JPH0827283B2 - Composition for immunoaggregation reaction - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antigens such as indirect carrier agglutination reaction and aggregation suppression reaction.
It relates to an aqueous composition containing an antigen (or antibody) -sensitized carrier for use in an antibody agglutination reaction, in particular, such a composition improved for stable use over a long period of time.

The so-called indirect carrier agglutination reaction, in which the antigen or antibody in the sample is measured by the agglutination reaction between the sensitized carrier sensitized with the corresponding antibody or antigen and the sample, is a method already established.

This method is easy to operate, easy to judge, can be judged in a short time, relatively high sensitivity, high reproducibility, low cost, no special equipment is required, and it can be performed easily anywhere. It is widely used due to its various advantages.

Generally, as a carrier for sensitizing an antigen or antibody,
Fixed or non-fixed red blood cells from mammals or birds,
Alternatively, artificial carriers such as latex, styrene polymer, gelatin, epoxy resin, cellulose resin and activated carbon powder are used. This is because the fixation of red blood cells is generally performed with aldehydes such as formaldehyde, glutaraldehyde, pyruvaldehyde, etc., to improve their storage stability. An aqueous suspension of a sensitized carrier prepared by sensitizing these carriers with an antigen or an antibody has tentative storage stability and can be used for about 2 to 3 months. However, in order to industrially manufacture the sensitized carrier, storage stability of one year or more is required.

Therefore, attempts have been made to freeze-dry an aqueous suspension of the sensitized carrier to further extend the storage stability. However, when freeze-drying is performed in the state of an aqueous suspension, deterioration of the appearance shape (shrinked state, carmel-like etc.), deterioration of reaction pattern, and storage stability after freeze-drying are less than those without freeze-drying. Problems such as poor quality and poor reproducibility occur. This is apparently because the freeze-drying process adversely affects the sensitized carrier.

Therefore, it is not necessary to freeze-dry, or even if freeze-drying, there is no change in the reaction pattern before and after freeze-drying,
There is a demand for a suspension of a sensitizing carrier which has a good appearance and can be used stably for more than one year.

As a result of intensive studies, the inventors of the present invention have found that the above problems can be solved all at once by allowing a specific amount of sugar and further selectively albumin to be present in an aqueous suspension or suspension of a sensitized carrier. Heading out, the present invention has been completed.

That is, the present invention provides a sensitized carrier for the purpose of enhancing the storage stability of the sensitized carrier when producing an aqueous suspension of the sensitized carrier used in an antigen-antibody agglutination reaction such as an indirect carrier agglutination reaction and an aggregation suppression reaction. A specific stabilizer substance is added to an aqueous suspension in a specific amount to provide an improved sensitized carrier-containing aqueous composition having high storage stability.

Such a specific amount of a specific stabilizer is 2 to 20 W / V%
Is lactose. If the amount of this lactose is less than about 2 W / V%, the effect of enhancing the storage stability is weak, and if it is more than 20 W / V%, the appearance tends to be very poor, and it becomes difficult to increase the viscosity or dissolve it. Not suitable for the purpose of the invention. A particularly preferred lactose concentration is 5-10 W / V%.

Also, by selectively adding albumin to the lactose, the effect is further amplified as compared with the case of adding lactose alone. This amplification effect is particularly pronounced in the preferred combination of lactose and albumin concentrations. The most preferred albumin concentration is 5-10 W / V%
It is 0.05-1 W / V% with respect to lactose. The albumin used may be any of those derived from hide, bovine, horse, goat, sheep, rabbit, chicken and the like, but it is particularly preferable to use bovine or human serum albumin.

An aqueous suspension containing a sensitizing carrier in a proportion of 0.5 to 10 W / V%, after well mixed with the above stabilizer, and mixed well, may be dispensed into a vial and stored as it is, but more preferably,
This vial is put on a shelf in a chamber of a freeze dryer which has been cooled to about -40 to -45 ° C in advance, and vacuum freeze dried for 24 to 48 hours. After that, a freeze-dried carrier is completed by sealing in a vacuum or after filling with nitrogen gas.

