JPH0534348A - Novel buffer for detecting rheumatism factor and method thereof - Google Patents

Abstract

PURPOSE: To dispense with troublesome pretreatment and improve the temperature stability and RF detecting accuracy by allowing a heparin for efficaciously restraining the interference of a polyvalent property to be contained in a buffer of a rheumatism factor (abbreviated to RF) immunity testing specimen. CONSTITUTION: In a RF immunity testing specimen containing a quantity of heparin for efficaciously restraining the interference of a buffer and Clq, the pepapri has at least a concentration of about 750.000USP unit/liter. The buffer has a buffering capacity in a range of pH from about 7.0 to about 8.5, and is selected from phosphoric acid, glycine, borate, TRIS, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, TRICINE, BICINE, TAPS, and a group consisting of these mixtures. Then, the specimen is brought into contact with an indicator system containing the exposure Fc part of a number of IqG antibodies in the presence of heparin and the existence of coagulation is detected through a nephelometry and turbidimetry.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to novel buffers and methods for the detection of rheumatoid factors.

[0002]

BACKGROUND OF THE INVENTION Rheumatoid arthritis is a systemic disease characterized by muscle soreness and stiffness, as well as joint inflammation and destruction. These manifestations are primarily the result of antibodies, sometimes called autoantibodies, that react with the individual immunoglobulin antibodies. Although the exact cause of rheumatoid arthritis is not known, there are three different factors: (1) genetic predisposition to rheumatoid arthritis, (2) environmental factors such as viral infection, (3) There is evidence that it involves a functional defect in T-lymphocytes. Genetic, environmental,
And the correlation between immunological factors is unknown. Evidence of altered immune function leading to signs of rheumatoid arthritis includes hypergammaglobulinemia and immunoglobulin G (IgG) autoantibodies that reduce T-lymphocyte reactivity in vivo and in vitro. Existence is mentioned.
These "antiantiboby" and "antiimmunoglobulin" have been named rheumatoid factors (RF) because they are associated with rheumatoid arthritis. RF has also been found in patients with variable but most connective tissue disease, many chronic and subacute infections and various disorders. Moreover, RF has been found in many apparently healthy people, especially the elderly.

Despite the lack of a clear diagnosis of rheumatoid arthritis, RF remains valuable as a precursor indicator. For example, studies have shown that high RF abundance or titer is associated with destructive joint disease, the presence of rheumatoid nodules and the progression of numerous similar systemic complications. Changes in RF titer during disease progression are not very effective in determining the disease progression of an individual. Nonetheless, in a study of a group of patients treated with pharmacological agents, the average titer of RF is generally reduced if amelioration of symptoms occurs. More than half of patients with seropositive rheumatoid arthritis and sedation are seronegative. However, about 25% of such patients continue to have high titers in their serum despite clinical recovery.

Multivalent RF autoantibodies specifically bind to the monovalent Fc portion or tail of IgG class immunoglobulin antibodies. The RF can thus bind the IgG antibody, while one or more hands of the IgG antibody bind its corresponding antigen. In fact, RF is IgG in its bound form due to the conformational changes that occur in IgG antibodies.
It is believed to be better attracted to the antibody, making the Fc portion of the antibody more accessible.

The most commonly used test method for RF is the latex agglutination method of Singer and Plots.
tination method), which is sometimes called the latex fixation method. Singer J. M. and Plots C. M. "Latex fixation test I. Application to serological diagnosis of rheumatoid arthritis", American Journal.
Of Medicine , 21 , 888-892 (1956). In this test, human IgG coated latex particles aggregate on a slide, usually with a 1:20 dilution of the test sample.
The test sample is typically human serum, and synovial fluid (taken from the suspected joint) is also sometimes analyzed. This test is generally considered a screening method and gives a yes or no result. Furthermore, titer can be evaluated using a solution obtained by sequentially diluting serum used for the original latex test tube test. In the latex test tube test,
Latex particles agglomerate in solution and the agglomerates are visually evaluated in response to various standards or controls. The tube dilution test has the advantage of being considerably more sensitive than the slide test, but is more cumbersome to operate.

The latex agglutination test, which is performed visually rather than mechanically (eg, nephelometric analysis), is the basis of most of the subsequently devised RF detection methods. All of these methods involve the use of indicator systems, such as latex, bentonite, red blood cells, to attach human IgG. The presence of RF is recognized by aggregation, coagulation or precipitation of various indicator systems.

