GB1577131A - Serological testing - Google Patents
Serological testing Download PDFInfo
- Publication number
- GB1577131A GB1577131A GB1106576A GB1106576A GB1577131A GB 1577131 A GB1577131 A GB 1577131A GB 1106576 A GB1106576 A GB 1106576A GB 1106576 A GB1106576 A GB 1106576A GB 1577131 A GB1577131 A GB 1577131A
- Authority
- GB
- United Kingdom
- Prior art keywords
- test
- cells
- serum
- antigen
- dilution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- G01N33/555—Red blood cell
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
(54) IMPROVEMENTS IN OR RELATING TO
SEROLOGICAL TESTING
(71) I, DONALD CHARLES WHITLEY, a
British Subject, of 4, Wellington Crescent,
Shipley, West Yorkshire BD18 3PH, do hereby declare the invention, for which I pray that a patent may be granted to me, and the method by which it is to be performed to be particularly described in and by the following statement:- The invention relates to the serological testing and has particular, but not exclusive, application to the treponema haemagglutination (THA) test for syphilis.
The THA test does not require microscope reading of its results and has a high sensitivity and specificity as reported by
P.J.L. Sequeira and A. E. Eldridge in the
British Journal of Venereal Diseases,
Volume 49, No. 3 published in June, 1973.
Basically, the test uses treated erythrocytes sensitised with a treponema antigen, specifically Nicholl's strain of treponema pallidum usually sonicated and adhered to tanned formalised avian erythrocytes. The principle of the test rests in mixing inactivated test serum, usually diluted, and the sensitised erythrocytes (test cells) in a well or tray, and then observing how the cells settle. A smooth ring or button pattern represents a negative result, and a thin granular mat-like pattern represents a positive result, a thin granular deposit together with a rough and/or irregular ring being considered weakly reactive.Unfortunately, the test cells are somewhat unstable and deteriorate with time even when kept at temperatures of about 4"C. This can result in reduced adhesion of the antigen to the erythrocytes exacerbating any tendencies towards indistinct pattern definition.
It is an object of the invention to facilitate such tests and, to this end, there is provided packs of concentrated test cells that have good antigen adhesion and give clear pattern definition with a reasonable shelf-life.
According to the invention there is provided a serological test kit comprising a container including a concentrated test cell serum suspension to be diluted for test purposes which serum comprises test cells having antigen adhered to erythrocytes admixed with untreated serum and buffered to a predetermined pH value, and a pack of control cells.
A first feature contributing to realising the object of the invention provides for the erythrocytes to be sensitised, say from fowl, to be subjected to sedimentation separation for selecting relatively heavy cells, thus reducing the range of cell size variations.
Preferably, a filtered batch of cells is suspended in a serum saline, allowed to settle by gravity in a shallow vessel, and then has the supernatant together with lighter cells removed, for example by suction, or, and prehaps preferably, by floating off in successive steps.
The selection of relatively heavy and more uniform cells gives good settling patterns and, in particular, leads to better negative patterns.
A second feature contributing to realising the object of the invention resides in sonicated antigen being fine-filtered to remove even very small particles of residual cellular debris.
Preferably, Nicholl's strain of pathogenic treponema pallidum harvested from rabbits testes as short unsensitised treponemes, typically after only six to seven days growth, are first centrifuged, usually repeatedly, to remove tissue debris prior to sonication of a suspension thereof in distilled water, and are then filtered via a fine pore membrane to remove residual small particles of cellular debris prior to addition of a preservative to form a concentrated antigen capable of storage for long periods.
Erythrocytes, preferably selected as above, will be sensitised by admixture with the antigen after being formalised and tanned. Shelf-life may be significantly increased in gluteral dehyde is used instead of formalin, though, due to process conditions, the antigen needs to be thermastable at about 30"C.
A third feature contributing to realising the object of the invention requires sensitised test cells to be absorbed with normal, i.e. uninfected serum. This also results in better settling patterns. Preferably, the resulting stock suspension is concentrated, that is requires dilution for use.
A fourth feature contributing to realising the object of the invention resides in suspension serum for the sensitised test cells being sampled and tested for treponema antibody in order to eliminate batches with even very small amounts of such antibody.
Preferably a quantitive THA test as specified hereinafter is made starting at a dilution of 1 in 8 and any serum showing even weak reactions, rather than clear button patterns, are discarded.
The third and fourth features both lead to the achievement of better settling patterns.
Preferably antigen of the second feature of the invention is mixed with tanned cells of the first feature, both suitably buffered, and allowed to react. After centrifuging and discharging the supernatant, the cells are then resuspended in a stabilising solution containing 4% to 12% of rabbit serum selected as above.
