JP2714572B2 - Incubation medium containing lactoferrin for solid-phase immunoassay assays - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a lactoferrin-containing ink medium for use in solid-phase immunoassay assays and the use of this medium. The composition of the incubation medium (“buffer solution”, “incubation / environment”) required for solid-phase immunoassay assays, for example, ELISA, requires non-specific binding of accompanying substances in the sample to the solid phase. Must be prevented. Additives known for this purpose are proteins such as albumin, casein, protein mixtures such as animal serum, hydrolyzed gentin or its derivatives and surfactants. Incubation medium containing such additives,
In particular, measurement errors that occur when the sample is heated cannot be prevented. For example, heating is a good measure to reduce the infectivity of specimens that originate from HIV (human immunodeficiency virus). Accordingly, an object of the present invention is to find an incubation medium that does not cause errors in measured values due to heating of a specimen during use. Surprisingly, it has been found that this objective is achieved by an incubation medium containing lactoferrin. The present invention relates to an incubation medium for solid-phase immunoassay assay, which contains lactoferrin as an agent for preventing non-specific binding of accompanying substances in a sample to a solid phase. The incubation medium referred to in the present invention is a liquid phase of a solid-phase immunoassay assay in which an immunochemical reaction is performed. Lactoferrin is added at a concentration of 0.05-20 g /.
A concentration of 0.15 to 0.25, especially 0.2 g / is preferred. Lactoferrin can be added to an incubation medium or buffer known per se for solid-phase immunoassay assays. An example of these is the West German Patent A
-27 448 36, page 24, which contains, besides buffer substances, 5 g / bovine serum albumin, 5 g / polyoxyethylene (20) sorbitan monolaurate (
^(R) Tweeen 20) in phosphate buffered saline (PBS). Another example is the German patent A-31 15
No. 115, page 10, which is ₂ g in 0.2 mol / NaH ₂ PO ₄ / Na ₂ HPO ₄ buffer (pH 6.5).
/ Bovine serum albumin and 200 ml / normal goat serum. One example of a preferred buffer solution is used in Example 1. Such a medium is conveniently a protein known per se,
It may also preferably contain hydrolysis products of casein and / or gelatin, or hydrolyzed and chemically treated gelatin. The lactoferrin used according to the present invention removes cream from mammalian milk, adjusts the pH of the cream-free milk to 3.5-4.5, removes the resulting protein precipitate and removes the supernatant with an ion exchanger. By extracting and isolating lactoferrin by chromatography, it can be collected from the milk. Lactoferrin is isolated from milk, preferably from milk. In a preferred method for collecting lactoferrin suitable for the purpose of the present invention, fat is removed from the milk, for example by centrifugation. Take out and discard the upper phase of the resulting two phases. The lower phase is adjusted to pH 3.5-4.5 with acid. The resulting precipitate is settled out, for example, by centrifugation and discarded. Adjust the conductivity to 6-7 mS / cm by diluting the supernatant with water. A cation exchanger is then introduced, for example CM-cellulose or SP-Sephadex (TM) which has been swollen and equilibrated with a buffer having a conductivity of pH 4 to 5.5 and preferably of 6 to 7 mS / cm. The suspension is filtered and the residue is washed several times with the same buffer, then suspended therein and packed into a column.
It is preferably eluted at pH 5 with a gradient of 0-1 mol / NaCl. NaC in the range of 500-650 mmol /
The fraction eluted as a homogeneous peak measured at 280 nm in l contains lactoferrin which is particularly suitable for the incubation medium. In SDS-PAGE electrophoresis, the lactoferrin appears as a band with a molecular weight of 80,000 ± 3,000. Anion exchangers such as DEAE- or QAE-Sephadex
Chromatography with (trade name) or with DEAE-cellulose adjusts the supernatant obtained from the above precipitation at pH 3.5-4.5 to pH 6.5-7.5 and conductivity 6-8 mS / cm. And applied to a column packed with an ion exchanger pre-equilibrated with a buffer of pH 6.5-7.5 and a conductivity of 6-8 mS / cm.
Lactoferrin then passes through the column or NaCl
When eluted with a concentration gradient, it is eluted as the first peak. Lactoferrin must be present when incubating the analyte or analyte with the reactants bound to the solid phase. It can be added to the solution containing the analyte or in a solution or buffer that dilutes the analyte. However, this one also
Prior to contacting with the analyte in the dissolved state, it may be contacted with a reactant bound to and / or dried on the solid phase. Suitable reactants that are bound to the solid phase are components of a bioaffinity binding system capable of binding such analytes. In the case of an immunological binding system, these reactants are antigens or antibodies. Solid-phase immunoassay assays
It can be either an antagonistic or a two-sided immunoassay (San Deutsch) assay, or even an indirect assay. In the latter case, the analyte is an antibody and the solid phase reactant is the corresponding antigen, and the amount of antibody bound to the solid phase is measured by a second labeled antibody. The assay system can be a single-stage or a multi-stage assay. Lactoferrin prevents binding of analyte components that do not belong to a particular bioaffinity binding system. In this way,
Lactoferrin avoids incorrect results in the detection and measurement of the analyte. The following examples are illustrative of the present invention and demonstrate that lactoferrin is suitable as a component of an incubation medium. These examples are intended to show that lactoferrin can be introduced and used in various amounts in various incubation media. Example 1 1. Isolation of lactoferrin Twenty milks were centrifuged at 3400 × g for 30 minutes and the milk fat in the supernatant was removed. A small amount of insoluble components was removed as a precipitate and discarded similarly. The pH of the milk from which fat has been removed is adjusted to pH 4.
Adjusted to 0. The resulting protein precipitate is reduced to 340
Removed by centrifugation at 0xg. After adjusting the pH to 5.0 with a sodium hydroxide solution, the supernatant having a conductivity of 12 mS / cm at 20 ° C. is diluted with water to 7 mS / cm, and then the swollen ion exchanger carboxymethylcellulose CM32 ( 200 ml (supplied by Whatmann) and the mixture was stirred at room temperature for 4 hours. The ion exchanger is 50 mmol / sodium acetate buffer (pH
5.0). The ion exchanger was then separated on a suction funnel, suspended in sodium acetate buffer and transferred to a chromatographic column. The column was then washed with 400 ml of the acetate buffer described above. The protein bound to the cation exchanger was eluted using a 1 mol / sodium chloride solution. The eluate was adjusted to pH 5.0 using 1N sodium hydroxide solution and dialyzed to 50 mmol / sodium acetate (pH 5.0). The protein mixture was chromatographed again on 200 ml of carboxymethylcellulose CM32. From 50 mmol / sodium acetate (pH 5) to 50 mmol / sodium acetate (pH 5)
And a 1 mol / sodium chloride linear gradient. The fourth peak of the elution diagram (the solution is 18-20
(having a conductivity of mS / cm) were collected as one fraction, concentrated by ultrafiltration and 0.5 g / P
Gels were run on Sepharose ™ AcA 34 equilibrated with BS. The main ingredient from this gel (the amount is 1.2g
Contained lactoferrin. 2. Comparison of lactoferrin-containing sample dilution buffer with lactoferrin-free sample dilution buffer Enzygnost (trade name) anti-HSV / ELISA from Behringwerke
The kit was used for solid-phase immunoassay. The assay was performed according to the method described in the manual inserted into the pack, and the outline is described below. The sample was diluted with 0.2 g / lactoferrin or the same sample dilution buffer (137 mmol / Na
Cl, 7.247 mmol _{/ Na 2 HPO 4 · 2H 2} O and 2.720 mmol / KH ₂ PO _4, and 2.7 mmol / KCl, 0.4 mmol / MgCl _2, 3 mmol / NaN _3, 10ml / fetal calf serum and 40 ml / Tween ( Diluted with 1:25 instead of the recommended 1:44, and then incubated with the antigen immobilized in the wells of the microassay plate to remove unbound antibody Are removed by washing, the enzyme is bound to the antibody / antigen complex formed using the conjugate solution, excess conjugate solution is removed by washing, and the color is developed via the enzyme using the chromogenic substrate solution. ,
