JPH01233298A - Antibody, its production, reagent for measuring hmlc and monocronal antibody - Google Patents

Abstract

NEW MATERIAL: Specific antibodies to heart muscle myosin L-chains(HMLC), each of which has a ≤5% cross-reactivity ratio to skeletal muscle myosin L-chains(SMLC).

USE: Diagnostic agents for myocardial infarction and reagents for measuring HMLC.

PREPARATION: The preparation of such an antibody, for example, comprises: blending a mixture of HMLC I (having a molecular weight of 26,000) and HMLC II (having a molecular weight of 18,000), which mixture is used as an immunogen, with a complete Freund's adjuvant; administering the resulting blend to an mammal such as sheep to immunize the mammal; thereafter, collecting blood from the mammal on the 7th, 14th and 30th day after the immunization to separate antiserum; then, subjecting the antiserum to purification by immunoadsorption using an SMLC-immunoadsorbent that consists of a silicate(s) such as Aerosil(R); and thereafter, subjecting the purified antiserum to dialysis against a buffer to prepare the objective antibody specific to HMLC.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a specific antibody against cardiac myosin light chain (HMLC), a method for producing the same, and the use of the antibody in an immunoreagent for measuring BMLO.

Prior Art Myosin is a protein with a molecular weight of approximately 500,000. Myosin has a molecular weight of about 200,000 2
2 E chains, 2 L chains with a molecular weight of about 26,000 1
(HMLC1) and two light chains B (HMLC1) with molecular weights of approximately 180 (70). Myosin is present in the thick quarters of cardiac and skeletal muscles.

Ryo! 1. HMLC reaches the plasma because myosin is degraded due to inevitable myocardial necrosis, and therefore it is considered suitable not only for the diagnosis and course control of myocardial infarction, but also for myosin disintegration beyond the degree of myocardial damage. (Circulation (C1rcul)
ation) 58 (1968) 11301. Ta
p, Cardio,! .

44 (1980) 185, Am, J, Cardi
ol.

41 (1978) 641, Circulation (01
rculatiOn) 60 C Addendum 1) (1979
)169).

Since the half-life of HMLC in the plasma is very short, approximately 70-80 minutes, a subsequent decline due to the stochastic event of infarction or a broad course of time can cause HMLC! It can be assumed that the release of is continuous. Therefore, one infarction enzyme 'OX, OK-MBX GOT and LD
Unlike H, reliable diagnosis and progress control are possible by measuring HMLC. Furthermore, it would also be possible to reliably state the diagnosis of unstable angina.

For in such cases the HMLC would likewise be wiped out by the myocardial infarction that would probably be present (Am, J, 0ardio
l 54 C1984) 964).

However, in this case only a small amount of BMLO reaches the plasma, so that sensitive and special measurement methods are required. Usually, in such cases, in particular immunoassays are suitable as a detection method.

BMLO's radioimmunoassay has already been used in Am.

J, 01ln, Path, 71 (1978) 3
09-318. This is a competitive radioimmunoassay in which HMLC and iodinated HMLC from a sample are competitively inhibited by a polyclonal antibody that is beaked onto a solid phase. However, a drawback of this method is that the cross-reactivity between HML (3) and skeletal muscle light chain (SMLC) is 3.8% with respect to the used antiserum.

In fact, it has been proposed to purify the antiserum against HMLC by removing SMLC antibodies or to purify it by fractionating it by chromatography; It has not been possible to provide antibodies against HMLC that exhibit sufficiently low cross-reactivity. That is, Jap,
01rcu1atiOn44 (1980) 185-18
In No. 6, it was recognized that the cross-reactivity rate was 8% in the immunoscan test. 01rculation 5
8 (1978) 1130-1136, it was reported that the cross-reactivity rate was 2.9-10% in the immunoscan test. The actual cross-reactivity rate is correspondingly high. For the value measured in the Immunosan V inch test of 2.9%, the actual cross-reactivity rate is (2,9)”
%, or 8.4%.

