Materials and Reagents

Casein (Hammarsten grade) (BDH Prolabo, VWR International, catalog number: 440203H )
Lipopolysaccharides from Escherichia coli O111: B4-FITC conjugate (Sigma-Aldrich, catalog number: F3665 )
Polymyxin B sulfate salt (Sigma-Aldrich, catalog number: P4119 )
Proteins that were used for the example below (see Representative data) but which are not necessary for the assay:
1. LBP human recombinant (Sigma-Aldrich, catalog number: SRP6033 )
2. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
3. Myoglobin from equine skeletal muscle (Sigma-Aldrich, catalog number: M0630 )
Salts
1. Na₂HPO₄ anhydrous (Merck KGaA, Merck Millipore, catalog number: 106586 )
2. NaH₂PO₄^.H₂O (Merck KGaA, Merck Millipore, catalog number: 106346 )
3. NaCl (Merck KGaA, Merck Millipore, catalog number: 106404 )
4. EDTA disodium salt 2-hydrate (AppliChem GmbH, PanReac AppliChem, catalog number: 131669 )
Sheep anti-FITC: HRP (Bio-Rad Laboratories, AbD Serotec, catalog number: 640005 )
SIGMAFAST^TM OPD (Sigma-Aldrich, catalog number: P9187 )
Tween^® 20 (Merck KGaA, Merck Millipore, catalog number: 822184 )
0.3 M NaOH (prepared from 50% w/w NaOH solution) (18.94 M) (Sigma-Aldrich, catalog number: 415413 )
7.4% HCl (37% commercial solution diluted 1/5 with distilled water) (Merck KGaA, Merck Millipore, catalog number: 100317 )
Phosphate buffered saline (PBS) containing 0.05% Tween^® 20 (PBS-T)
3 N H₂SO₄ (1.5 M)
Casein hydrolysate solution (see Recipes)
Concentrated PB (see Recipes)
PBS (see Recipes)
PBS-EDTA (see Recipes)

Equipment

96-well microplate (polystyrene with clear flat bottom wells) (Greiner Bio-One GmbH, catalog number: 762071 )
Automatic (mono- and multichannel) pipettes and pipette tips
Calibrated beakers, graduated cylinders and conical flasks
Magnetic stir bars and magnetic stirrer
Manual or automatic microplate washer (e.g., DAS plate washer)
Microcentrifuge tubes (e.g., Eppendorf; Kartell, catalog numbers: 297, for 1.5 ml , and 1298 , for 0.5 ml tubes)
Microplate adhesive sealing films (e.g., AlumaSeal^® II film; Sigma-Aldrich, catalog number: A2350 )
Microplate reader (equipped for measuring absorbance at 492 nm) (e.g., Beckman Coulter, model: Biomek^® plate reader )
Microtitre plate shaker with a small orbit diameter (preferably 1.5 mm) set to 750 rpm (e.g., Stuart, model: SSM5 )
pH meter
Reagent reservoir for multichannel pipettes
Scales
Shaking incubator set to 37 °C and 150 rpm (e.g., Panasonic Corporation, SANYO Electric, model: Orbi-Safe ^TM)

Procedure

Determine the number of wells needed for the assay. The test should be performed at least in duplicate. Following the ELISA worksheet example below, if you test in duplicate 8 serial dilutions of an unknown protein/peptide plus the same dilutions of polymyxin B (positive inhibition control) and a known protein/peptide with no binding ability to LPS (negative inhibition control) you need 52 wells: 3 x 8 x 2 = 48 wells for protein/peptide dilutions, two wells with LPS-FITC (reference control) and two wells without LPS-FITC (negative control).
Add 200 µl of casein hydrolysate solution to the wells of a 96-well microplate. Seal the microplate with the adhesive film and incubate overnight at 4 °C without shaking.

Separately, prepare a 2x solution of LPS O111: B4-FITC conjugate (5 µg/ml) and 2x serial dilutions of the protein/peptide to be tested (starting dilution 80 µg/ml) in PBS-EDTA. Prepare the same dilutions of polymyxin B (positive inhibition control) and a negative inhibition control (e.g., ovalbumin). Eight serial ¼ dilutions are optimal for most proteins. For the ELISA worksheet example prepare:

2x solution of LPS-FITC: 3.5 ml of LPS-FITC at 5 µg/ml in PBS-EDTA.

