JP3368531B2 - Indirect agglutination immunoassay - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is an indirect method capable of performing an agglutination reaction on one sample under three different conditions, and determining the positive or negative of the sample from the obtained agglutination result regardless of the non-specific reaction. An agglutination immunoassay method. Specifically, for one specimen, a first well containing a specimen solution, a second well containing a specimen solution containing a reaction-inhibiting substance, and a third well containing a specimen solution having a higher dilution ratio than the first well are provided. The present invention relates to an indirect agglutination immunoassay method in which an immunoreaction with a measurement reagent is prepared in these three wells, and positive / negative of a sample can be determined from the obtained agglutination result without depending on a nonspecific reaction.

[0002]

2. Description of the Related Art Conventionally, an indirect agglutination immunoassay method has been used to measure immunoassay substances such as antigens and antibodies in a sample. The indirect agglutination immunoassay method is a method of measuring an immunoassay substance by an antigen-antibody reaction from an agglutination image obtained after the reaction, but sometimes even a negative specimen may cause agglutination by a nonspecific reaction. That must be taken into consideration.

Conventional measuring methods include a method using one well and a method using two wells. The method using one well is a measurement method using a sample solution having a dilution ratio that does not cause aggregation due to a non-specific reaction.

The method using two wells is a measuring method in which a solution containing sensitized particles is dropped in one well and a solution containing unsensitized particles is dropped in the other well.
Here, since the dilution ratio of these two wells is low, aggregation due to non-specific reaction may occur. In both the measuring method using one well and the measuring method using two wells, the positive / negative of the measurement sample is judged from the agglutination result of each well after sufficient reaction. Positive / negative was judged by performing such a measuring method.

[0005]

SUMMARY OF THE INVENTION In the prior art, 1
The two-well assay method can facilitate positive and negative measurements and determinations, but dilute the sample to a concentration that does not cause nonspecific reaction (usually a dilution factor of 20 times or more that of the original sample). Therefore, the sensitivity was low. In addition, the two-well measurement method has high sensitivity because it uses a higher concentration of sample than the one-well measurement, but this measurement method has a high concentration, which may cause nonspecific reaction. If there is aggregation due to a non-specific reaction, it is possible to determine whether the aggregation in the well in which the antigen-sensitized particles have been dropped is due to a non-specific reaction or a positive sample. It was not possible, and there was a drawback that it was not possible to judge positive / negative at this stage.

As described above, further improvement in sensitivity is required in the case of the assay method using one well in the qualitative test, and in the case of the assay method using two wells, positive / negative in the qualitative test. Since it is not possible to make a complete determination, it is necessary to perform a quantitative test by performing an absorption operation using a specific reaction-suppressing substance even for a sample that is originally a negative sample but cannot be determined due to a nonspecific reaction. It was In this case, a final determination cannot be made without conducting a quantitative test for negative samples that cause aggregation due to non-specific reactions. It has been a problem that the measurement time increases with the increase of.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the conventional problems, the present inventors have found a novel indirect agglutination immunoassay method and completed the present invention. The present invention is an indirect agglutination immunoassay method capable of determining positive / negative of a sample by a single measurement.

To carry out the measurement of the present invention, first, for one sample, a first well containing a sample solution, a second well containing a sample solution containing a reaction-inhibiting substance, and a dilution ratio higher than the first well And a third well containing a high analyte solution. The reaction suppressing substance is not added to the first and third wells.

Particles sensitized with an antigen or an antibody that specifically reacts with the substance to be measured are added to the solution thus prepared and stirred. The combination of the agglutination results of each well after this reaction enables efficient determination of the sample. If each well is determined to be positive, it is expressed as +, and if it is determined to be negative, it is expressed as −, and the samples can be classified as follows based on the obtained aggregation result.

First, positive samples can be classified into the following three types. In the case of a positive sample with a titer of 3 wells or more (high titer), the 1st well is +, the 2nd well is not completely suppressed due to the high titer, and the 3rd well is also high. Since the titer is a titer equal to or higher than the dilution ratio, it becomes +. At this time, even if aggregation due to non-specific reaction occurs, all become +,
The final decision remains the same. When the titer is 3 wells or less (low titer) and the agglutination due to non-specific reaction does not occur, the first well is +, the second well is completely suppressed-, the third well. Since it has a low titer, it becomes − upon dilution. In the case of a positive sample with a titer of 3 wells or less and agglutination due to non-specific reaction, the first well is +, and the second well is completely suppressed, but + due to agglutination due to non-specific reaction. Yes, the third well is suppressed by dilution in both reactions.

