NEW KITS FOR THE SEROLOGICAL DIAGNOSIS OF SARS-CoV-2 INFECTION - - PowerPoint PPT Presentation

NEW KITS FOR THE SEROLOGICAL DIAGNOSIS OF SARS-CoV-2 INFECTION

Founded in 1980 as a spin-off of Sclavo Diagnostici, and part of the Sienese district of life sciences, one of the most important in the world in the field of immunology and vaccine development, DIESSE has a long tradition on the development of immunoassays. Since 1990 DIESSE has being manufacturing ELISA kits for the serology of infectious diseases, producing in- house all the antigens (native and recombinant) and the raw materials (monoclonal and polyclonal antibodies) needed for developing the kits.

THE PROJECT COMES FROM A COLLABORATION BETWEEN DIESSE AND THE NATIONAL INSTITUTE FOR INFECTIOUS DISEASES LAZZARO SPALLANZANI IN ROMA WHERE THE NEW CORONAVIRUS RESPONSIBLE FOR COVID 19 WAS ISOLATED FOR THE FIRST TIME IN ITALY.

THE VIRUS HAS BEEN CULTIVATED IN OUR HIGH BIOLOGICAL CONTAINMENT BSL3 LABORATORIES USING A PATENTED PROPRIETARY PROTOCOL THANKS TO THE COLLABORATION WITH A CONSULTANT COMING FROM SIENA’S VACCINE DISTRICT AND EXPERT IN THE DEVELOPMENT OF VIRAL VACCINES

PATENTED TECHNOLOGY FOR VIRUS PRODUCTION

Electrophoresis

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

SARS CoV 2 batch 001 (left) and batch 002 (right) Gel a gradiente 4-15% - Silver Stain SARS CoV 2 batch 003 inreducing conditions (left) and NON- reducing (right) Gel 10% - Silver Stain

SARS-CoV-2 Proteins

SARS-CoV-2 Spike Glycoprotein: 1281 aa protein (≈ 140 kDa) Nucleocapsid protein: 419 aa (≈ 46 kDa) Membran Protein : 222 aa (≈ 24 kDa) Envelope Protein: 75 aa (≈ 8 kDa)

https://www.ncbi.nlm.nih.gov/protein

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

Western Blotting IgG

Western Blotting - SARS CoV 2 bacth 001, detected anti-human IgG. Sieri 1-5 positivi Spallanzani, 6-11 negativi Spallanzani; Sieri Abbaltis cross-reactives with Sars Cov; Sieri CD: negative generic sera pre-pandemia

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

Western Blotting IgG negative

Western Blotting – SARS-CoV-2 batch 001, detected with anti-human IgG. Samples: generic pre-pandemia negative sera

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

Western Blotting IgG

non reducing conditions (conformational epitopes)

Western Blotting – SARS-CoV-2 batch 003, detected with anti-human IgG. Samples Ab: Abbaltis cross-reactive with SARS CoV; Samples 194 – 165 generic pre-pandemia negative sera

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

Western Blotting IgA

Western Blotting – SARS-CoV-2 batch 001, detected with anti-human IgA. Sieri: Sp 1 Spallanzani positive, Sp 9 Spallanzani negative; Sieri Ab: Abbaltis cross- reactives for SARS CoV; 187 – 170: generic pre-pandemia negative sera

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

Western Blotting IgM

Western Blotting – SARS-CoV-2 batch 001, detected with anti-human IgM. Sieri: Sp 1 Spallanzani positivo, Sp 7-10 Spallanzani negativi; Sieri Ab: Abbaltis cross reattivi per SARS CoV; 246 and 256 generic pre- pandemia negative sera

S A R S C O V 2 – A N T I G E N C H A R A C T E R I Z A T I O N

THE NATIVE ANTIGEN IS USED TO SENSITIZE THE MICROTITER WELLS OF ELISA PLATES THE SPECIFICITY FOR THE DETECTION OF IgG, IgM AND IgA IS GIVEN BY THE CONJUGATE USED IN THE VARIOUS KITS IN THE IgM KIT A SORBENT IS ADDED IN THE SAMPLE DILUENT TO ELIMINATE RF INTERFERENCE

RATIONALE OF THE CHOICE OF THE NATIVE ANTIGEN?

FOR TWO CLEAR REASONS:

• 1. SARS-CoV-2 IS A "NEW" VIRUS, WITH EXTENSIVE CROSS-REACTIONS WITH OTHER

CORONAVIRUSES, FOR WHICH THE DEVELOPMENT OF RECOMBINANT PROTEINS “IN A HURRY” DOES NOT ENSURE ADEQUATE LEVELS OF SPECIFICITY AS THE MOST SUITABLE EPITOPES COULD HAVE NOT BEEN IDENTIFIED. SEE FOR EXAMPLE THE SPECIFICITY DATA OF THE EUROIMMUN IFU

RATIONALE OF THE CHOICE OF THE NATIVE ANTIGEN?

