S1 in the mild cases. The antibody response patterns in our population suggest a second infection pattern, leading us to hypothesize that cross-reactivity occurs between SARS-CoV-2 and past infection with other human coronaviruses.

Comparison of enzyme immunoassay and immunoprecipitation for creatine kinase MB in diagnosis of acute myocardial infarction.

1985 ◽  
Vol 31 (3) ◽  
pp. 470-474 ◽  
Author(s):  
A H Wu ◽  
T G Gornet ◽  
J P Bretaudiere ◽  
P R Panfili
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Creatine Kinase ◽  
Enzyme Immunoassay ◽  
Clinical Performance ◽  
Analytical Sensitivity ◽  
Clinical Sensitivity ◽  
Receiver Operating Characteristic Curves ◽  
Creatine Kinase Mb ◽  
Roche Diagnostics

Abstract We compared the clinical performance of measuring creatine kinase (EC 2.7.3.2) isoenzyme MB by use of an enzyme immunoassay (Enzygnost CK-MB, Behring Diagnostics) with an immunoprecipitation method (Isomune-CK, Roche Diagnostics) for the diagnosis of acute myocardial infarction. Sera from 80 patients admitted to the coronary care unit because of chest pain were examined: 40 who had this diagnosis of myocardial infarction, and 40 in whom it was ruled out. In addition, sera from 40 apparently healthy individuals were examined. The clinical sensitivity and specificity of these methods were evaluated by use of receiver operating characteristic curves. We conclude that for clinical efficiency, this enzyme immunoassay is slightly superior to the immunoprecipitation assay we used, because of its greater analytical sensitivity and precision for measuring the mass of the isoenzyme.

scholarly journals Development of a PTHrP Chemiluminescent Immunoassay to Assess Humoral Hypercalcemia of Malignancy

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1015-A1015
Author(s):  
Susan Louise Ashrafzadeh-Kian ◽  
Joshua Bornhorst ◽  
Alicia Algeciras-Schimnich
Keyword(s):  
Limit Of Detection ◽  
Reference Interval ◽  
Cross Reactivity ◽  
Humoral Hypercalcemia Of Malignancy ◽  
Clinical Sensitivity ◽  
Hypercalcemia Of Malignancy ◽  
Humoral Hypercalcemia ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Acridinium Ester

Abstract Background: Measurement of parathyroid hormone related peptide (PTHrP) is helpful in the diagnosis and clinical management of patients suspected of humoral hypercalcemia of malignancy (HHM). In these patients uncontrolled release of PTHrP by tumor cells is responsible for the hypercalcemia and PTH concentrations are typically suppressed. Objective: Develop a sensitive and specific assay for quantitation of PTHrP in plasma. Method: Calibrators (PTHrP 1-86) and samples (50uL) were incubated with an anti-PTHrP goat polyclonal acridinium ester labeled antibody. Complexes were transferred and incubated in a microplate coated with an anti-PTHrP polyclonal rabbit antibody. After washing, the acridinium ester generated signal, which is directly proportional to the amount of PTHrP in sample, was quantified. Results: In this assay PTHrp was stable for 24 hours ambient, 3 days refrigerated, 34 days frozen and through 3 freeze/thaws. Intra and inter-assay imprecision in EDTA plasma (~0.16-35.0 pmol/L) ranged from 2.2-8.6% and 5-15%, respectively. The limit of detection was 0.04 pmol/L and the limit of quantitation was 0.16 pmol/L (15% CV). The analytical measuring range was 0.39-50.5 pmol/L (slope of 1.07 and r2 of 0.99). Average spike recovery was 98% (range 85-108%). The assay was not affected by hemoglobin of ≤500 mg/dL, triglycerides of ≤2000 mg/dL, or bilirubin of ≤50mg/dL. No hook effect was noted up to 500 pmol/L. PTH (1-84) did not cross-react in the assay. C-terminal PTHrP(107-139), and N-terminal PTHrP(1-36) had no significant cross-reactivity (≤1.1%). Mid-PTHrP(38-94) had 8.3% cross-reactivity. Comparison with an in-house PTHrP assay (n=267) showed an r2 of 0.96, and slope of 2.25 by Passing-Bablok regression fit. The 97.5% reference interval for PTHrP (n=114) was ≤0.7 pmol/L, however a higher concentration (≤4.2 pmol/L) was identified as a more specific clinical cut-off. A retrospective clinical validation study showed that using ≤4.2 pmol/L resulted in a 91% clinical sensitivity and a 98% clinical specificity. Conclusion: We have developed an analytically and clinically sensitive and specific PTHrP immunoassay. A cutoff of ≤4.2 pmol/L is clinically useful in the evaluation of patients suspected of hypercalcemia of malignancy.

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