Next, the negative samples can be classified into the following two types. In the case of a negative sample in which agglutination due to a non-specific reaction has not occurred, neither the target reaction nor the non-specific reaction has occurred, so that the value is − in all wells. In the case of a negative sample in which agglutination has occurred due to a non-specific reaction, agglutination has occurred in the first well due to a non-specific reaction +,
The reaction-suppressing substance in the second well inhibits the desired reaction but does not inhibit the non-specific reaction, so that it also becomes +, and in the third well, the agglutination due to the non-specific reaction does not occur, so that it becomes −.

In the above results, and seem to be the same as each other at first glance, but it is possible to judge which is actually based on the difference in agglutination image between the first well and the second well. The final judgment was made when there was no difference in the agglutination images of the first and second wells, and when the first and second wells were compared, the first well had a strong positive image and the second well had a weak image. When the image is positive, it is determined. The final judgment is made from the combination of the aggregation results as described above, and Table 1 shows a summary. At this time, the difference between the first well and the second well of one tube or more in the agglutination method using 2 n-th power dilution is recognized.

[0013]

[Table 1] * Indicates that there is almost no difference in the agglutination image between the first well and the second well, and indicates that there is a difference of 1 or more tubes in the agglutination image between the first well and the second well.

In the present invention, it is preferable that the reaction is controlled by adjusting the content of the reaction-inhibiting substance in the second well and the dilution ratio of the third well so that the agglutination of the specific reaction occurs to the same extent. The relationship with the magnification is the antigen used,
It is appropriately adjusted depending on the antibody and the reaction suppressing substance.

Particles used as the measuring reagent are particles generally used in indirect agglutination immunoassay, such as animal red blood cells such as human, sheep, chicken, goat and the like, artificial solid particles coated with gelatin, latex, magnetic particles and the like. Mention may be made of particles which are biologically inert.

As the indirect agglutination immunoassay method, depending on the particles used, a red blood cell agglutination immunoassay method, a latex agglutination immunoassay method, an agglutination immunoassay method of promoting sedimentation by magnetic force, and the magnetic force described in JP-A-3-191864. An agglutination immunoassay method in which the particles are forcibly collected at the bottom of the container and then the particles are made to flow out can be mentioned.

As the reaction-suppressing substance used in the present invention, an antibody can be used in the antigen measuring system. This antibody may be a polyclonal antibody or a monoclonal antibody, and may be the same as the antibody bound to the sensitized particles, but it is usually preferable to be different. Further, in an antibody measuring system, any antigen may be used as long as it is an antigen against the antibody to be measured. As the antigen used for this purpose, any of natural antigen, recombinant antigen and synthetic antigen can be used, and it is preferable to be different from the sensitizing antigen as in the antigen measurement system.

In the present specification, the term "concentration" refers to a concentration that also includes a measurement reagent for actually performing the reaction, that is, a final concentration. In the expression using a multiple, the magnification based on the original sample is expressed as the dilution ratio. The concentration of the third well diluted to such a degree that non-specific reaction does not occur can be appropriately selected depending on the type of immunoassay substance or sample in the sample to be measured. Usually, the dilution ratio is preferably 20 times or more, and particularly preferably 32 times or more that of the original sample.

[0019]

The present invention will be described in more detail with reference to Reference Examples and Examples below.

Reference Example 1 Preparation of Sensitized Particles for Indirect Aggregation Immune Reaction In accordance with the method described in Japanese Patent Publication No. 3-17103, gelatin, gum arabic, sodium hexametaphosphate and ferric colloid were insolubilized to obtain an average. Gelatin magnetic particles having a particle size of 2.0 μm were obtained. The gelatin particles were treated with polyquinone to sensitize a polyclonal anti-HBs antibody to prepare anti-HBs antibody-sensitized magnetic particles.

Reference Example 2 Preparation of Diluting Solution Containing Reaction Inhibiting Substance Diluting Solution (0.15 M Phosphate Buffer Solution pH 7.2,
0.1% NaN ₃ , 0.25% gum arabic, 1% NR
S, 1% SH-NRS, 1% Heat-Gerati
n, 0.2 mg / mL M38B2) to which a monoclonal anti-HBs antibody (HB135) was added so as to have a concentration of 937.5 ng / mL, which was used as a sample diluent for the inhibition test of the present invention.

