Overjanje in primerjava seroloških metod za merjenje koncentracije specifičnih protiteles proti SARS-CoV-2

Colja, Sara (author), Jerin, Aleš (mentor), Skitek, Milan (comentor)

Leta 2020 je Svetovna zdravstvena organizacija razglasila pandemijo bolezni COVID 19 in pozvala države k sprejemanju ukrepov za preprečevanje širjenja virusa SARS CoV 2. Hitra in učinkovita diagnostika je bila na začetku pandemije ključnega pomena pri preprečevanju širjenja bolezni, zato so se na tržišču pojavili številni testi. Kot zlati standard določanja virusa SARS-CoV¬ 2 je bila izbrana metoda RT-PCR, ki daje najbolj zanesljive rezultate, saj omogoča detekcijo nukleinskih kislin. Žal pa so slabosti metode čas izvedbe, cena testa in zahteva po izobraženem kadru. To je vodilo do razvoja hitrih antigenskih testov, ki omogočijo detekcijo virusa SARS CoV 2 v zgolj petnajstih minutah, so preprosti za uporabo in jih lahko izvaja vsak posameznik. Kljub temu pa detekcija antigena ni dovolj za spremljanje pandemije. Pomembni so tudi serološki testi, ki omogočajo detekcijo protiteles. Namen magistrske naloge je bil overjanje nove serološke metode za kvantifikacijo protiteles proti virusu SARS CoV 2 na Kliničnem inštitutu za klinično kemijo in biokemijo Univerzitetnega kliničnega centra Ljubljana. Uporabili smo vzorce 63 anonimnih polnoletnih preiskovancev, izmed katerih je bilo 36 RT PCR pozitivnih in 27 RT PCR negativnih. Metoda, ki smo jo overili za nadaljnjo uporabo v klinični praksi, je Elecsys® Anti SARS CoV 2 S proizvajalca Roche Diagnostics, ki omogoča merjenje celokupne koncentracije protiteles proti proteinu S virusa SARS CoV 2. Za overjanje metode smo izračunali natančnost in pravilnost metode, ki sta ustrezali zahtevam laboratorija. Izračunali smo tudi diagnostično specifičnost, katere vrednost je bila 96,30 % (95-% CI: 81,03–99,91 %), in diagnostično občutljivost metode, katere vrednost je bila 97,22 % (95-% CI: 85,47–99,93 %). Metodo smo primerjali z metodama COVID 19 S1 RBD protein Human IgM ELISA Kit in COVID 19 S1 RBD protein Human IgG ELISA Kit proizvajalca Ray Bio. Diagnostični specifičnosti obeh metod sta imeli vrednost 100 % (95-% CI: 87,23–100 %). Diagnostična občutljivost COVID 19 S1 RBD protein Human IgM ELISA Kit je imela vrednost 8,33 % (95-% CI: 1,75–22,47 %), za metodo COVID 19 S1 RBD protein Human IgG ELISA kit pa 33,33 % (95-% CI: 18,56–50,97 %). Vse tri metode smo na koncu primerjali še s pomočjo Cohenovega koeficienta kappa, s katerim smo opazili komaj zaznavno ujemanje med metodami. Če povzamemo, je metoda Elecsys® Anti SARS CoV 2 S optimalna izbira za kvantifikacijo protiteles v klinični praksi, medtem ko metodi proizvajalca Ray Bio ne dajeta dovolj zanesljivih rezultatov.

• Language: Slovenian
• Keywords: SARS CoV 2, imunski test, protitelesa, primerjava metod, overjanje, COVID-19, ELISA, ECLIA
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-135420
• Publication date in RUL: 12.03.2022

Secondary language

• Language: English
• Title: Verification and comparison of serological assays for quantification of specific anti-SARS-CoV-2 antibodies

Abstract

In the beginning of 2020, the World Health Organization announced a pandemic of COVID 19. They called on states to take on measures to prevent the spread of SARS CoV 2 virus. A rapid and effective diagnostic was a key for having a virus spread under control, so many new tests became available on the market. The RT PCR method was chosen as a golden standard for diagnosis of COVID 19. It gives the most reliable results, because it is based on detection of nucleic acid of the virus. This method also has a few cons, such as performance time, price of the test and a need for educated personnel. That has led to the development of rapid antigen tests that can detect the SARS CoV 2 virus in only fifteen minutes, are easy to use and can be performed by anyone. However, antigen detection is not sufficient for a pandemic monitoring. Serological tests are also of a big importance. The purpose of the master’s thesis was a verification of a new serological method for quantification of antibodies against SARS CoV 2 at the Institute of Clinical Chemistry and Biochemistry University Medical Centre Ljubljana. We used 65 anonymous samples, collected form adults – 36 of them were RT-PCR positive and 27 RT PCR were negative. The method chosen for the implementation in clinical practice was Elecsys® Anti SARS CoV 2 S, manufactured by Roche Diagnostics, which allowed us to measure a total concentration of antibodies against S protein of SARS CoV 2. For the verification, we calculated precision and accuracy of this method, and both of them met the laboratory requirements. The next step was to calculate the diagnostic specificity that was 96.30% (95% CI: 81.03–99.91%) and sensitivity that was 97.22% (95% CI: 85.47–99.93%). This method was then compared to COVID 19 S1 RBD protein Human IgM ELISA Kit and COVID 19 S1 RBD protein Human IgG ELISA Kit, manufactured by Ray Bio that were developed for research use only. Diagnostic specificity of both methods was 100% (95% CI: 87.23–100%). Diagnostic sensitivity for COVID 19 S1 RBD protein Human IgM ELISA Kit was only 8.33% (95% CI: 1.75–22.47%), and 33.33% (95% CI: 18.56–50.97%) for COVID 19 S1 RBD protein Human IgG ELISA Kit. The last step was to compare all three methods with the Cohen’s coefficient Kappa, which showed only slight agreement between verified methods. In conclusion, the Elecsys® Anti SARS CoV 2 S method is the optimal choice for quantification of antibodies against SARS CoV 2 in clinical practice, while products manufactured by Ray Bio are not reliable enough for clinical use.

• Keywords: SARS-CoV-2, immunoassay, antibodies, method comparison, verification, COVID-19, ELISA, ECLIA