2x serial dilutions of the proteins/peptides in PBS-EDTA:

Table 1. Preparation of serial dilutions

Dilution (dil) number	Volume and source of proteins/peptides	Volume of PBS-EDTA	Protein concentration
1	3.2 μl from a 5 mg/ml stock	196.8 μl	80 µg/ml
2	50 μl of dil 1	150 μl	20 µg/ml
3	50 μl of dil 2	150 μl	5 µg/ml
4	50 μl of dil 3	150 μl	1.25 µg/ml
5	50 μl of dil 4	150 μl	0.312 µg/ml
6	50 μl of dil 5	150 μl	0.078 µg/ml
7	50 μl of dil 6	150 μl	0.020 µg/ml
8	50 μl of dil 7	150 μl	0.005 µg/ml

Mix 120 µl of LPS-FITC solution (2x) with 120 µl of each dilution (2x) or PBS-EDTA (reference control) in individual microcentrifuge tubes, mix well, and incubate the samples for 1 h at RT.
Aspirate the content of the wells coated with the casein hydrolysate and wash 3 times with 200 µl of PBS (no soak) at RT without shaking.
Immediately, add 100 µl of the preincubated solutions or PBS-EDTA (negative control) to each well in duplicate. Seal the microplate and incubate for 30 min at RT under shaking at 750 rpm.
Wash the plate 5 times with 200 µl of PBS-T (no soak) at RT without shaking.
Add 100 µl of sheep anti-FITC: HRP diluted 1/4,000 in PBS-T. Seal the microplate and incubate for 30 min at RT under shaking at 750 rpm.
Wash the plate as described in step 7.
Prepare the chromogen [o-phenylenediamine dihydrochloride (OPD)] solution following the manufacturer’s instructions.
Add 100 µl of the OPD solution to each well and incubate for 20 min at RT, without shaking and in the dark.
Add 25 µl of 3 N H₂SO₄ to each well to stop the reaction.
Read the absorbance of the wells at 492 nm within 30 min.

The percentage of inhibition for each protein dilution is calculated according to the formula: [(average OD reference control) - (average OD protein dil + LPS-FITC)]/ (average OD reference control) x 100. OD: Optical density.
*Alternatively, incubations in steps 6 and 8 can be performed in an incubator for 1 h at 37 °C without shaking, after sealing the plate with the adhesive film.

Table 2. ELISA worksheet example

	1	2	3	4	5	6	7
A	100 µl LPS-FITC (reference control)	100 µl Protein dil 1 + LPS-FITC		100 µl Polymyxin B dil 1 + LPS-FITC		100 µl Ovalbumin dil 1 + LPS-FITC
B		100 µl Protein dil 2 + LPS-FITC		100 µl Polymyxin B dil 2 + LPS-FITC		100 µl Ovalbumin dil 2 + LPS-FITC
C	100 µl PBS-EDTA (negative control)	100 µl Protein dil 3 + LPS-FITC		100 µl Polymyxin B dil 3 + LPS-FITC		100 µl Ovalbumin dil 3 + LPS-FITC
D		100 µl Protein dil 4 + LPS-FITC		100 µl Pol ymyxin B dil 4 + LPS-FITC		100 µl Ovalbumin dil 4 + LPS-FITC
E		100 µl Protein dil 5 + LPS-FITC		100 µl Polymyx in B dil 5 + LPS-FITC		100 µl Ovalbumin dil 5 + LPS-FITC
F		100 µl Protein dil 6 + LPS-FITC		100 µl Polymyxin B dil 6 + LPS-FITC		100 µl Ovalbumin dil 6 + LPS-FITC
G		100 µl Protein dil 7 + LPS-FITC		100 µl Polymyxin B dil 7 + LPS-FITC		100 µl Ovalbumin dil 7 + LPS-FITC
H		100 µl Protein dil 8 + LPS-FITC		100 µl Polymyxin B dil 8 + LPS-FITC		100 µl Ovalbumin dil 8 + LPS-FITC

Representative data

Figure 1. Example of inhibition curve obtained with several molecules using the reported method to analyze LPS binding. LPS-FITC (0.25 µg/well) was incubated with four-fold dilutions of polymyxin B (circles), LBP human recombinant (squares), bovine serum albumin (BSA) (inverted triangles) and myoglobin from equine skeletal muscle (triangles). It is noteworthy that an excess of protein/peptide might have a “zone effect” in the assay, as occurs with polymyxin B.