Molecular immunology
munolOg7) 19 (1982) 451~
455 attempts to overcome said drawbacks by using monoclonal antibodies instead of polyclonal antibodies. However, this antibody also shows only an unsatisfactory cross-reactivity rate (17%). Furthermore, the monoclonal antibody described in European Patent No. 205177 is unsuitable for specific 11MLO measurement. All antibodies described in the European patent specification which react with HMLC from patient serum also react well with KIII MLCi.

Means for Accomplishing the Invention The object of the present invention is to provide an antibody against HMI, (:!), which overcomes the above-mentioned drawbacks and has a low cross-reactivity rate compared to SMLCIc, and a method for producing antibodies of this type. .

A subject of the invention is a H
This is an antibody against MLC. Antibodies of this type can be obtained by the method of the invention described below.

Another object of the present invention is to immunize one animal species with human HMLC and obtain crude serum so that HMI, O
A method for producing antibodies against crude hemocytogens, which may be purified by negative immunoadsorption with a R#A-based SMLC-immunoadsorbent, optionally after preliminary preparation. It is characterized by

Surprisingly, the method according to the invention provides cross-reactivity rates for SMLC of less than 5%, preferably less than 2%, particularly preferably less than 1
It has been found that it is possible to obtain antibodies against HMLC of less than %.

Cross-reactivity measurements were performed using HMLC or EIMLO as the stationary phase and using up to 0.1 μμ of antibody against SMLC per sample. The detection reaction was performed with a conjugate of an antibody directed toward the Fcr portion of the l(MLC!-antibody) and a measurable group (eg, POD).

HMLC and SMLC are, for example, C1rcu1. R
ag.

19 (1985) 611 and Bioehem, J.

119 (1970) 31, by methods well known to those skilled in the art, such as from cardiac or skeletal muscle. For immunization, HMLCl,
Immunization can be carried out in any animal by methods well known to those skilled in the art.

The crude serum obtained in this case is a silicate-based S
It can be used directly for negative immunoadsorption with MLC-immunoadsorbents.

However, for the time being it is advantageous to carry out a preliminary purification, for example by ion exchange chromatography and dialysis.

Optionally, the crude serum thus pretreated is negatively immunoadsorbed with a silicate-based SMLC-immunoadsorbent. For this purpose, crude serum is applied onto a column, in which case it is surprising that antibodies against HMLCK have a cross-reactivity of less than 5%, preferably less than 2%, particularly preferably less than 1%. , the more conjugated, the more antibodies with higher cross-reactivity can be conjugated. Subsequently, the column is filled with the reagents customary for this purpose (e.g. phosphate buffer-7,
5. Wash with PBS to make it protein-free.

The eluate thus obtained can be used to prepare reagents for HMLC tests. However, it is advantageous to carry out further purification of the eluate, in particular by positive immunoadsorption with an HMLC immunoadsorbent.

Suitable carriers for the BMLO immunoadsorbents are silicate-based adsorbents which are well known to those skilled in the art. This medium can be present in powdered or granular form, for example as a glue.

Suitable carriers include, for example, spherosil.
, 1 ■)
'The-pou18nc) ), selecti spell (13rlecti 8pero) (Pierce Eurochemie)
) or Baker-Hiem (J, T, Baker
Chem) silica gel. SMLC is covalently bonded to the carrier by a conventional method, optionally via a spacer/linker.

In order to enable the bonding of BMLO to the silicate, the silicate must advantageously have a bondable group. For this purpose, silicates can be used, for example trichlor7m7
It is advantageous to react with a reactive silane such as 7'''c2 virdine or 3-(triethoxysilyl)propylamino, in which case the functional groups thus formed on the support have It is possible to attach 8MLO directly. In one preferred configuration, a difunctional reagent is attached as a spacer to the functional group, and subsequently BMLO is attached to it.

In this case, SMLCl and! It has proven advantageous to use a mixture consisting of 3MLo1.

