JPS5992354A - Reagent for immune serological diagnosis - Google Patents

Abstract

PURPOSE:To obtain a reagent for immune serological diagnosis keeping high sensitivity for a long period and does not cause self-flocculating with a long time preservation, by adding penicillamine to latex suspension. CONSTITUTION:The titled reagent for immune serological diagnosis has special features that 250mug-5,000mug gamma-globulin is sensitized per 10mg latex particles having 0.08-0.80mum diameter and containing 0.08-2.0wt% said latex particles further 0.01-10wt% penicillamine is added to this suspension. As latex particle, for example, polystyrene or copolymer of a monomer having phenyl radical and an anionic monomer are suitable. Further, either D-, L- or DL- penicillamines can be used.

Description

[Detailed description of the invention] The present invention relates to immunoserological diagnostic reagents.

Conventionally, latex particles are used as carriers, and antigens or antibodies are sensitized, and the agglutination reaction of the latex particles is caused by the antigen-antibody reaction specifically with antibodies or anti-antigens in serum. Diagnosis of L11 severe disease has been carried out in the field of clinical testing as an immunoserological diagnostic method, such as rheumatoid factor, HBs antigen, HBs antibody, anti-streptolysin-○ (ASO), C-reactivity. Protein compensation (C
RP), α-fetoprotein, oncofetal anti-Ai ((c
Such immunological diagnostic reagents for mBpy are also used for tests such as aA).

As such a diagnostic reagent, for example, a mixture of gamma globulin mixed with polystyrene particles and adsorbed onto the particle surface in a latex suspension in which polystyrene particles are dispersed is generally used. In such a diagnostic reagent, gamma globulin exists in an equilibrium state in which gamma globulin is adsorbed and desorbed between the book that is not adsorbed to the latex particles in solution and the gamma globulin that is on the latex surface. Gamma globulin is mainly composed of immunoglobulin G (IIIG), which accounts for more than 90% of gamma globulin. IIG molecules tend to associate with each other and form durable bodies such as double bodies and Bfi bodies, but
The long-lasting body has a strong tendency to collect latex particles t#, which may cause deterioration of the reagent over time.

For this reason, for example, in JP-A-54-26327, changes over time are suppressed by adding purine chloride and naccarose to a latex suspension. However, choline chloride,
Addition of saccharomyces causes an increase in the viscosity of the suspension, resulting in a decrease in sensitivity during the aggregation reaction of latex particles, that is, an increase in the lower limit of the concentration of the detectable analyte, and a decrease in the aggregation reaction rate. arise anew.

The present invention eliminates these drawbacks and improves the a
The object of the present invention is to provide an immunoserological diagnostic reagent that maintains high sensitivity during aggregation reactions over a long period of time and does not cause self-aggregation even after long-term storage.

The gist of the present invention is to use latex particles toWg with a particle size of 008 μm to 0.80 μm, and gamma globulin of 250 μm.
50,007 days sensitized from 1 to 50,007 days, and the suspension contains 0.08 to 2.0 weight of the latex particles, and the suspension contains 0.01 to 2.0 weight of the latex particles. Heavy 1
The invention consists of an 11 loaf reagent for immunological serological diagnosis, characterized in that from 1 to 100% of penicillamine is added.

Next, the immunoserological diagnostic reagent of the present invention will be explained in more detail.

The particle size of the latex particles used in the present invention ranges from 0.08 μm to 0.80 μm. This is because when gamma globulin sensitized to latex particles reacts with antigens or antibodies in the sample and causes an agglutination reaction, several to dozens of latex particles stick together and form an agglutinated image. This is a suitable particle size range for determining the particle size with the naked eye, optically detecting it by changes in scattered light, and detecting it by changing the intensity of transmitted light.

Ditex particles, all particles are from 0.08 μm to 0.8
Preferably, it exists in the range of 0 μm, but it should be as small as not to affect the agglomerated image, preferably within 5 μm.
It is also falsified that the particle size slightly deviates from the specified particle size.

Suitable latex particles include, for example, polystyrene particles and copolymer particles of a monomer having a phenyl group and an anionic polymer.

In the case of copolymer particles, phenyl group-containing monomers such as styrene, divinylbenzene, ethylstyrene, α-methylstyrene, etc. are present, and anionic monomers include, for example, styrene sulfonic acid. salt, divinylbenzenesulfonate, ethylstyrene sulfonate/acid, α
-Methylsulfonate, etc.

In addition, in order to obtain the above-mentioned copolymer particles, for example, 100 parts by weight of a monomer having a phenyl group and 0.1 parts by weight of an anionic monomer are added.
~70 parts by weight can be obtained by causing a copolymerization reaction in water using a persulfate as a polymerization initiator in the absence of an emulsifier. The copolymer particles thus obtained are given surface charges based on the anionic monomer as a copolymerization component and the anion of the polymerization initiator fragment. The surface charge density of the copolymer particles is determined by the dissociation concentration of anions in the latex suspension. OXIO mol/d ~8
0. OX10 is assumed to be within the range of /d, and if it is within the range of It will be done.

In the present invention, 250 μm to 5000 pf of gamma globulin is sensitized per 10 latex particles.

In the present invention, the gamma globulin sensitized to latex particles may be gamma globulin itself, or 11! It may be an iG fraction, or it may be an antigen or antibody itself obtained by further fractionating JG.

For example, when detecting il' CRP, JG is further fractionated and 9F (ab')2 is used.
Interference with other RA factors and interference with complement can be removed. In this case, it is assumed that the sensitized scene of F (s b' )2 exists within the above range.

Gamma globulin sensitization occurs with latex particles at 10'Vfi.
! 17250μm to 5000μm is 2
If the diameter is as small as 50 μm, the aggregation rate will be slow, and the aggregation image after the agglutination reaction will be difficult to detect.
This is because if the amount is too large, a large amount of gamma globulin free in the system reacts, which tends to result in a decrease in sensitivity in the a group reaction.

The amount of latex particles dispersed in the latex suspension ranges from 0.08 to 2.0 weight Jl. The amount of latex particles is kept within the above range because if the amount of latex particles is as small as 0.08 weight iqb, it becomes difficult to detect an agglomerated image after the agglutination reaction. This is because if the amount is too large, a large number of small groups will occur due to the agglutination reaction, which will make it difficult to judge the agglutination image.

In the present invention, tit o, oxffiJ in latex suspension.
1000 wt. of penicillamine was added from 100 ml. Penicillamine is a sulfur-containing α-amino acid represented by the following formula.

It was also found that penicillamine has the function of suppressing the association of IJG and preventing a decrease in the sensitivity of latex diagnostic reagents. Benishi 2ξ/ can be used in 0 body, L body, and DL body. The amount of penicillamine added in the 2tex suspension ranges from 0.01% by weight to xo, oi@chi. If the amount is less than 0.01% by weight, there is a risk that the effect of suppressing IQG association and preventing a decrease in sensitivity when used as a diagnostic reagent will not be expressed. This is because the edges of the resin tend to dry when the resin is expanded, which is not preferable because crystals tend to form due to the relationship with solubility.

In the present invention, the sensitivity of the diagnostic reagent, the agglutination reaction time, etc.
For example, purine chloride, saccharose, etc. may be added in addition to 1 penicillamine to obtain f1.

According to the present invention, it is possible to obtain an immunoserological diagnostic reagent that maintains high sensitivity during the agglutination reaction of latex particles over a long period of time and does not cause self-aggregation even after long-term storage.

Example (1) Preparation of 2tex reagent for detection of hepatitis B virus surface antigen, O, OI 5M phosphate saline buffer (, rt
t-Kji was prepared by dispersing polystyrene latex particles having a particle size of 0.46 μm in t-Kji (liquid A) to contain 2.0 g of polystyrene latex particles having a particle size of 0.46 μm.

IjG fraction components containing anti-HB@antigen antibodies purified from antiserum obtained by immunizing guinea pigs with HRs antigen,
A solution (B
liquid) was adjusted.

The A and B solutions were mixed at a volume ratio of i:IK, and the IjG fraction was sensitized to 10 nJl of polystyrene latex particles for 250 μy and heated at 37° C. for 120 minutes.

Penicillamine and sodium azide as a preservative are further added to this latex suspension to finally form polystyrene latex particles. O heavy J! :%, penicillamine 0.5
A diagnostic reagent containing 0.1 weight percent of sodium azide and 0.1 weight percent of sodium azide was obtained.

(21r & degree determination and storage stability test 50 μt of the diagnostic reagent obtained as above and purified 1
50μ sample of 18s antigen diluted with normal human pool serum
# after 3 minutes of mixing on a slide glass for aggregation determination.
The sensitivity was determined by observing ffft with the naked eye. In addition, the storage stability was evaluated by comparing the sensitivity of the diagnostic reagents immediately after preparing them and those stored at 4° C. for one year.

Table 1 Evaluation criteria You... Big #! Image gathering is observed.

10.../J% Small or medium agglutination images are observed.

B: The agglomerated image is unclear.

-... No agglomerated image was observed and the sample was transparent.

As is clear from Table 1, the diagnostic reagents immediately after preparation and those after being stored at 4°C for 1 year after conditioning at 1t11 have the highest quality. It was confirmed that there was no change in the sensitivity of the fruit image, and that the storage stability was excellent.

Comparative Example 1 For comparison with Example 1, a similar test was conducted on the product in which penicillamine was not used in Example 1, and the results showed that the Ins antigen concentration after purification at 4° C. was 1θ
μ! In the case of 7 mg, it was determined as →-.

In this case, the latex port is used as a negative control (n n
There was a risk that the test would be incorrect even for normal human serum (which does not contain s antigen), so it could not be used as a diagnostic reagent.

Example 2 +11') Latex reagent for detecting equine factor In a latex suspension in which polystyrene latex particles having a size of 0.20 pm are dispersed in a glycine-caustic soda buffer, 5 aoooμy of human body per 107v of polystyrene latex particles. The cells were sensitized to gamma globulin and left overnight at 4°C. This latex suspension contains penicillamine,
Bovine serum albumin is added, sodium azide is added as a preservative, and finally polystyrene latex particles 0.5% by weight, penicillamine 10.01i, bovine serum albumin 1, OM member, sodium azide 0 Diagnostic reagents each containing .1-fold were obtained.

(2) Sensitivity determination and storage stability test Sensitivity determination and storage stability test were performed on 10 serum samples in the same manner as in Example 1. The results are shown in Table 2.

Table 2 As is clear from Table 2, the diagnostic reagent was stored at 4°C for 1 year after 811 sterilization and after 1 year of storage at 4°C.
It was confirmed that there was no change in image sensitivity and that storage stability was excellent.

Example 3 (1) The particle diameter of the latex reagent for CRP detection was 0.
Polystyrene latex particles toI in a latex suspension in which 12 μm polystyrene latex particles are dispersed in glycine-caustic soda buffer (pH 8,6)
Per lv, 500 μl of the IjG fraction separated at RT from goat-produced anti-Human CRP antiserum was sensitized.

Next, penicillamine, sucrose, bovine serum albumin, choline chloride, and sodium azide were added to this latex suspension, and finally polystyrene latex particles were
．． 8-JL%, penicillamine 0.0 weight, sucrose 1.0 weight, bovine serum albumin 1.0 weight,
Choline chloride2. s weight j 1%, sodium azide o, t
Diagnostic reagents containing 1 ft.% and 1 ft., respectively, were obtained.

(2) #1 degree determination and storage stability test For the diagnostic reagent obtained as above, $1 Mc
Example 1 for different serum samples containing #1 up
As a result of conducting sensitivity determination and storage stability test in the same manner as above, it was confirmed that the storage stability was excellent as shown in Table 3.

jg3 Table Comparative Example 3 For comparison with Example 3, the reagent in Example 3 from which penicin-2mine was removed was prepared, and the sensitivity determination and storage stability test were conducted in the same manner as in Example 3. As a result, as shown in Table 4, it was observed that the storage stability to quality deteriorated.

Table 4 Example 4 (1) Preparation of latex reagent for CRP detection Styrene-sodium styrene sulfonate copolymer latex particles with an Rffl of 0112 μm were 5.0 μm Ji: % (50ff
/a+/) The copolymer latex particles were dispersed in a phosphate saline buffer (, II 6.5 ) (solution A) and adjusted to v4.

IJG semen isolated from goat-produced anti-human) CRP antiserum
500071 to phosphate saline buffer (H6,5)
The dissolved 77me (liquid B) was prepared.

The A and B solutions were mixed at a volume ratio of 1:1 and heated at 37°C.
The copolymer latex particles were stirred with SO for 1 oyy.
In addition, 6 rlc minutes 1i1jlo00μy'II:l& was made. Further, Benicitumine and sodium azide were added as a solution in phosphate saline buffer (H6,5), and finally the copolymer latex particles were 1.0 ffit%, 50% by weight of Benicitsumi, and 0.1% by weight of sodium azide. A diagnostic reagent containing the following was obtained. The surface charge density of ceramic latex particles L2 is 1 at the dissociated concentration of anions in the tex suspension.
It was 6.4 X I O-7 mol/-.

(2) Ripeness determination and storage stability test' Regarding the diagnostic reagent obtained as above,
Example 1 Regarding serum samples with CRP content concentration
Sensitivity determination and storage stability tests were conducted in the same manner as above, and as shown in Table 5, it was confirmed that the product had excellent storage stability.

Table 5

Claims (1)

[Claims] 1. Particle size O, OSμm to 0.80μm latex particles 1O ~ Our gamma globulin is 250μm to 50μm
001tf sensitized, the latex particles are 00
The latex suspension contains from 8 to 2.0% by weight, and from 0.01% to 1% by weight in the suspension.
1. An immunoserological diagnostic reagent characterized in that 000 heavy-duty penicillamine is added.