Urine-based Enzyme-Linked Immunosorbent Assay to detect anti-SARS-CoV-2 nucleocapsid protein antibodies

Abstract

An in house urine-based ELISA (Enzyme-linked immunosorbent assay) were developed to detect anti-SARS-CoV-2 nucleocapsid protein (N) IgG antibodies in urine samples, providing a noninvasive diagnostic tool to assess the humoral response against COVID-19. The presence of anti-SARS-CoV-2 N protein antibodies was detected with 94% sensitivity and 100% specificity with urines collected from the second to the 60^th day post symptoms onset with this protocol.

Background

Many different serological assays are available for COVID-19, which preferentially uses plasma or serum as sample. Drawing blood can be unpleasant and difficult to perform in some circumstances, requiring sterile equipment and trained staff. Body fluids such as urine have been suggested as an alternative for the detection of immunoglobulins against several microbial agents (1-10). In this context, we develop an in-house ELISA to detect anti- SARS-CoV-2 nucleocapsid protein antibodies in urine samples. The protocol was based on a pre-validated serum-based ELISA with some modifications. Urine-based and paired serum-based ELISA achieved a very similar qualitative profile. Some of the advantages of the urine-based ELISA include ease of sample collection, biological sample stability and high levels of accuracy. Since most vaccine platforms are based on immune response against SARS-CoV-2 Spike (S) protein, anti-SARS-CoV-2 N ELISA may differentiate in this case individuals previously infected with SARS- CoV-2 from those vaccinated (11,12). So, this test may contribute to epidemiological studies by helping to determine previous exposure to SARS-CoV-2 in a population or individual level.

Materials and Reagents

1. High binding 96-well polystyrene microplate, (Corning, Merck, Germany, catalog number: CLS2592).
2. SARS-CoV-2 Nucleocapsid Recombinant protein (in house production). *¹
   *¹ Note: alternative N antigen from Fapon Biotech Inc, China, catalog number: FP0516.
3. Coating buffer: 0.2 M sodium carbonate/bicarbonate, pH 9.6 (Merck, Germany, catalog number: 222321 and S6014).
4. Wash buffer: 0.1 M sodium phosphate (Merck, Germany, catalog number: 7558-79-4), 0.15 M sodium chloride (Merck, Germany, catalog number): S9625, pH 7.4 containing 1% Tween 20 (Merck, Germany, catalog number: P2287), pH 7.4.
5. Blocking buffer: 1% (w/v) Bovine Serum Albumin (BSA, Merck, Germany, catalog number: A7030) in wash buffer pH 7.4.
6. Conjugated antibody: peroxidase-conjugated anti-human IgG antibody (Sigma- Aldrich, A0170) diluted (1:10,000) in wash buffer.
7. Substrate: TMB substrate (Scienco, Brazil, Reference: One Step).
8. Stop solution: 0.5M sulfuric acid (Merck, Germany, catalog number: 258105).
9. 50 mL reagent reservoir sterile polystyrene (Merck, Germany, catalog number: CLS4870).
10. Tips: 10 µl, 200 µl and 300 µl.

Equipment

1. Vortex Mixer (Thermo Fisher Scientific, USA, catalog number: 88882012).
2. Incubator at 37°C (Fanem, Brazil, catalog number: 502/2-C).
3. ELISA Microplate Washer (Wellwash™ Microplate Washer, 5165000).
4. Microplate Spectrophotometer (Multiskan Go, Thermo Fisher Scientific, USA, catalog number: N10588, 450 nm).
5. Microplate Washer (not compulsory, Wellwash Versa, Thermo Fisher Scientific, USA, catalog number: 5165010).
6. Monochannel 1-10 µl pipette (Thermo Fisher Scientific, USA, catalog number: 4641040N).
7. Monochannel 10-100 µl pipette (Thermo Fisher Scientific, USA, catalog number: 4641070N).
8. Multichannel 30-300 µl pipette (Thermo Fisher Scientific, USA, catalog number: 4661070N).

Software

1. Excel (Microsoft).
2. GraphPad Prism (version 8.0,USA).

Procedure

1. Plates

1. Coating: Distribute 100 µl of coating solution containing the antigen in each well of the plate (400 ng/well). Incubate at 2-8°C overnight;

2. Blocking: Remove the coating solution from the wells by aspiration (ELISA microplate washer) or by decanting (manual). Add 250 µl of blocking solution to each well of the plate. Incubate at room temperature for two hours;

3. Drying: Remove the blocking solution, tap the plate inverted for a few seconds on absorbent paper until dry.

2. ELISA

1. Place all reagents at room temperature and wait for 30 minutes until stabilize;
2. Add 100 μL of each sample (undiluted urine) to the wells of the plate;
3. Cover the wells with plate sealer;
4. Incubate the plate for 60 minutes at 37°C;
5. Remove the plate sealer;
6. Discard the contents of the cavities by suction (washing machine) or by decanting (manual);

7. Add 300ul of Wash Solution to each well (and then remove) and repeat it for 5 times. To ensure plate drying, at the end of the wash, tap the plate inverted for a few seconds on absorbent paper;

8. Add 100 μL of Conjugated antibody into each well;
9. Cover the wells with plate sealer;
10. Incubate the plate 60 minutes at 37°C;
11. Remove the plate sealer;
12. Repeat the Wash step (step g);
13. Add 100 μL of Substrate (TMB) into all wells;
14. Protect the plate from light;
15. Incubate the plate at room temperature until the desired color intensity is reached; *²
    *² As established by the manufacturer, for Scienco TMB we used 30 minutes.
16. Add 100 µl of the Stop Solution into all wells;
17. Measure the absorbance at 450nm.

3. Data analysis

a. Calculation of ELISA cut-off value.

The optical density (OD) of the negative control samples *³ was determined and used to calculate the Cut-off as determined by the formula (13):
*³ We used n=30 negative samples.

𝑐𝑢𝑡 − 𝑜𝑓𝑓 = 𝐴_𝑛𝑒𝑔 + 2 (𝑆𝐷_𝑛𝑒𝑔)

𝐴_𝑛𝑒𝑔: Average of negative samples

𝑆𝐷_𝑛𝑒𝑔: standard deviation of negative samples

Use the cut-off to evaluate the diagnostic performance by estimation of sensitivity, specificity, area under the curve at GraphPad Prism (Analyses>Column analyses>Roc Curve).

b. Index calculation: calculate the index (I) of sample absorbance (Abs) over the value of cut-off, according to the equation:

The results can be classified as follows:

I < 0.8: negative

0.8 ≤ I < 1.1: borderline (indeterminate)

I ≥ 1.1: positive

Use conditional formatting in Excel to classify the results.

In the case of an indeterminate result, the sample must be reanalysed. Samples that repeatedly obtain indeterminate results should be retested using an alternative method. If results remain indeterminate, a new sample should be collected in one week.

Acknowledgments

We acknowledge the help of all students, nurses, and clinicians involved in the collection and organization of the samples. We also thank Hospital das Clínicas/UFMG and Hospital Santa Helena for allowing the collection of samples. Last, we thank CT-Vacinas/UFMG for technical support. The study was supported with the following grants: Secretaria de Educação Superior do Ministério da Educação (SESU/MEC) - Grant 04/2020 - Enfrentamento da COVID-19; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Grant APQ-408675/2018-7; Brazilian Ministry of Science, Technology and Innovation (MCTI) - Rede Virus thought its many iniciatives ; Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CNPq and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) - fellowships/scholarships.

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