FOR TWO CLEAR REASONS:

• 2. THE METHOD USED AS A REFERENCE AT THE INMI SPALLANZANI IS THE

IMMUNOFLUORESCENCE (IFA) THAT USES VERO CELLS INFECTED WITH THE VIRUS (THEREFORE NATIVE ANTIGEN) AS A SUBSTRATE ON THE SLIDE. WE HAVE DEVELOPED OUR KITS WITH THE PURPOSE OF PROVIDING RESULTS THAT ARE COMPARABLE WITH THE IFA.

IgG IgA IgM

RATIONALE OF THE CHOICE OF THE NATIVE ANTIGEN?

ANTIBODIES RECOGNIZE PROTEINS IN THEIR NATIVE STATE (FOLDING), WHILE THE RECOMBINANT PROTEINS (ESPECIALLY IF OBTAINED) IN Escherichia coli ARE LINEAR.

• SAME DILUTION OF THE SAMPLES FOR

THE 3 KITS (1:101)

• SAME INCUBATION TIME

(1 ORA + 1 ORA + 15 MIN)

• INCUBATION AT ROOM TEMPERATURE
• LIQUID, READY-TO-USE REAGENTS
• COMMON REAGENTS

INTERCHANGEABLE BETWEEN KITS

MAIN FEATURES OF THE NEW KITS

Performance Kit SARS-CoV-2 IgA

S E N S I T I V I T Y A N D S P E C I F I C I T Y

• Total

• 150

• Total

• 150

Sensitivity 94% CI 89-98% Specificity 96% CI 87-96% CONCORDANCE 96%

202 patients from the the virology laboratory of the L. Spallanzani Institute characterized by PCR and IFA were analyzed

kit SARS-CoV2 IgA IFA IgA

+

• Totale

+

64 5 69

• 4

129 133 Total 68 134 202

DIAGNOSTIC MEANING IgA

THE SARS-CoV-2 ENTERS THE HUMAN BODY FROM THE HIGHER AIRWAYS (NOSE AND OROPHARINGE) TRANSPORTED BY AEROSOL DROPLETS EMITTED BY INFECTED PEOPLE.

DIAGNOSTIC MEANING IgA

IgA IS THE MOST PRESENT IMMUNOGLOBULIN CLASS IN MUCOSAL SECRETIONS, INCLUDING RESPIRATORY ONES. THE SURFACE OF THE MUCOSAE IN THE HUMAN BODY IS 400 SQUARE METERS. IgA ARE THE SECOND CALSS OF IMMUNOGLOBULINS IN SERUM AFTER IgG. THE HUMAN BODY SPENDS A SIGNIFICANT QUANTITY OF ENERGY FOR THE SYNTHESIS OF THE IgA WHOSE FUNCTION IS TO CREATE A FIRST LINE OF DEFENSE FROM THE PENETRATION OF MICROORGANISMS AT THE LEVEL OF THE MUCOSAE.

Performance Kit SARS-CoV-2 IgG

• Total

• 150

• Total

• 150

Sensitivity 94% CI 86-98% Specificity 93% CI 87-96% CONCORDANCE 93%

209 patients from the virology laboratory of the L. Spallanzani Institute characterized by PCR and IFA were analyzed

kit SARS-CoV2 IgG IFA IgG

+

• Total

+

67 10 77

• 4

128 132 Total 71 138 209

S E N S I T I V I T Y A N D S P E C I F I C I T Y

Performance Kit SARS-CoV-2 IgM

• Total

• 150

• Total

• 150

Sensitivity 88% CI 63-84% Specificity 83% CI 87-97% CONCORDANCE 85%

135 patients from the virology laboratory of the L. Spallanzani Institute characterized by PCR and IFA were analyzed

kit SARS-CoV2 IgM IFA IgM

+

• Total

+

57 8 65

• 12

58 88 Total 69 76 135

S E N S I T I V I T Y A N D S P E C I F I C I T Y

Seroconversions IgA IgG

S E R O C O N V E R S I O N A S S E S S M E N T S

ID samples Acceptance date Identification patient PCR IFA IgA lotto 001/s IgG lotto 001/s IgA IgG IgM media index media index 5 31/01/2020 LX RILEVATO 20 80 20 0,203 0,6 0,747 1,4 6 04/02/2020 LX RILEVATO 40 320 40 0,889 2,5 1,437 2,6 7 07/02/2020 LX RILEVATO 640 320 80 1,438 4,1