In this case, BMLOl: 8ML (the ratio of 3l is IK
Not necessary. However, it is advantageous to use a ratio of more than 1:1 or an excess of SMLCl.

As the medium, the following are particularly suitable: niaminoprogyl group, succinoylaminopropyl group, hexamethylene diamino group, diol group, diazofluoroborate group, p-nitrophenyl group, epoxyzorobyl group, glutaric group. Silicates with aldehyde groups and/or glycidoxyprobyl groups. Optionally, a spacer/linker is introduced prior to bonding to the carrier material. For example, difunctional linkers well known to those skilled in the art such as nigertaldialdehyde, N-succinimidyl 6-(2-pyridyldithio)propionate, ethyl 4-azidophenyl-1,4-dithioptyl imide are suitable. D) X HOI, 4-fluoro-6-nitrophenyl group sil.

N-hydroxysuccinimidyl-4-azidobenzoate, m-maleimidobenzoyl-N-hydroxysuccinimir ester, N-succinimidyl-6 (4'-azido-2'-nitrophenyl-amino)=? f'/ate, succinimidyl (4-iodoacetyl) aminobenzoate or succinimidyl-4-(p-maleimidophenyl) butyrate.

It turns out that the distance between the silicate support and the SMLC is preferably less than or equal to 20 atoms.

A distance of 7 to 12 atoms has been found to be particularly favorable.

As the silicate, we used spherosyl activated with trichloraminopropylsilane or 3-()ethoxysilyl)propylamino, and combined this aminos 7erosyl with glutardialdehyr as a bifunctional reagent. It has proven particularly advantageous to react and to bind BMLO to a support prepared in this way.

After purification with the BMLO column, it may prove advantageous to additionally connect the immunosorbent with the BMLO immunoadsorbent to the intermediate connection of the dialysis outlet. As the carrier, carriers well known to those skilled in the art are suitable.

However, here too it is particularly advantageous to use silicate-based immune adsorbents. For HMLCt-conjugation to the carrier, the BMLO-immunoadsorbent is meaningfully structured.

Another subject of the invention is HM with a cross-reactivity of less than 5%, preferably less than 2%, particularly preferably less than 1%, relative to SMLC.
This is an immunoreagent for measuring HMLC using LC-antibody.

With reagents of this kind it is possible, for example, to carry out sandwich tests or competitive tests.

In the case of sandwich tests, HMLC-passivated antibodies and conjugates of HMLC-antibodies and measurable groups are used. In this case, only one or two of the antibodies to be used can be obtained by the method of the present invention.

Measurable groups are groups well known to those skilled in the art, such as enzymes, fluorescent dyes and radiolabels.

Using this sandwich test, HMLC concentrations of 2 HE/Lt can still be detected with incubation times of 5 or less, but in this case the cross-reactivity with SMLC cannot be measured. Can not.

The reagents can be in solution as well as on or in dry reagent carriers.

This allows shorter incubation times to be achieved since there is no need to dilute the sample for dry reagent carriers.

One preferred configuration of the reagent contains an HMLC-passivated antibody obtained by the method of the invention and a conjugate of a monoclonal antibody and a measurable group. Surprisingly, it was found that a reagent containing this food combination produced a high theoretical sensitivity in the case of testing and a low blank value in the HML○ measurement.

Monoclonal antibodies can be prepared by methods well known to those skilled in the art.
Obtained after immunization with LC. Antibodies MAK4D12 and 1B10 were found to be particularly suitable. The cell line corresponding to this monoclonal antibody is available from the European Collection of Animal Cell Cultures.
Animal Ce1l Cultures ) (KO
AO (1!), Bourtondon, UK (Porto
n Down) Currently, Deposit number 4D12-hill chca
88[]22503 and I B 10-wcAo
.

It has been deposited under No. 88022504.

Another object of the invention is a method for measuring HMLC,
This method is characterized in that the sample is combined with a passivated antibody obtained according to the invention against HMLC and a conjugate consisting of a monoclonal antibody and a measurable group.