Notes

The ideal OD range is 0.6-1.2 for the reference control, which was obtained using LPS-FITC at 0.25 µg/well. As the FITC content may vary between batches this concentration might need to be adjusted before performing the inhibition assay. The extent of labeling of the LPS-FITC used to develop this method was of 7.20 µg FITC/mg LPS.
Binding of a protein to LPS does not imply that it is linked to a biological effect. Further experiments should be done to test the activity in vitro and in vivo.
Depending on several factors (amino acid composition, type of interaction with LPS, number of binding sites, etc.) each protein or peptide needs a different optimal concentration to obtain the maximal inhibition. Hence, several dilutions of the proteins or peptides need to be assayed.
The main advantage of using a competitive ELISA instead of an indirect ELISA is that in the latter, the protein/peptide to be tested is coated to the wells and blocking agents may interfere with LPS binding (e.g., binding LPS per se, causing steric hindrance). Casein hydrolysate is commonly used as a blocking agent in ELISA and also serves as LPS binding agent, enabling its use in the competitive system reported.
Inhibition will occur if the protein or peptide to be tested binds to the same or a close LPS region as the casein hydrolysate. Although casein hydrolysate is composed of several derived peptides and therefore the mechanism of binding to LPS is poorly understood, as polymyxin B inhibits LPS binding to casein, this suggests that the casein hydrolysate binds at least to the lipid A region of LPS.
LPS is incubated in PBS-EDTA in the absence of detergents as both LPS and detergents have an amphipathic nature and may give unexpected results difficult to interpret when incubated together.

Recipes

Casein hydrolysate solution
Note: The preparation is based on the method described by Pearce-Pratt and Roser (2010) with some modifications.
1. Dissolve 2.5 g of casein in 80 ml of 0.3 M NaOH and incubate overnight at 37 °C under shaking at 150 rpm
2. Adjust pH to 8 slowly with 7.4% HCl
  Note: Aggregation of casein fragments occurs transiently while the pH is adjusted. Keep the pH meter electrode out of the solution when you add HCl. Read the pH or add more HCl dropwise always after the aggregates are dissolved.
3. Add 2 ml of concentrated PB (the pH lowers to approximately 7.5)
4. Adjust pH to 7.2 with 7.4% HCl
5. Made up to 200 ml with distilled water and readjust the pH if necessary
Concentrated PB (0.75 M, pH 7.2)
For a 50 ml solution:
Dissolve 4.08 g of Na₂HPO₄ anhydrous (Mw: 141.96) and 1.22 g of NaH₂PO₄^.H₂O (Mw: 137.99) up to 50 ml with distilled water
PBS (150 mM, pH 7.2)
For a 1 L solution:
Dissolve 1.63 g of Na₂HPO₄ anhydrous (Mw: 141.96), 0.49 g of NaH₂PO₄^.H₂O (Mw: 137.99) and 7.89 g of NaCl (Mw: 58.44) up to 1 L with distilled water
PBS (150 mM, pH 7.2)-EDTA (1 mM)
Add EDTA disodium salt 2-hydrate at a concentration of 1 mM in PBS
As EDTA lowers the pH, readjust the pH to 7.2 with NaOH

Acknowledgments

This work was supported by grants AGL2011-30563-C03 (Ministerio de Ciencia e Innovación, Spain) and CN 2012/155 (Xunta de Galicia, Spain). Victoria Martínez-Sernández holds a predoctoral fellowship (Programa de Formación del Profesorado Universitario) from the Spanish Ministerio de Educación, Cultura y Deporte. This protocol was modified from Martínez-Sernández et al. (2014).

References

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