A fifth feature contributing to realising the object of the invention resides in a concentrated test cell serum suspension including a stabilising agent buffered to a desired pH value, typically 7.4 to 7.6.
This aids achievement of good shelf-life.
Combining some or preferably all of the above features enables the preparation of a concentrated test cell package as a liquid for dilution and use in micro as well as macro testing techniques.
This invention thus enables the presentation of kits comprising a pack of concentrated test cells together with a pack of control cells and, preferably, a pack or packs of standardised positive and/or negative control serum. A special diluent for successive dilutions on quantitative testing, described below using saline, may be preferred in giving a predetermined protein level at each dilution, say equivalent to that resulting from the first dilution in the ensuing description. The range of protein variation from beginning to end of the successive diluting is then greatly reduced and the test patterns enhanced.
The preparation features, although preferably applied to chicken erythrocytes are generally applicable to avian and other, for example sheep, erythrocytes, and, although specifically disclosed in relation to THA test (and control) cell production, are of more general application in supplies of materials for serological testing purposes.
One specific process for the preparation of the test cells for THA tests will now be described in detail, by way of example.
Chicken erythrocytes are obtained from a day-old admixture of fresh chicken blood and sodium citrate solution, and are repeatedly coarse-filtered, say using layers of muslin, washed in saline, and centrifuged.
The resulting cells are then suspended at 5% in 1% rabbit serum saline, shaken well, and allowed to settle by gravity in a shallow vessel.
The supernatant together with the relatively lightweight cells are then removed, preferably by suction, to leave a layer of relatively heavy cells which are then washed and finally resuspended at 10% in phosphate buffered saline having a desired pH value, specifically 7.4.
The resulting suspension is then warmed and admixed with similarly buffered formalin and incubated, say for one hour.
After such incubation, more formalin is admixed and the suspension is incubated for a longer period, say one day, after which the supernatant is discarded and the formalised cells thoroughly washed in saline, for storage as a 10% stock suspension in saline including 0.1% sodium azide.
The formalising process has been found to be best carried out at a temperature of 37"C with successive additions of 15% buffered formalin in quantities of onequarter of the volume of the original suspension.
The cells in the stock suspension are tanned in a weak solution (say 1 in 20,000) of tannic acid in saline, and incubated for a short period. The resulting cells are centrifuged and washed in phosphate buffered saline (specifically at pH 6.4) and suspended, preferably in a measured volume for storage at a cool temperature, say of 4"C, for periods of up to about a week.
The treponema antigen is prepared from
Nicholl's strain of pathogenic treponema pallidum grown in rabbits testes in conventional manner but harvested as short unsensitised treponemes after six or seven days. Treponemes are extracted from dissected testes by stirring in an azide saline which is subsequently centrifuged to remove tissue debris. The resulting deposit of treponemes may be resuspended in azide saline for repeated centrifuging as required.
Finally the treponema are washed and suspended in distilled water preferably at a high concentration, for example, with an
International Opacity standard of 40 to 80 units, whereupon they are disrupted by sonication in a cooled vessel and the result ing antigen is fine-membrane filtered to remove even smaller particles of cellular debris before being preserved by the addition of 0.1 % sodium azide. This concentrated antigen can be stored at 4"C for at least a year.
A measured quantity (say 68 ml) of the tanned cells in phosphate buffered saline are eyenly suspended and mixed with a measured quantity (say 100 ml) of antigen diluted in a similar buffering solution. The precise dilution may be determined by trial and error, taking successive small samples until a dilution is reached where a THA test as described later herein yields standard positive and negative sedimentation patterns with standard positive and negative sedimentation patterns with standard positive and negative control sera. After being allowed to react the mixture is centrifuged and the supernatant discarded.
The resulting cells are resuspended and incubated in a measured quantity (say 100 ml) of 2% negative serum in saline, following which a further centrifuging and washing process is performed using a measured quantity of 0.1% azide saline solution.
Finally the test cells are suspended in a measured quantity (say 133 ml) of stabilising solution including 4% to 12% of selected rabbit serum and buffered to a pH value of 7.4 to 7.6. A concentrated test cell suspension results which needs to be diluted for use by the addition of saline, typically 1.5 ml of saline to 0.75 ml of test cells, for the specific quantities mentioned above.
The resulting test cells have been found to have a good shelf-life, for example three or four weeks. Positive control cells may be prepared as the test cells except that no antigen solution is used.
