10  Antigens

The following list of purified antigens are used in the multiplex to produce standard curves which are present on each plate of samples tested. These antigens were used in cross-reactivity tests to establish which pairs of antibodies were specific for binding to the target analytes.

This protocol describes how to prepare and aliquot antigens in advance of standard curve preparation, and for future use.

Manufacturer Part No. Pack size Product name
bio-techne 923-AN-025 25 µg Recombinant Human Angiopoietin-1 Protein
bio-techne 623-AN-025 25 µg Recombinant Human Angiopoietin-2 Protein
bio-techne 2200-SE-050 50 µg Recombinant Human Azurocidin/CAP37/HBP Protein
bio-techne 2599-CH-050 50 µg Recombinant Human Chitinase 3-like 1 Protein
bio-techne 206-IL-010 10 µg Recombinant Human IL-6 Protein
bio-techne 208-IL-010 10 µg Recombinant Human IL-8/CXCL8 Protein
bio-techne 1064-IL-010 10 µg Recombinant Human IL-10 (aa 19-178) Protein
bio-techne 266-IP-010 10 µg Recombinant Human CXCL10/IP-10 Protein
Insight Biotechnology TP307418 20 µg MX1 (NM_002462) Human Recombinant Protein
bio-techne 636-R1-025 25 µg Recombinant Human sTNFR1/TNFRSF1A Protein
bio-techne 375-TL-010 10 µg Recombinant Human TRAIL/TNFSF10 Protein
bio-techne 1278-TR-050 50 µg Recombinant Human TREM-1 Fc Chimera Protein

NOTE : Expiry dates are three months from re-hydration, according to the manufacturer.

Antigens tested but later removed from the final multiplex are listed below. These are not needed in order to run the multiplex and are therefore not included in the Shopping List document. They are provided here for completeness.

Manufacturer Part No. Pack size PRODUCT DESCRIPTION
bio-techne 1707-CR-200 200 µg Recombinant Human C-Reactive Protein/CRP Protein
bio-techne 321-FL-050 50 µg Recombinant Human VEGFR1/Flt-1 Fc Chimera Protein
bio-techne 9607-PN-050 50 µg Recombinant Human Procalcitonin Protein CF

10.1 Protocol

⌚ Timing: ~3 hours, but can be split into shorter periods if needed (see Tip 2).

This protocol takes place at ambient temperature. All incubations are at ambient temperature.

Tip: Keep the proteins as cold as possible, for as long as possible, to avoid degradation. Work with tubes on ice or in chilled racks. Likewise, thaw proteins that are in solution on ice.

Tip 2: If time is limited, prepare a smaller number of proteins at one time, completing the full process of rehydration, aliquotting and storage for these, and return another time to complete the next batch.

  1. Prepare filter-sterlised PBS as per Buffers and Reagents protocol.

  2. Label protein lo-bind tubes with antigen name, catalogue number, concentration, volume and expiry date. The approximate number of tubes needed for each antigen is shown in column 6 in Table 1 below.

  3. Rehydrate lyophylised antigens using the volumes of PBS listed in column 2 of Table 1.

    NOTE: MxA comes in solution and does not need rehydrating. See manufacturer’s data sheet for concentration.

  4. Leave vials to sit for 60 seconds to rehydrate, then vortex gently and spin down.

  5. Aliquot into the volumes and number of tubes indicated in columns 5 and 6 of Table 1.

  6. Store aliquots at -70 or below immediately.

Table 1. Recombinant antigen stock aliquotting.

  1. Antigen
  1. PBS to add to vial to make stock (ul)
  1. Stock concentration (ug/ml)
  1. Volume needed each time (ul)
  1. Aliquot size (ul)
  1. Number of aliquots
Ang1 250 100 6 27 9
Ang2 250 100 12 20 9
Azu 250 200 12 26 9
CHI3L1 250 200 6 27 9
IL-6 100 100 4 10 9
IL-8 100 100 4 10 9
IL-10 100 100 4 10 9
IP-10 100 100 4 10 9
MxA none (already liquid) See data sheet Depends on stock conc. e.g. 13 e.g. 4
sTNFR1 250 100 4 10 9
TRAIL 100 100 4 10 9
TREM1 500 100 4 54 9