Example 1 Measurement of HBs Antigen 40 μL and 25 μL of a sample diluent in the first and third wells of a V-type microplate, 40 μL of a sample diluent for an inhibition test in the second well, and use for dilution 25 μL of the sample diluent was added to each well. Then, a diluted sample containing the HBs antigen, a RIA positive sample,
Each well was prepared as follows for each of the RIA-negative sample and the RIA-negative sample (which cannot be determined by the indirect agglutination immunoassay method). 10 samples in the first and second wells
μL was added to each well, and after stirring, 25 μL was taken from the first well, added to the well for dilution, stirred, and 25 μL was taken from the well for dilution, added to the third well, stirred, and 25 μL was taken from the third well and discarded. . The second well is 25μ
L was taken and discarded. After standing for 10 minutes (absorption time), 25 μL of a dispersion containing 0.2% of anti-HBs antibody-sensitized magnetic gelatin particles prepared in Reference Example 1 was added to the first to third wells. After stirring for 5 minutes and leaving it on the sedimentation promoting table using a magnet for 90 seconds, the microplate is moved to 90 on the pattern judging table.
The presence / absence of immune reaction was determined by inclining at 60 degrees for 2 seconds to flow out particles from the bottom of the well of the microplate to form an aggregation pattern. The combination of the aggregation patterns of the first to third wells was used to determine whether the measurement sample was positive or negative. Here, it was determined that the flow start length ≦ 3.00 mm was positive (+) and> 3.00 mm was negative (−). The final judgment was made from the results of the first to third wells, and the results are shown in Table 2. The length in Table 2 is the length of the flow start, and the unit is mm. For samples in which the first and second wells are + and the third well is-
The final determination was made by determining whether or not aggregation due to a non-specific reaction was included, based on the difference in the flow lengths of the first well and the second well. Here, the difference in the length of the flow start is 0.6 mm
When the above is the case, it is determined that the first well contains agglutination due to a non-specific reaction. (0.6 mm corresponds to a reproducibility width ± 1 tube in the agglutination method.) For all of the measured samples (1) to (8), the same measurement results as RIA were obtained as shown in Table 2.

[0023]

[Table 2] * (1), (2): HBs antigen-containing diluted sample (3), (4): RIA positive sample (5), (6): RIA negative sample (7), (8): RIA negative However, specimens that cannot be determined by conventional indirect agglutination immunoassays

[0024]

INDUSTRIAL APPLICABILITY The indirect agglutination immunoassay method of the present invention is a highly sensitive assay method capable of determining positive / negative of a sample by a single measurement regardless of non-specific reaction. According to the present invention, it is possible to make a determination in a single measurement even for a sample that could not be determined as positive or negative in a conventional qualitative test, and accordingly, a quantitative test may be performed only for a necessary sample. Can be shortened.

Continuation of front page (56) Reference JP-A-8-327628 (JP, A) JP-A-62-15464 (JP, A) JP-A-8-327629 (JP, A) JP-A-60-256057 (JP , A) JP-A-11-101803 (JP, A) JP-A-9-89893 (JP, A) JP-A-55-111839 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB) Name) G01N 33/53-33/579

Claims (4)

(57) [Claims] 1. For one sample, a first well containing a sample solution, a second well containing a sample solution containing a reaction-inhibiting substance, and a third well containing a sample solution having a higher dilution ratio than the first well. An indirect agglutination immunoassay method capable of determining the positive / negative of a sample based on the obtained agglutination result without depending on the nonspecific reaction, by preparing an immunoassay with a measurement reagent in these three wells. 2. The concentration of the sample in the second well is the same as that of the first well, and the concentration of the sample in the third well is a concentration at which aggregation due to a non-specific reaction does not occur.
The indirect agglutination immunoassay method according to. 3. The indirect agglutination immunoassay according to claim 1, wherein the content of the reaction-inhibiting substance in the second well and the dilution ratio in the third well are adjusted in advance so that the agglutination occurs to the same extent in the positive sample. Method. 4. The indirect agglutination immunoassay method according to claim 1, wherein the dilution ratio of the third well is 32 times or more that of the original sample.