The rabbit serum for use in the stabilising solution is subjected to testing so that even those with a very small amount of treponema anti-body are removed. This test is carried out using the quantitative THA test to be described but starting at a dilution of one in eight. Any batches showing even weak reactions at this strength are discarded and only those yielding clear button negative patterns are selected. The precise percentage in the range of about 4% to 12% of rabbit srum in stock test and control cells is determined by testing positive and negative settling patterns.
The ease of use for screening and the quantitive tests will be realised from the following example. Weak reactors in this screen test sometimes prove to have a strong infection with a partial prozone in the initial dilution.
A specific screening test can be made by adding .025 ml of test cells to .075 ml of a one in sixty dilution of patients' sera, typically in a well of a "Microtiter" plate.
Selection of reacting patients' sera can be made after about 30 mintues.
A quantitive test will use the same initial dilution of both test and control cells, with the test cells being subjected to three further dilutions of patients' serum, namely 1 in 240, 1 in 960, and 1 in 3840. The use of quadruple dilutions and a single drop (.025 ml) of reagent cells allows these tests to be performed rapidly with simple equipment and ensures easy reading with the naked eye in a period of only 30 to 45 minutes.
Tests may use either macro or micro techniques, the requirements being for a supply of test cells, positive control cells, and standard negative and positive control serum. These are readily packaged with at least the control and test cells in concentrated form. Preferably the positive control serum used will give a weakly reactive result at a 1/960 dilution.
The dilution stage at which the samples first go weakly reactive indicates the strength of the positive report. As usual, of course, initial weak reactions may indicate primary syphillis or a previously treated syphilis or yaws.
A preferred test pack includes two 0.75 ml vials of concentrated test cells and one vial of concentrated control cells giving a capacity for some 175 screening tests or 44 quantitative tests where drops are added to five wells, one for control cells and the other for the initial and quadruple dilutions.
Experience shows that an increase in concentration of the packs may provide increased shelf-life, say 10% to 20% for the antigen and 10% for the cells compared with the above.
The above-mentioned special diluent may be protein-enhanced by rabbit, horse, or even human serum. This improves test patterns as the samples have a substantially constant viscosity.
Another improvement results if the proportions of 1:3 for test serum to a 1:60 dilution of patients serum are reversed, i.e.
made 3:1 at a 1 in 20 dilution of patient's serum.
WHAT I CLAIM IS:
1. A serological test kit comprising a container including a concentrated test cell serum suspension to be diluted for test purposes which serum comprises test cells having antigen adhered to erythrocytes admixed with untreated serum and buffered to a predetermined pH value, and a pack of control cells.
2. A serological test kit according to claim 1, wherein the untreated serum comprises selected rabbit serum.
3. A serological test kit according to
**WARNING** end of DESC field may overlap start of CLMS **.
Claims (11)
1. A serological test kit comprising a container including a concentrated test cell serum suspension to be diluted for test purposes which serum comprises test cells having antigen adhered to erythrocytes admixed with untreated serum and buffered to a predetermined pH value, and a pack of control cells.
2. A serological test kit according to claim 1, wherein the untreated serum comprises selected rabbit serum.
3. A serological test kit according to
claim 1 or claim 2, wherein the untreated serum comprises 4% to 12% of the total serum suspension.
4. A serological test kit according to claim 1, 2 or 3, wherein the predetermined pH value is in the range 7.4 to 7.6.
5. A serological test kit according to any preceding claim, wherein the test cell serum suspension comprises erythrocytes of at least a desired weight having been selected by sedimentation separation.
6. A serological test kit according to claim 5, wherein the erythrocytes having antigen adhering thereto are from fowl.
7. A serological test kit according to any preceding claim, wherein the antigen adhered to the test cells had first been subjected to fine filtration to remove small particles of residual cellular debris.
8. A serological test kit according to claim 7, wherein the antigen comprises
Nicholls strain of pathogenic treponema pallidum harvested from rabbits' testes.
9. A serological test kit according to any preceding claim, wherein the untreated serum is substantially free of antigen antibody having been prior sampled and tested.
10. A serological test kit according to any preceding claim, also including a protein diluent for test purposes.
11. A THA test kit substantially as herein specifically described and according to any of the preceding claims.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1106576A GB1577131A (en) | 1977-03-18 | 1977-03-18 | Serological testing |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1106576A GB1577131A (en) | 1977-03-18 | 1977-03-18 | Serological testing |
Publications (1)
Publication Number | Publication Date |
---|---|
GB1577131A true GB1577131A (en) | 1980-10-22 |
Family
ID=9979356
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB1106576A Expired GB1577131A (en) | 1977-03-18 | 1977-03-18 | