Squalene Antibodies with Lipid Antigens

ELISA Development

The previously reported assay for measuring antibodies to SQE used PVDF Multi-Screen-IP plates (Matyas et al., 2000).  They were cumbersome to use, requiring a time-consuming hand washing and orbital-shaker incubation protocol.  Therefore, a new plate that could be washed with an ELISA plate washer was needed to run the assay.  Seven types of plates including Immulon II U-bottom, Immulon II flat bottom, Costar tissue culture U-bottom, Costar tissue culture flat bottom, Immulon 4HBX, and Nunc-Immunoplate F96 Maxisorp were compared with PVDF plates.  The primary goal was to find a plate that yielded high SQE absorbance values and low isopropanol absorbance values (background). 

Costar tissue culture U-bottom plates with PBS-4% FBS blocking buffer met both requirements: High SQE values and low isopropanol values. The Immulon II U-bottom with PBS-4% FBS, Immulon II flat bottom with PBS-0.3% gelatin, Immulon II flat bottom with PBS-4% FBS, and Costar tissue culture U-bottom with PBS-0.3% gelatin plates gave high background values compared to the PVDF plates. The Immulon 4HBX with PBS-4% FBS, Nunc-Immunoplate F96 Maxisorp with gelatin, and Immunoplate F96 Maxisorp with PBS-4% FBS plates yielded moderately high background values. The Immulon II U-bottom, Costar tissue culture flat bottom, and Immulon 4HBX with PBS-0.3% gelatin plates gave mixed background values, with both high and low background values (Tables I and II).

The Costar tissue culture U-bottom plate with PBS-4% FBS blocking buffer and the Costar tissue culture flat bottom plate with PBS-4% FBS blocking buffer had acceptable backgrounds. However, the Costar tissue culture flat bottom plate had lower absorbance values for wells coated with SQE as opposed to the absorbance values returned by the Costar tissue culture U-bottom plate (Tables I and II). Therefore, the Costar tissue culture U-bottom plate with PBS-4% FBS blocking buffer was used in the ELISA.

Monoclonal Antibody Cross-Reactivity with Lipid Antigens

The monoclonal anti-SQE antibodies cross-reacted with lipid antigens other than SQE. The mAbs reacted well with DMPC, DMPG, Lipid A, hexadecane, stearylamine, cholesterol, arachidic acid, behenic acid, palmitic acid, stearic acid, lignoceric acid, and myristic acid. The mAbs also reacted fairly well with pristane, though reacted insignificantly with lauric acid. The mAbs reacted to a lesser extent with SQA than with SQE (Table III, Figs. 1 and 2).

Clones 11 and 14 reacted weakly with DMPC; clones 2, 3, 9, and 10 reacted moderately with DMPC; and clones 1, 4-7, 16, 18, and 19 reacted strongly with DMPC. Clones 1, 2, 5, 7, 9, 10, 16, 18, and 19 reacted weakly with DMPG, and clone 3 reacted moderately with DMPG. Clone 8 reacted weakly with Lipid A, and clones 1-3, 5, 6, 9-11, 14, 16, 18, and 19 reacted strongly with lipid A. Clones 1, 3, 9-11, and 19 reacted weakly with hexadecane, and clones 16 and 18 reacted moderately with hexadecane. Clones 7, 16, 18, and 19 reacted weakly with pristane. Clones 1-6, 9-11, 14, and 16 reacted weakly with stearylamine, and clones 7, 18, and 19 reacted moderately with stearylamine. Clones 1, 2, 8-11, 14, 16, 18, and 19 reacted weakly with cholesterol, and clones 3-7 reacted strongly with cholesterol. Clones 8-11, 14, 16, 18, and 19 reacted strongly with arachidic acid. Clones 4 and 6 reacted weakly with behenic acid; clone 2 reacted moderately with behenic acid; and clones 1, 3, 8-11, 14, 16, 18, and 19 reacted strongly with behenic acid. Clones 4 and 11 reacted weakly with palmitic acid; clones 2, 5, and 6 reacted moderately with palmitic acid; and clones 1, 3, 7-10, 14, 16, 18, and 19 reacted strongly with palmitic acid. Clones 1-3, 5-6, 8, 10, and 11 reacted weakly with lignoceric acid; clones 7, 9, 16, and 19 reacted moderately with lignoceric acid; and clones 14 and 18 reacted strongly with lignoceric acid. Clones 3, 8, 9, and 11 reacted weakly with myristic acid; clones 1, 2, and 10 reacted moderately with myristic acid; and clones 14, 16, 18, and 19 reacted strongly with myristic acid. Only clone 9 reacted weakly with lauric acid, and all clones reacted strongly with stearic acid (Table III).

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Last modified: May 20, 2001