In this case, the order of addition is arbitrary. In some cases,
After incubation, the solid and liquid phases are separated and the measurable groups are measured in one of the two phases.

EXAMPLES The invention will now be explained in more detail with reference to examples and drawings.

Example 1 Mio'/7-LQ (HMLC Oyohi 13MLO) no ED
Preparation and isolation HMLC and SMLCf: 01rcul Rea,
i 9 (1965) 611 andzu・biochemical・
Journal (Biochem, J, ) 119(1
It was isolated from human skeletal muscle tissue and human cardiac muscle tissue by the method described in 970) 31.

Example 2 Obtaining antiserum HMLCl (molecular weight 26000) and 11MLOl
A mixture of [(molecular weight 18000) was used as an immunogen. Sheep were completely treated with HMLCl in 70 indoor adjuvant (OVA) and! The first with 55μj of 0.1 da/- solution
Immunized. Additionally, immunization was carried out on days 7, 14 and 6.
This was done on day 0.

Additionally, immunization was carried out for a total of 60 days. In this case of sequential injection, HMI, CO, CI 51 in OVA each time.
1Mj/*o solution 28117& was used. After 6 months, crude serum was obtained.

Example 6 Isolation of polyclonal sheep engineered gG-antibody against HMLCK from crude serum Crude serum 11 was mixed with Aerosil 159 (manufacturer: Degutsu
(Degussa), ) was added at room temperature for 1 hour.
Stir for an hour and centrifuge. Subsequently, 1.7 mol/l of ammonium sulfate was added to the supernatant, and this was diluted at room temperature for 2 hours.
Stir slowly for an hour. Subsequently, the precipitate was centrifuged, homogenized in dialysis buffer 0.2j (potassium a-acid 15 mmol/l, sodium chloride 50 mmol/l, pH 7.0) and diluted 4 times with dialysis buffer 101 at 4°C. Dialyze.

After centrifugation again, the dialyzed γ-globulin was transferred to px
Purify with dialysis buffer under elution through 52-cellulose.

PAK purified in this way, SMLC of (HMLC)
The cross-reactivity rate for iC is up to 100% (Example 6
(measured by).

, Example 4 PAK to SMLC-Immunodsorbent (HMLC〉 Negative Immunoadsorption of PAN purified by Example 6 <HMLC!) solution with 50 mmol potassium phosphate/1. ．． ...7゜5; Sodium chloride 150 mmol/z (pns); Azide 0.1
%, and H at room temperature at a concentration of about 15 Da/d.
Place on top of the MLC-immunodsorbent-column (manufactured, see Example 88) and wash protein-free with PBS/AzylO 11% (PBS: phosphate buffer).

Example 5 PAK against EMLO-immunodsorbent (HM'LrO
) was subjected to HML under the conditions described in Example 4.
C-K11m cloth pull on immunoadsorbent (manufactured as in Example 8)
. After washing with ABB/AdiPO31%, propionic acid 1 mol/! Elute at room temperature.

Subsequently, the eluate is dialyzed against VK-water for 2 hours at 4°C and then against carbonate/l carbonate buffer 1 mmol/1 (pH 9,5) overnight at 4°O. .

Example 6 Measurement of cross-reactivity ratio In order to measure the cross-reactivity rate, a test system consisting of passivated HMLC bound to POD and a polyclonal antibody against Sheep-Far (Sheep-Far)-PAW) was used. used.

a) Ostar (pvc) as solid phase production carrier 2
) was used. per cavity, carbonate buffer 0.2 mol/V, H in PJ49.4
A total of 0.11 of a solution of MLC 5 μ/l/l was added and the temperature was kept constant at 4° C. overnight. After washing twice with PBS, phosphate buffer 50 containing 1% R8A and 0.9% Mail at 0.21117 per cavity.
mmol/l, -7,a was added and kept at room temperature for 1 hour.

1:i) Production of POD-conjugate Polyclonal antibody from domestic rabbit against Fcγ
Far) -PAK) in Knzymo'logy
) 7 Q (1980) 104-142 to horseradish root-POD (peroxidase).

