JPS5926066A - Amplification method of solid phasing antibody - Google Patents

Abstract

PURPOSE:To increase easily the number of solid phase molecules per unit area of a solid phasing antibody without changing the surface area and shape of an insoluble solid phase carrier, by forming the composite body of the solid phasing antibody and antibody on the surface of said carrier. CONSTITUTION:In the case of using, for example, a glass beam, as an insoluble solid phase carrier, an antihuman IgE rabbit antibody and protein A are first brought into reaction to form a composite body of the antihuman IgE rabbit antibody-Protein A. A glass beam for converting the antihuman IgE rabbit antibody into a solid phase is put into the reacting liquid thereof and is brought into reaction to form the composite body of the antihuman IgE rabbit antibody- Protein A-antihuman IgE rabbit antibody on the surface of the glass beam, that is, the composite body of the solid phasing antibody-antibody. The increase in the number of solid phase molecules per unit area of the solid phasing antibody is thus made possible and the antihuman IgE rabbit antibody on the surface of the glass beam is amplified.

Description

Detailed Description of the Invention: A reaction between antigens and antibodies occurs even at extremely low temperatures. Therefore, this fact can be used to extract antigens from blood or body fluids for extraction analysis, diagnosis, and clinical moxibustion. Kokichi, who performs 1 Sai, is 6-cho Noh, and 4-CI is several: II!
JII example ir and many (1 no [dead publications are reported.

However, for the identification of antigens stimulated by conventional antibodies: for example, radioimmunoassays using radioactive isotopes, enzymes, etc. Methods using firefly proboscis, sae, pigments, etc. are known. In the method of 1, et al. (IL), there was no test 4 in which the J antigen and antibody were immobilized on the surface of a nonfermentable carrier to facilitate the antigen-antibody reaction and the removal of unreacted antibody antigen after the reaction. Therefore, it is not 6 sex body table 101
Technology to immobilize antigens and antibodies on a solid phase is essential.

For this reason, such solid-phase technology is good and bad for the surface of bamboo.
Reactive groups such as carboxyl groups, amine groups, hydroxyl groups, etc. (and antigens, antibodies, etc.)
'Chemical crosslinking method using hydrocarposiimide etc. 4. +1' Kimiaki 5 (i 110052 No. l Samurai Kimiaki", + (5-1 1 0 0 5 4 each in No. 3)
The mixture of polymerizable monomers and antigens or antibodies is exposed to light or ionizing radiation,
There are methods to immobilize antigens or anti-J-antigens by polymerizing the body, or methods using anal adsorption.

In this way, by making full use of the 6R methods, antibodies in the sample can be extracted. Although it is possible to perform constant pulsation of antigen, historically, insoluble 1'JE
In order to increase the surface area of the homologous antibody on the surface of the 4Q body, operations such as washing t71 can be performed effectively to increase the surface area of the 4Q body. Effective in-phase methods have been considered, such as changing the shape of the insoluble carrier to make it easier to understand, but it is unclear which of these methods has the best effect. .

On the other hand, Juto's solid phase θξ is also 1. There are inherent disadvantages in itself; - For example, "CI adsorption method is unstable after solid phase formation, and the antibody (1) can cut solid phase C by hitting the surface."
However, although methods using chemical cross-linking agents are still in use today, it is not necessary to use antibodies (4) that are sufficiently active.
The formation of a solid phase for antibodies (antigens) involves the formation of a solid phase using light or ionizing radiation, and the immobilization of antibodies (anti-1. Effective l, [
Although a solid-phase moiety (antigen) can be obtained, there are drawbacks such as the need for special equipment.

This invention was made in view of the above circumstances.

The object of the present invention is to provide a method for increasing immobilized antibodies by which the number of solid phase molecules per unit area of immobilized antibodies can be easily increased.

(Z, ``,This expression 1s'' -Real Mli example is d((,Explain.

Here, in such an embodiment, the glass beam is replaced with an insoluble solid phase 4
+7d when using 11 bodies and C.

4', iα treated with soluic acid and thoroughly dried with distilled water
Prepare a glass beam with a diameter of 7 oyn and connect it to the 2nd 3rd -
,++n1oopro Kyl triethoxys
Amine groups were introduced onto the surface of the glass piece by applying a solution of ilone acetone at 45° C. for 24 hours, and then applying it to a 5% glucuraldehyde solution for 1 hour. Another 0.
Washed with 25M monolithium phosphate buffer T1 (0
．． The cells were treated with 1 Q25M sodium phosphate buffer containing anti-sheep and IglJ sheep antibodies (both IgU components) at a ratio of 5 mg/Ille at 4°C for 1 week.

After that, remove the reaction solution and add 0.1M NaCg, 1mM
fgcr, 0.1% 1xialbumin, 0.1
After washing with oIM phosphorus thorium hydroxide solution containing % NaN, it was left to stand at 4°C for 24 hours to remove 1 anti-human IgF.
C. Sheep antibody-immobilized glass beats were obtained.

Until the above-mentioned over-maintenance, conventional glutaraldehyde was used.
This is the same as the solid phase method using cross-linking 2H.

Next, we will describe a method for testing anti-human Igl on the surface of Rusby 1 and sheep antibodies. First, anti-human sheep antibody (IgCJ fraction) l mg/m/? ,
I Il+/and P r o t e in AQ, 0
28 nvAner 101+1/! and react at 4°C for 1 hour. Then, J'rotcinA becomes anti-Human)
r, g 1. 'j t:-Tsuji antibody (, [g(] fraction) Ii' c, reacts with the fragment, anti-human 1 g
IJ sheep antibody-1'rotefA complex formation 4
-ru.

This reaction solution + 1 = i previously prepared anti-human 1. ITh
To immobilize the hinge antibody, insert a last piece and invert at 4°C for 16 hours. (This one-time ProteinΔ may be allowed to react with the added antibody after the surface effect of the antibody already immobilized.) In this reaction, one part of the surface of the glass piece is exposed to anti-human IglJ. The number of solid phase molecules per unit iTneR of the immobilized antibody can be increased, and as a result, the single human IRI'3 sheep antibody on the surface of the glass piece can be amplified.

By the way, the anti-leaching l-Jgl was determined by the 1N of the Rotc order using the Lein-Didge method.
>Increase in sheep antibodies +13 in row 4 (Glass Peas 7,
Increased f1] and use glass beads each using Vr future method without C? When I created the IT f arrow lit line, I got the same result as the drawing. Here C (arrow 1j″L line 1
``0'' (ei n Aζ, (2)
The Igl.; sheep antibody was increased by 11J, but the Rasvino and Ed-2 were prepared using conventional methods (using glass beads).

Therefore, it is clear from 171 that the calibration curve 1
J'rote is a sensitive curve with a large C change rate compared to that of J'rote 11-line 2.
1 by inA! Confirm the 1st river antibody II+
Second is done.

In addition, in Example C, glass beads were used as the insoluble factor Iff, j; Cells, etc., 1'r't, C of any shape, Table 1j (1's solid + 1'l) (Then, the increase in antibody iJ is iiJ ability. It is also possible to amplify antibodies immobilized with a 4fT method using physical adsorption, a solid 4fT method using a chemical cross-linking agent, a method using light or rfE radiation, etc. In addition, insoluble phases with 11 antibodies are not limited to Enri'i, Inno, Assay, but Radio Ino, Ino, Inno, and Fluo's ImmunoAra1! are also useful. .11 can be done. Sara (Kosuta Masu 11" cut as jJ
Protcin A alone is thought to be 4" insoluble, 1 antibody on the body surface 7), or cross-linking/X j) to chemically crosslink between antibodies.

Anti-Haj(, antibody is easy to react with 1ΣU) on soluble 411,
And if there is more than one operation example, cleaning the unreacted substances is effective (this?
il, the shape of C is made, direction 1, jl≠11-ruko6
- is 'C'. Talented. Solid phase t, l:,
Lid, 7 yen, 1' more effective reagent (Ice is in phase with 1 day i12 and/or 1: 1: As a result, a reagent with high sensitivity and short reaction time is used. It's easy to get r(L).

[Brief explanation of drawings]

The drawings are for explaining one embodiment of the present invention. 1.2...Sasei Line Ic+E (Ll/ml ・I"+1!'l Agency Director Kazuo Wakasugi) 1 Indication of °lii No. I Patent Application No. 1982-127202 2 Name of the Invention Immobilized antibody width enhancement Method l fli il: 4 iris 11 leaf bolts (71' Patent applicant (037) Olympus Optical Industry Co., Ltd. 4th generation, 1:, l
j person 5, spontaneous correction''-゛■peha (i, ?11i 11', sentence, j elephant layer γ 3
% Clearance 1￥lJ: 7. Contents of amendment (1) “3-nm
1nopro pyltrietl+oxyBilon
eJ [3-; uninopropyl trietb
oxysilane J (!: Correct ilJ. (2) Same order specification, page 4, line 12, day 5, 3rd
"Sheep antibody" written in line 5, line 6, line 7 is corrected to "rabbit antibody." (3) Line 17 of page 4 of detailed description 1 in the same order, line 1 of page 5
Line to; ゝl\2nd line, 14th line to 15th line, 1st line
Line 9, page 6, lines 1 to 2, line 6, "sheep antibody" is changed to "woolly antibody". (4) Lines 9 to 10 of page 5 of the same specification []
1. The [hinge antibody] described here has been corrected to ``Wooligi antibody''. (5) Kaihanmei #III W middle tube 4R line 12, 5th
Delete l' (], gG, both months written in the 3rd and 6th lines of the page. (6) l written in the 14th line of the 4th page of retrospective details ν4
'l+nMMg(shi/', 0.1chi') to l-1mM
MgCl, , Q, 1%” is corrected. (Power Reminiscences of Meibu I Evening, page 5, line 20, page 7, line 1 [Correct ``enzyme im assay'' in 11th and 11th iv to ``enzytono assay''. (8) Same order. "1 Radioimmunoassay" written in the second line of page 7 of the specification is corrected to "Radioimmunoassay."

Claims (2)

[Claims] (1) Is Kokichi characterized by having a means for forming a solid-body-antibody complex on the surface of an insoluble solid phase? 1-11J method for increasing immobilized antibodies. (2) The above-mentioned solid A (A C body-Abdominal union formation increases by 1 rl)
1-IJ) 5 method for increasing in-phase antibodies as described in the patent, which is characterized by using agent J.