A method of determining analyte concentration
A technology for analysis and concentration, which is applied in the analysis of materials, material analysis by optical means, and measurement devices, etc. It can solve the problems of inapplicable flow cell inspection methods, inability to precipitate analytes, and inability to measure the total concentration of analytes, etc. question
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Embodiment 1
[0070] Measurement of small amounts of anti-HSA antibodies in buffer in the presence of high concentrations of HSA
[0071] Human serum albumin (Sigma-Aldrich, Missouri, USA) was diluted to 50 μg / ml in 10 mM acetate pH 5.0 and immobilized on Flow cell 3 (Fc3) to about 9kRU in T100.
[0072] 100 μl samples containing 110 μg / ml HSA (Sigma-Aldrich) and different concentrations of anti-HSA (internal reagent) were prepared. As a control, samples not containing HSA were used.
[0073] Samples were acidified with 50 μl of 0.2M glycine pH 2.8.
[0074] Each sample is then injected into T100, using prototype injection Mix the sample with HBS-EP+ (0.1M HEPES, 0.15M NaCl, 3mM EDTA and 0.05% v / v Surfactant P20, pH 7.4-Biacore AB) at a ratio of 15:85, and in sequence Neutralize samples before passing through all flow cells of the IFC. Detection is carried out in Fc3 (previous experiments have shown that the mixing of acidic sample and neutralizing solution is optimal in the flow cel...
Embodiment 2
[0079] Anti-2-microglobulin was measured in buffer in the presence or absence of β-2-microglobulin by standard amine coupling (amine coupling kit, Biacore AB) with 10 mM acetate pH 4.5 (Biacore AB). 20 μg / ml β-2-microglobulin (β2μ) (internal reagent) in Flow cell 3 (Fc3) in T100 to about 1.7 kRU.
[0080] Prepare 100 μl buffer samples containing 100 μg / ml anti-β-2-microglobulin (anti-β2μ) (internal reagent) and different concentrations of β-2-microglobulin in HBS-EP+ (Biacore AB) ( β2μ) (internal reagent). Buffer samples were then acidified with 50 μl of 0.2M glycine, pH 2.8. Control samples were not acidified.
[0081] Each sample is then injected into T100, sample was mixed with HBS-EP+ (Biacore AB) at a ratio of 15:85 using prototype injection and neutralized before passing sequentially through all flow cells of the IFC. Regeneration was performed with glycine pH 1.5 (Biacore AB). The results are shown in Table II below.
[0082] Table II
[0083] β2μ(μg / ...
Embodiment 3
[0086] Measurement of anti-2-microglobulin in human plasma in the presence or absence of beta-2-microglobulin
[0087] 20 μg / ml β-2-microglobulin (β2μ) (internal reagent) in 10 mM acetate pH 4.5 (Biacore AB) was immobilized using standard amine coupling (Amine Coupling Kit, Biacore AB) on Flow cell 3 (Fc3) in T100 to about 1.7 kRU.
[0088] 100 μl human plasma samples were prepared containing either (i) 50 μg / ml anti-β2μ, or (ii) 50 μg / ml anti-β2μ and 5 μg / ml β2μ, and 1% v / v surfactant P20 (Biacore AB). (Human plasma samples typically contain about 1 μg / ml of β2μ). Plasma samples were then acidified (to give pH 2-3) with 15 μl of 1M HCl.
[0089] Each sample is then injected into T100, sample with 0.1MK using prototype injection 2 HPO 4 , pH 9.0, and 1% v / v surfactant P20 (Biacore AB) were mixed in a ratio of 15:85 and the samples were neutralized before passing through all the flow cells of the IFC in sequence. Regeneration was performed with glycine pH 1.5 (Biacore A...
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