C) Carrying out the measurement The sample was diluted with phosphate buffer 50 mmol/l, pk 47.4 and according to Example 6 and Example 4 PAN < HMI, O
> 1.0βI/Go or according to Example 5, P
Change A to <HML□) 0.1μ, 9/1.

The sample 0°1a thus prepared is added to each cavity and kept constant at room temperature for 1 hour. After washing twice with PBS, one sheep-Far
〉-PAK-POD-conjugate Q, 1d (POD-
0.2 U/'ILt) was added and the mixture was incubated at room temperature for 1 hour.

After washing twice with PB13, ABTS■ (2,2'-7
Ginogy [6-niterubenztheazoline-sulfonic acid (6))-diammonium salt] 0.1 at/(phosphoric acid/citrate buffer 100 mmol/A', pJ(4,4, Totally add 1.9 mmol/l in 3.2 mmol/j of sodium peroxate. After 45 minutes of incubation, the difference in absorbance from the blank value was 405.
Measured in nm.

The cross-reactivity rate is determined from the absorbance value, which is the same as that of the solid phase inoculated with HMLC and HMLC for the same sample.
obtained using a solid phase seeded with

In this case, due to the negative immunoadsorption on SMLC and the positive immunoadsorption on HMLC, no cross-reactivity of PAK<HMLc) with respect to SMLC was found to be undetectable (Table 1).

Table 1: Immunoadsorption Example 7 EL, HMT, O-measurement by TdayA-sa/r-inch test To measure the fully passivated <nMLa)-PAK (obtained according to Example 5) Ta)
A test system consisting of conjugates from (NiMLO, , > -PAK and POD) was used (preparation of conjugates similar to Example 6b).

a) Solid phase near vo-micro titration plate, Kostar (0oatar)b
) Inoculation (< HMLC) - PAN passivation) 20,
1 mu/cavity Citric buffer 0.1 mol/l! , pJ (< HMLC in 4-0) -PAW 5 μμ/1 (Example 5) are incubated overnight at 4° C. and washed twice with PBS (fa acid buffered saline).

0) Afterload: 0.27/Cavity EP: (phosphate buffer 50 mmol/l,...7.4.1 Wash with Jao'l 0.9%, R8A 1% [bovine serum albumin], twice with PBB d) Carrying out the test: Add the sample (EMLO or sMLc O~100 n, 19.Ad) in 0.1g/cavity IP and incubate for 2 hours at room temperature. Wash twice with P13B.

0, I II//Caviar E Air (HMLC>
- Add the conjugate (activity Q, 1ty /rsJ) of KET (Example 5) and POD (prepared analogously to Example 6b) and incubate for 2 hours at room temperature. Wash twice with PBB.

0.1 go/cavity group 3 [solution (citrate it
s solution 0.1 mol/J, p) i4.4, ABTB (1.9 mmol/l) in sodium perborate 3, 2 mmol/l) is kept constant at room temperature for 45 minutes.

Wavelength: 405 with KItISA-bladder removal machine
/ Measured at 490 nm.

The results can be seen from Table 2.

Table 2 Example 8 EIMLO - Preparation of immunoadsorbent Spherosil (8
pherosil) (O-Nebouran (Rho)
After drying, react with 10 (v/v')% 6(triethoxysilyl)propylamino in DMSO at 85° C. overnight to convert to 7-erosilyl-NH2. DM reaction wave, free reagent
Wash and remove with 80 and Improper Tools and absorbent t-
Dry at 50°C.

Su7erosilu NH2t IQ % nof Mix with rutaraldehyde solution pH 3.7 and heat at 55° C. for 2 hours. Subsequently, the suspension is filtered under vacuum through a glass filter. Next, Su7 Erosil's 7
Wash with twice the volume of distilled water and 5 times the volume of potassium phosphate 10 mmol/l.

Postwash with pH 8.0/sodium chloride 0.1 mol/l.