IgG heat agglutination can also be used directly as an antigen or indicator system in an assay. Some believe that the heat agglutination step results in a change in the structural morphology of the IgG, giving better detection results. It is believed that the structural conformational changes simulate the structural conformational changes that occur when IgM binds to the corresponding antigen with one or more of its linkages.

An early test method known as the sensitized sheep cell agglutination test (Waller-Rose test) is still used in some clinical laboratories. The sheep cells, whose red blood cells have been coated with the rabbit antibody, aggregate by some kind of RF. However, in order to prevent false positive test reactions from occurring, it is necessary to first remove the anti-sheep cell antibody from the test sample by a suitable absorption method. In addition, standardized fresh sheep cells must be used daily before use. Sheep cell agglutination test for RF of rheumatoid arthritis
Although some believe that it is more specific for, the need for fresh cells is somewhat inconvenient for performing this method as a routine procedure.

Other tests used include methods which are a direct modification of the original latex agglutination test. For example, a coagulation test using bentonite particles instead of latex to apply aggregated human IgG as an indicator system is used in some laboratories. Formalin- and tannin-treated sheep cells, ie, preserved cells, are used to coat aggregated human IgG as an indicator system. These cells are then aggregated by RF. This test is very sensitive,
It is slightly more difficult to operate than the latex test and does not provide any practical advantage for routine research. An IgG-RF radioimmunoassay (radioimmunoassay) has also been developed, which uses insolubilized IgG as an immunosolvent and from which IgG can be eluted and confirmed. Immunoassays using the double and single diffusion methods have been developed as well.

Automated nephelometric or turbidity analysis can generally be performed using various indicator systems. Turbidity analysis measures the reduction in light passing through a suspension of particles or aggregates. Light reduction is caused by the reflection, scattering and absorption of light by the agglomerates. It is the scattered or reflected light that enters the detector, which is oriented in a non-light path, as measured by the nephelometric analysis. Where automation is not applicable or impractical, simplified nephelometric and turbidity analyzes are used in which the amount of aggregates observed in the sample is visually compared to a single standard or series of standard controls.

In addition to the indicator system described above, heat coagulation I
GG can be used as an indicator system. The first drawback of using heat agglutinated IgG as an indicator system is the Fc of IgG obtained in the heat agglutination step.
That is, the arrangement of parts becomes uncontrollable and random. If latex, bentonite or similar particles are used for the core of the indicator system, either intact IgG antibody or Fc fragment can be used, F
The number and sequence of c-binding sites can be easily controlled depending on the binding method used. Nefero analysis and turbidity analysis methods, regardless of the type of indicator system selected,
It provides the great advantage of objectively analyzing by instrument that the particle agglomeration expressed in the titration stage was conventionally highly subjectively evaluated.

The RF test serum or synovial fluid must usually be heated to 56 ° C. to inactivate the reactive complement component Clq prior to the assay. The Clq component often present in serum samples has been referred to as a multivalent “tulip bundle”, but each “tulip” can bind to the same Fc of IgG as RF. Therefore, any Clq present in the test sample can also agglomerate the IgG coated particles, resulting in a false positive reaction.

The total duration of the heat inactivation process is about 45 minutes. Each sample is first incubated at 56 ° C ± 1 ° C for 30 minutes ± 1 minute. The sample is then microcentrifuged for 5 minutes. In addition, the remaining approximately 10 minutes are used for sample processing. This required sample preparation imposes on the RF assay an additional time consuming operation that generally must be incorporated into each test.

Pretreatment, or heat inactivation treatment, has in some cases been avoided when latex slide agglomeration and / or latex test tube tests are used. The repelling of heat inactivation treatments in these tests generally required the use of standard glycine buffer and a large dilution of Clq.
For example, standard latex tube tests use serial dilutions from 1:20 to 1: 10240. Glycine compounds are believed to have some inhibitory effect on Clq.

However, each RF test has various requirements for characteristics such as the accuracy of the test. For example, most nephelometric and turbidity tests provide objective numerical data as opposed to visual judgment within some given concentration range. These automated forms of the nefero and turbidity assays do not allow significant dilution of serum samples as required to sufficiently dilute interfering Clq from test samples in visual latex agglutination and latex tube tests. . Therefore, this parameter adjustment is unavoidable with typical Nefero and Turbidity RF assays.

The universal application in automated Nefero and Turbidity RF assays using only glycine buffer has proven ineffective. Other known Clq inhibitors, such as diaminobutane and deoxyribonucleic acid disclosed in U.S. Pat. No. 4,153,417, have likewise been proved unsatisfactory in these nefero and turbidity assays. These compounds inhibit RF activity or at the same time do not impair the assay results.
It has no effect on either inhibiting activity.

Due to the high cost of the latex test tube test in terms of glassware, reagents and technician turnaround time, and the drawbacks of the slide agglutination test, automated nephero and turbidity tests have become highly desirable. It was
It would be advantageous to provide a test method of this kind that does not require a separate pretreatment step to inactivate Clq.

[0018]

SUMMARY OF THE INVENTION Novel buffers for use in RF assays in serum and synovial fluid are provided by the present invention.
The new buffer contains heparin. Surprisingly, heparin acts as an interference suppressor of Clq without significantly suppressing RF activity, thus eliminating the cumbersome and time consuming heat inactivation pretreatment step required in prior art procedures. Was discovered. The buffers of the present invention are particularly useful for Nefero and Turbidity assays,
It has been surprisingly discovered that it gives the RF assay a high degree of temperature stability and improved accuracy.

Heparin is a mucopolysaccharide sulfate that prolongs blood clotting time. Commercially available sodium heparin is a mixture of active ingredients that has the property of prolonging the clotting time of blood. Sodium heparin is normally obtained from the lungs, intestinal mucus, and other suitable tissues of livestock mammals used for bovine, ovine, porcine and other human food.

Heparin activity is measured in the United States in units based on the United States Pharmacopeia (USP), which is traditionally different from the International Unit (IU). The potency of sodium heparin, based on dry matter, is generally less than 120 USP Units in 1 mg when obtained from the lung and generally less than 140 USP Units in 1 mg when obtained from other tissues. 90.0
% Or more and less than 110.0%.

According to the present invention, at least 750,0 per liter of buffer to suppress Clq during the RF assay.
00 USP units of sodium heparin are added. When the preferred phosphate buffer of the present invention is used, it is optimally at least about 100 per liter.
10,000 USP units are added.

Sodium heparin can be effectively added to any buffer that can be used in the RF assay. However, the optimal amount of heparin will vary depending on the buffer selected. For example, the optimal amount of sodium heparin used in glycine buffer is reportedly generally low due to glycine's intrinsic inhibitory effect on Clq. The upper limit of sodium heparin added to each buffer is generally determined by economic considerations.

Buffers used in RF assays are generally pH
It has a buffer capacity in the range of about 7.0 to about 8.5. Traditional buffers of this type include phosphate, glycine, borate and TRIS (tri- [hydroxymethyl] aminomethane) and their derivatives such as glycylglycine. Also used are several new biological buffers, MOPS (3-
[N-morpholino] propanesulfonic acid), TES
(N-tris- [hydroxymethyl] methyl-2-aminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid),
DIPSO (3- [N-bis- (hydroxyethyl)-
Amino] -2-hydroxy-propanesulfonic acid), T
APSO (3- [N- (tris-hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid),
POPSO (piperazine-N, N'-bis- [2-hydroxypropanesulfonic acid]), HEPPSO (N-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid), TRICINE (N-tris- [hydroxymethyl]) Methylglycine), BICINE
(N, N-bis- [2-hydroxyethyl] glycine)
And TAPS (N-tris- [hydroxymethyl] methyl-3-aminopropanesulfonic acid).

The formation of scattering centers, ie aggregates, is achieved by removing a significant fraction of the water such as dextran or polyethylene glycol (PEG).
This can be facilitated by the use of hydrophilic ionic polymers which increase the potential for protein interactions. Also, the use of polymers in the nephelometric assay offers the advantages of increased sensitivity and reduced antiserum consumption.

Heparin is surprisingly an ideal Clq inhibitor for use in RF assay buffers because of its greater specificity for Clq than some compounds proposed in the prior art, such as diaminobutane and DNA. It was discovered. In particular, heparin was found to have very little inhibitory effect on RF when concentrations suitable for effective Clq suppression were used. Furthermore, heparinized phosphate buffer exhibits the same curve fit as that of prior art borate buffers using heat inactivation pretreatment,
It has shown excellent interrelationships with prior art methods.

[0026]

EXAMPLES Next, the present invention will be described in more detail based on examples.

Reference Example 1 Preparation of heparin buffer 200 liters of heparin-containing RF buffer was prepared as follows.

[0028]

[Table 1]

The components were dissolved in an excess amount of deionized water using a magnetic stirrer, and the resulting solution was further adjusted to a predetermined amount with the remaining amount of deionized water. Heparin did not dissolve easily, and a stirring time of 30 minutes was required for complete dissolution.

Example 1 A typical RF nephel immunoassay was performed using standard RF dilutions, heat-aggregated IgG and the heparin buffer obtained in Example 1. These results were compared to the same assay results using prior art borate buffer with another heat inactivation step.

The RF concentrate was obtained from Alto Scientific, Inc. of San Marcos, Calif. And used as an internal standard. This concentrate was diluted to the concentration shown in the first column of Table 2. Nefero analysis is performed by using an ICS (trademark) nephelometer (Beckman Instruments), and 500 ml of heparin buffer solution is put into each ICS (trademark) glass bottle (Beckman Instruments), and each R
100 ml of the appropriate sample was injected from the F standard dilution. A device gain setting of manual mode M33 was used. After the transient infusion transient had subsided and baseline had been obtained, 42 ml of heat-aggregated IgG (RF Antigen ™, Beckman Instruments) was added to initiate peak rate signal recording on the instrument. The results are shown in the second column of Table 2.

Then, the diluted sample of the RF standard solution was placed at 56 ° C. (±
After heat inactivation treatment at 1 ° C. for 30 minutes (± 1 minute), centrifugation was performed at 11,000 rpm for 5 minutes. Nefero assay measurements were performed in the same manner as above, except that the heparin buffer was changed to the prior art borate buffer.

[0033]

[Table 2]

The results in Table 2 are shown graphically in FIG. 1, from which the heparin buffers of the present invention have the same curve fit as the data obtained by the heat inactivation method using the prior art standard borate buffer. It can be seen that the data possessed is obtained.

Example 2 Temperature Sensitivity The RF nephelometric assay in Example 2 was run at three different temperatures,
That is, the same internal standard was repeated at 18 ° C, 25 ° C and 32 ° C. The crude data obtained from these tests were used as the internal standard for the College of American Pathologist's RF reference preparation (CAP.
RPRF) or standardized against a target value created by matching to an external control system created at the Discies Control Center (CDC). The CAP RPRF Control System is from the World Health Organization (WHO). CAP
The RPRF activation unit is standardized on a scale operating from about 20 to about 125 units. CDC activity units are reported as International Units (IU) and have been standardized on a scale operating from about 60 to about 400 units.

The standard used in the RF nephero immunoassay using the heparin buffer of the present invention was standardized to the CAP / RPRF unit. The target rate value of 2084 is C
Beckman I while checking the AP / RPRF unit
The concentration was set to 85.3 units / ml on a CS ™ nepherometer. The results of these temperature sensitivity studies are shown in Table 3. The same result is shown in FIG.

[0037]

[Table 3]

The same RF internal standard was used for the RF nepheroimmunoassay with heat inactivation and prior art borate buffer. However, it was standardized by the CDC International Unit. For all others, the same procedure was followed as for heparin buffer except as described in Example 2. The target rate value of 1470 matches the CDC system,
Beckman ICS ™ Nepherometer concentration 2
It was set to 23.0 IU / ml. The results of temperature sensitivity studies of these prior art operations are shown in Table 4 and graphed in FIG.
Shown in.

[0039]

[Table 4]

A comparison of the graphs shown in FIGS. 2 and 3 reveals the surprising temperature sensitivity of the heparin buffer of the present invention. In particular, the Nefero RF assay using the heparin buffers of the present invention shows an increase of about 29% rate units from 18 ° C to 32 ° C, whereas prior art methods using borate buffer and heat inactivation. An increase of about 75% was observed.

Example 3 An aliquot of the precision RF standard was diluted to the low level range and the nephelometric RF assay readings were repeated 20 times each for heparin buffer and borate buffer with heat inactivation. The measurement was performed at 18 ° C. and 2 as in Examples 2 and 3 above.
The same sample was run at 5 ° C. ICS (trademark)
Using a nephelometer (Beckman Instruments), heparin buffer was programmed to obtain results in CAP / RPRF units, and borate buffer was obtained in CDC international units. The results are shown in Table 5.

[0042]

[Table 5]

Then, an aliquot of the same RF standard solution was applied to the RF.
Diluted to a moderate concentration considered positive. Six replicate measurements using the prior art heat inactivation method with heparin buffer and standard borate buffer were performed on the same sample at 18 ° C, 25 ° C and 32 ° C, respectively. Results for heparin buffer samples are shown in CAP RPRF units and borate buffer results in CDC international units. The comparative data is shown in Table 6.

[0044]

[Table 6]

In most cases, heparin buffer samples showed significantly lower standard deviation. This data confirms the unusual finding of improved accuracy that results from using the heparin buffers of the present invention.

Since the present invention can be embodied in various forms without departing from the essential spirit thereof, the present invention should be made clear by the scope of the appended claims as opposed to the above description. is there.

[0047]

INDUSTRIAL APPLICABILITY The present invention provides a novel buffer containing heparin for use in RF assay in serum and synovial fluid. Heparin does not significantly inhibit RF activity but Cl
Acts as an interference suppressor of q and thus eliminates the cumbersome and time consuming heat deactivation pretreatment step required in prior art operations. The buffers of the present invention are particularly useful in nephero and turbidity assays, imparting a high degree of temperature stability and improved accuracy to RF assays.

[Brief description of drawings]

FIG. 1 is a data comparison graph of a nephelometric RF assay without heat inactivation with the heparin buffer of the present invention and a similar assay with heat inactivation using prior art borate.

FIG. 2 is a graph showing the temperature stability of a nephelometric RF assay using the heparin buffer of the present invention.

FIG. 3: Nefero analysis R using prior art borate R
It is a graph which shows the temperature stability of F test.

Claims (20)

[Claims] 1. A reagent for RF immunoassay comprising a buffer and an amount of heparin that effectively suppresses the interference of Clq. 2. The heparin is at least about 750,00.
2. Present at a concentration of 0 USP units / liter.
Described reagents. 3. The buffer has a pH range of about 7.0 to about 8.
The reagent according to claim 1, which has a buffering capacity of 5. 4. The heparin is at least about 750,00.
The reagent according to claim 3, which is present at 0 USP unit / liter. 5. The buffer is phosphate, glycine, borate, TRIS, MOPS, TES, HEPES, DIP.
SO, TAPSO, POPSO, HEPPSO, TRI
The reagent according to claim 3, which is selected from the group consisting of CINE, BICINE, TAPS and a mixture thereof. 6. The heparin buffer is about 1,000,00.
6. Present at a concentration of 0 USP units / liter.
Described reagents. 7. The reagent according to claim 6, wherein the buffering agent is a phosphate buffering agent. 8. The reagent according to claim 7, which further comprises an effective amount of polyethylene glycol. 9. The reagent of claim 8 wherein said polyethylene glycol is present at a concentration of at least 7.5 g / liter. 10. A. Contacting a test sample with an indicator system comprising exposed Fc portions of multiple IgG antibodies in the presence of heparin, b. F in a test sample consisting of detecting the presence of agglutination
A method of measuring R. 11. The method according to claim 10, wherein the detection is performed instrumentally.
The method described. 12. The method according to claim 11, wherein the detection is performed by nephelometric analysis or turbidity analysis. 13. The heparin is at least about 750,0.
13. The method of claim 12, wherein there are 00 USP units / liter. 14. The test sample is further subjected to a pH range of about 7.0.
14. The method of claim 13, wherein the method is contacted with a standard buffer having a buffering capacity of from about 8.5 to about 8.5. 15. The standard buffer is phosphate, glycine borate, TRIS, MOPS, TES, HEPES, D.
IPSO, TAPSO, POPSO. HEPPSO, T
15. The method of claim 14 selected from the group consisting of RICINE, BICINE, TAPS and mixtures thereof. 16. An Ig produced by heat-aggregating the indicator system.
The method according to claim 15, which is a G antibody. 17. The heparin buffer is about 1,000,0.
17. The method of claim 16, which is present at a concentration of 00 USP units / liter. 18. The method of claim 17, wherein the standard buffer is a phosphate buffer. 19. The method of claim 18, further comprising contacting the test sample with an effective amount of polyethylene glycol. 20. The method of claim 19, wherein the polyethylene glycol is present at a concentration of at least 7.5 g / liter.