The freeze-dried sensitized carrier can be reconstituted by adding water or an appropriate diluent, and can be used for the indirect carrier agglutination reaction or the like by adjusting the concentration as it is or by adjusting the concentration by dilution if necessary.

The sensitized carrier freeze-dried using the stabilizer according to the present invention can be stored at 4 ° C. for 2 years or more.

The addition of the stabilizer according to the present invention can enhance the storage stability of not only the sensitized carrier but also the carrier itself when fixed blood cells are used as the carrier. Therefore, the present invention can be applied extremely advantageously when blood cells are used as a carrier. Further, even when the stabilizer of the present invention is added to the sensitized carrier suspension in a liquid state without freeze-drying, the sensitized carrier is sufficiently stable at 4 ° C. for 6 months. It can be used for a long time and is extremely effective for applications where the disadvantages of freeze-drying must be avoided.

Thus, according to the present invention, it becomes possible to industrially produce a sensitizing carrier capable of stably maintaining excellent quality for a long period of time, and for daily clinical laboratory work such as hospitals and clinical laboratory centers. Can be supplied at low cost.

As the substance to be sensitized to the carrier, any antigenic substance or antibody can be used as long as it can be sensitized to the carrier by some method.

The solution for suspending the sensitized carrier may be any solution as long as it does not adversely affect the sensitized carrier, and physiological saline, PBS (phosphate buffered saline) or the like can be generally used.

Hereinafter, the present invention will be described more specifically by way of examples.

Example 1 [Fixation of sheep red blood cells] 100 ml of sheep blood was collected and mixed with 100 ml of Orseva solution,
Gauze filtration was performed, and the concentration of blood cells was measured by hematocrit. Wash 5 times with physiological saline, then “Methods in l
mmunology and lmmuno chemistry, vol.VI pp.33〜34 19
77 years (WILLIAMS, CHASE, Academic Press New York) ”
Formalin-fixed sheep blood cells were prepared as described by Abram B, Stavitsky.

That is, 8 times the amount of cold 3V as the precipitate of erythrocytes washed and centrifuged.
Add / V% formaldehyde saline and stir slowly at 4 ° C for 24 hours. Furthermore, twice the amount of cold 40V / V% formaldehyde physiological saline was added and the mixture was stirred for 24 hours. Then, the red blood cells were filtered with gauze, washed with physiological saline eight times, and suspended with PBS (M / 15 phosphate buffered saline pH7.2) to a concentration of 2.5 V / V%, sodium nitride 0.1 W / % And added and stored at 4 ° C.

[Preparation of carrier for sensitizing thyroglobulin]

The fixed sheep red blood cells prepared above were further washed twice with PBS and suspended so that the red blood cell concentration in PBS was 2.5 V / V%. “Methods in lmmunology and immunochemistry, vo
The antigen was sensitized to fixed sheep red blood cells with reference to l.IV pp.36 "(supra). Thyroglobulin purchased from Seikagaku Corporation was used as the antigen.
The mixture was diluted to 0.001 W / V% with S, mixed with 600 ml of 2.5 V / V% sheep fixed erythrocytes in an equal amount, and reacted at 37 ° C for 45 minutes. Then 3 with PBS
It was washed twice and resuspended in 600 ml PBS. To 600 ml of this tannic acid-treated red blood cell, 50 μg / ml PBS solution of thyroglobulin 60
0 ml was added and sensitization was performed at 37 ° C for 30 minutes. After washing 3 times with PBS, it was suspended in 1500 ml of PBS containing 0.1 W / V% sodium nitride to obtain 1500 ml of thyroglobulin-sensitized blood cells for indirect carrier aggregation reaction at a concentration of 1 V / V%.

〔freeze drying〕

To the thyroglobulin-sensitized blood cell 1V / V% suspension obtained above,
Add each stabilizer in Table 1 to a 10 ml vial for lyophilization.
Dispense 2.5 ml aliquots into the chamber of the vacuum lyophilizer that had been previously cooled to -45 ° C, and then at -50 ° C for 2 hours,
Lyophilization was carried out at a temperature of −50 ° C. to + 15 ° C. for 10 hours and + 20 ° C. for 12 hours. After drying, the vial was filled with nitrogen gas and then sealed. The results of comparison of the properties of the freeze-dried sensitized hemocytes thus obtained are shown in Table 1. The appearance after drying was judged based on the degree of shrinkage. The reaction pattern is
2.5 ml of water was added to each lyophilized sensitized hemocyte to reconstitute a 1V / V% sensitized hemocyte suspension, and anti-thyroglobulin antibody-positive serum and negative serum were used as specimens and indirect carriers (hemocytes ) Judgment was made based on the positive and negative patterns when the agglutination reaction was performed. Those judged as x are those in which a positive negative pattern was observed. The reaction pattern was tested for its stability over time.
As a result, the samples evaluated as “good” in Table 1 have significantly improved storage stability.

Example 2 Thyroglobulin-sensitized blood cells prepared in Example 1 1 V / V%
Add each stabilizer as shown in Table 2 to the suspension,
The results of lyophilization performed in the same manner as in Example 1 below and the properties of the lyophilized sensitized hemocytes thus produced were compared in the same manner as in Example 1 are shown in Table 2. Table 1 shows the effects when the stabilizer is used alone, and Table 2 shows the effects when the albumin and sugar are added in combination.

Example 3 In Tables 1 and 2 of Example 1 and Example 2, stabilizer sample Nos. 3, 4, 10, 16, 22, 31, 3 which were remarkably effective.
Table 3 shows the results of testing the titer of freeze-dried sensitized blood cells after storing the 11 samples of 3, 35, 37, 39 and 41 for a long time at 4 ° C. All samples were stable at 4 ° C for 2 years.

Example 4 With respect to 11 samples of the protective agent which were remarkably effective in Examples 1 and 2, the same storage stability as in Example 3 was examined for the product in a liquid state without freeze-drying. The result is the fourth
As shown in the table, the stability was 6 months to 1 year.

Example 5 An experiment was conducted on thyroglobulin-sensitized latex.

A known method for sensitizing latex (for example, Takahiro Oda et al .: Detection of Staphylococcal Enterotoxin from Foods Using Latex Agglutination Reaction, Fukuoka Ichiei Test Report, 4, 33-3)
7, 1979). That is, latex (polystyrene latex SDL-59, Takeda Pharmaceutical Co., Ltd.) was used in PBS at 0.025 W / V%.
The mixture was diluted with 25 μg / ml of thyroglobulin PBS solution and sensitized at room temperature for 1 hour. After centrifuging and washing 3 times with PBS, suspend with PBS and add 0.1W / V% sodium nitride.
A 0.025 W / V% sensitized latex suspension was prepared. 5th to this
The storage stability was investigated with time when the suspensions shown in the table were added as suspensions and stored as a liquid as they were, or when freeze-dried in the same manner as in Example 1 and stored in a dry state. . The determination method for the reaction pattern was according to Example 1, and the titer was according to the conventional method of sensitized latex agglutination reaction microtiter method. The results are shown in Table 5, which is stable for 6 months to 1 year in liquid state and 2 in dry state.
It had been stable for more than a year.

Claims (2)

[Claims] 1. A composition for immunoaggregation reaction, which comprises an aqueous suspension of an antigen- or antibody-sensitized carrier, contains 5-10 W / V% lactose as a stabilizer, and is freeze-dried. 2. A stabilizer containing selectively 0.05 to 1 W / V% of albumin as a stabilizer.
The composition according to the item.