The reaction was then stopped using a stop solution. The specimens thus prepared and colored depending on the content of the virus-specific antibody were evaluated by measuring the absorbance (E) at 405 nm photometrically. A portion of each of the sera of the nine subjects was heated at 56 ° C. for 30 minutes. Other parts were left untreated. In particular, in the case of a heated sample in a dilution buffer to which lactoferrin was not added, it was found that the absorbance measured in the control well was extremely high, but the absorbance in the antigen-coated well was also high. As a result, five cases were false negative results. Control wells have been treated ("coated") with a preparation of non-viral infected cells used for virus harvesting. When nine heated specimens were assayed using a lactoferrin-containing dilution buffer, no false negative results were observed. Unheated specimens were tested using dilution buffer without lactoferrin. The results helped check the results with the heated serum. Example 2 The effect of the addition of lactoferrin was tested in a one-step San Deutschland. In this type of HBsAg assay,
When 7.5 g / lactoferrin was added, all 14 unheated critical samples were definitely found to be negative, whereas without lactoferrin all were false positive measurements . Example 3 The effect of the addition of lactoferrin was tested in a three-step San Deutschland to detect influenza A antigen. Lactoferrin was used in the second and third steps of this solid-phase immunochemical method. In these two steps, antibody / lactoferrin initially containing (> 0.05 g /) and diluted for use /
Biotin conjugate, then streptavidin containing lactoferrin (0.05 g / min) and diluted for use
When the / POD conjugate was incubated, the background and influenza B foreign antigen were used as indices.
Non-specific reactions could be reduced by more than 80%. 450n
The ELISA results measured in m are shown here as representative examples: Non-specific binding without lactoferrin = 2.892 U Non-specific binding with lactoferrin = 0.231 U

   ────────────────────────────────────────────────── ─── Continuation of front page    (56) References JP-A-62-29533 (JP, A)                 JP-A-62-34059 (JP, A)                 JP-A-62-159047 (JP, A)                 JP-A-56-42142 (JP, A)                 J. Lab. Clin. Med. , 88               (1), p. 156-166, (1976)                 Clin. Chim. Acta, 151               (1), p. 61-69, (1985)

Claims (1)

(57) [Claims] An incubation medium for solid-phase immunoassay assay containing lactoferrin as an agent for preventing non-specific binding of accompanying substances in a sample to a solid phase. 2. 2. The incubation medium according to claim 1, wherein lactoferrin is present in a concentration range of 0.05 to 20 g /. 3. a) removing the cream from the milk of the mammal; b) adjusting the pH of the cream-free milk to 3.5-4.5; c) removing the protein precipitate formed in (b) above; 2. The ink incubation according to claim 1, comprising lactoferrin which can be collected from said milk by extracting and isolating lactoferrin from the supernatant produced in c) by chromatography on an ion exchanger. medium. 4. 2. An ink medium as claimed in claim 1, comprising a buffer salt, lactoferrin and, if appropriate, protein, and, if appropriate, a surfactant, and, if appropriate, a neutral salt. 5. 2. The incubation medium according to claim 1, comprising a buffer salt, lactoferrin, bovine serum albumin, a surfactant and, if appropriate, a neutral salt. 6. Claims containing buffer salts, lactoferrin, gelatin hydrolysis products, or gelatin that has been hydrolyzed and then chemically treated or chemically treated and then hydrolyzed, and surfactants and, where appropriate, neutral salts 2. The ink medium according to claim 1, wherein