For SMLC 101n9t Supherosil 1N, add 10 mmol/l of potassium phosphate, pJ (8,0/0.1 mol/l of sodium chloride) in approximately 50% of the volume of Supharosil used in a round bottom flask.

The flask is spun on a rotary evaporator overnight at room temperature.

After filtration, the spherosil is washed several times with a 0.9% IJaO1 solution as well as an ethanolamino solution. Continuing,
Add 6 parts by volume of ethanolamino solution and 1 volume at room temperature.
Maintain constant temperature for an hour. Pass through the tube again and wash again with NaCl solution.

Subsequently, the pH was adjusted to 7.5 with immunoabsorbing agent f: PB8,
I#I appearance: MAK, IB1 [] and PB8 /su) Balance with IJ Umuazi V. Storage takes place at 4°C.

Example 9 EL engineered f3A using monoclonal antibodies - HMLC-measurement by sandwich probing
prepared according to Example 5) and (HMLC)-MAK(4
A test system was used consisting of a conjugate from D12 or lB10) and POD (conjugate preparation similar to Example 6b).

The solid phase, coating, afterloading and measurement implementation were similar to Example 7.

The results can be seen from FIG.

The curves show that high sensitivities can be obtained with small sample blank values.

[Brief explanation of the drawing]

Figure 1 shows curve 1 with two different MAK;
AK4D 12 ng/mL HMLC

Claims (1)

[Claims] H having a cross-reactivity rate to SMLC of 1.5% or less
Specific antibody against MLC. 2, having a cross-reactivity rate to SMLC of 2% or less;
The specific antibody according to claim 1. 3. Has a cross-reactivity rate to SMLC of 1% or less;
The specific antibody according to claim 1. 4. Mammals with HMLC I and/or HMLC II
according to any one of claims 1, 2 or 3, which can be obtained by immunization with, obtaining crude serum and immunoadsorbent purification with a silicate-based SMLC-immunadsorbent. specific antibodies. 5. In the method of producing a preferential antibody against HMLC, the mammal is treated with HMLC I and/or HMLC II.
to obtain crude serum and silicate-based S
A method for producing a specific antibody against HMLC, which is characterized by immunoadsorption purification using an MLC-immunodsorbent. 6. The method according to claim 5, wherein silica gel is used as a carrier for the immunoadsorbent. 7. The method according to claim 5 or 6, wherein the SMLC is attached to the carrier via a bifunctional linker. 8. The method according to any one of claims 5 to 7, wherein the SMLC is coupled via glutardialdehyde to a support activated by amino groups. 9. The method according to any one of claims 5 to 8, wherein the SMLC is bonded to the support with a distance of less than or equal to 20 atoms. 10. A reagent for measuring HMLC, characterized by containing a specific antibody for HMLC having a cross-reactivity rate for SMLC of 5% or less.
reagent for measuring. 11. The reagent according to claim 10, which contains said antibody in passivated form. 12. The reagent according to claim 11, which contains a conjugate of an HMLC-antibody and a measurable group. 13. Claim 10 or 1, which contains a conjugate consisting of a monoclonal antibody against HMLC and a measurable group.
1. The reagent according to 1. 14. Claim 3 containing a conjugate consisting of monoclonal antibody 4D12 or 1B10 and a measurable group.
Reagents listed. 15. Hybridoma cell line ECACC880225
Monoclonal antibody 4D12 which can be obtained from 03
. 16. Hybridoma cell line ECACC880225
Monoclonal antibody 1B10 which can be obtained from 04
. 17. In the method of measuring HMLC, the sample is combined with a passivated specific antibody against HMLC having a cross-reactivity with SMLC of 5% or less and a conjugate consisting of a monoclonal antibody against HMLC and a measurable group. , an HMLC measurement method, characterized in that the phases are separated and the measurable group is determined in one of the two phases. 18, cell line ECACC88022503 or ECA
18. The method according to claim 17, wherein an antibody obtainable from CC88022504 is used as monoclonal antibody.