• ver
• ver

8 13/02/2020 LX RILEVATO 320 2560 160 0,337 1,2 3,605 6,6 9 02/03/2020 LX RILEVATO 640 1280 320 0,755 2,2

• ver
• ver

10 31/01/2020 HY RILEVATO neg neg neg 0,212 0,6 0,173 0,3 11 04/02/2020 HY RILEVATO 20 1280 20 0,306 0,9 0,212 0,4 12 10/02/2020 HY RILEVATO 320 2560 80 0,938 2,7 0,907 1,6 13 14/02/2020 HY RILEVATO 2560 1280 80 0,979 2,8 1,766 3,2 14 02/03/2020 HY RILEVATO 320 640 80 1,320 3,8 1,967 3,6

Cross-reactivity with other Coronaviruses

ID samples acc_dat PCR IFA IgA lotto 002/s IgG lotto 002/s IgM lotto 002/s IgG media index media index media index 1n cov 13/02/2020 HKU neg 0,607 1,6 0,269 0,5 0,264 0,8 2n cov 14/02/2020 HKU neg 0,595 1,6 0,322 0,6 0,287 0,8 3n cov 12/02/2020 HKU neg 0,529 1,4 0,269 0,5 0,248 0,7 4n cov 12/02/2020 HKU neg 0,106 0,3 0,131 0,2 0,393 1,1 5n cov 14/02/2020 NL63 neg 0,208 0,5 0,330 0,6 0,201 0,6 6n cov 29/01/2020 HKU neg 0,117 0,3 0,191 0,3 0,284 0,8 7n cov 25/01/2020 229E neg 0,155 0,4 0,147 0,3 0,406 1,2 8n cov 31/01/2020 HKU neg 0,196 0,5 0,290 0,5 0,188 0,5 9n cov 01/02/2020 HKU neg 0,226 0,6 0,328 0,6 0,179 0,5 10n cov 31/01/2020 NL63 neg 0,365 1,0 0,317 0,6 0,394 1,1 11n cov 31/01/2020 NL63 neg 0,198 0,5 0,213 0,4 0,162 0,5 12n cov 14/02/2020 HKU 160 0,195 0,5 0,160 0,3 0,226 0,6 13n cov 04/02/2020 HKU 320 0,301 0,8 0,229 0,4 1,071 3,1 14n cov 14/01/2020 HKU neg 0,229 0,6 0,250 0,5 0,150 0,4 15n cov 11/02/2020 HKU neg 0,191 0,5 0,170 0,3 0,163 0,5 16n cov 04/02/2020 NL63 neg 0,163 0,4 0,174 0,3 0,735 2,1 17n cov 04/02/2020 NL63 neg 0,168 0,4 0,172 0,3 0,575 1,6 18n cov 06/02/2020 HKU neg 0,110 0,3 0,197 0,4 0,191 0,5 19n cov 10/02/2020 HKU neg 0,112 0,3 0,182 0,3 0,190 0,5 20n cov 04/02/2020 HKU neg 0,151 0,4 0,554 1,0 0,237 0,7

S E R O C O N V E R S I O N A S S E S S M E N T S

Cross-reactivity with SARS CoV samples

Sera AbE IgA lotto 001/s IgG lotto 001/s IgM lotto 001/s media index media index media index 12575 0,201 0,4 0,941 2,0 0,270 0,6 16692 0,388 0,9 0,491 1,0 0,254 0,6 17150 0,705 1,6 0,492 1,0 0,258 0,6 17466 0,756 1,7 0,486 1,0 0,249 0,6 17515 0,204 0,5 0,531 1,1 1,109 2,5 17653 0,239 0,5 0,542 1,1 0,372 0,8 18116 0,311 0,7 1,108 2,3 1,070 2,4 18142 0,634 1,4 2,382 5,0 0,215 0,5 15379 0,049 0,1 0,300 0,6 0,356 0,8 15707 0,441 1,0 0,377 0,8 0,203 0,5 15719 0,244 0,5 0,266 0,6 0,323 0,7 17118 0,530 1,2 0,255 0,5 0,540 1,2 17295 0,656 1,5 0,178 0,4 0,897 2,0 17315 0,235 0,5 0,159 0,3 0,610 1,4 17754 0,204 0,5 0,229 0,5 0,317 0,7 17978 0,259 0,6 0,209 0,4 0,222 0,5 18062 0,191 0,4 0,331 0,7 0,178 0,4 18147 0,423 0,9 0,293 0,6 0,387 0,9

S E R O C O N V E R S I O N A S S E S S M E N T S

THE ANALYTICAL PROTOCOL IS COMMON TO THE THREE KITS, ALLOWING EASY AUTOMATION ON THE MICRO-PLATE PROCESSORS DIESSE HAS SELECTED THE SKYLAB 752 HIGH-PRODUCTIVITY INSTRUMENT FOR THE TENDER OF THE TOSCANA REGION THIS MACHINE CAN PROCESS UP TO 7 PLATES SIMULTANEOUSLY

ITALYONLY

THE EC CERTIFICATE OF CONFORMITY WILL BE OFFICIALLY OBTAINED BY THE 24th APRIL 2020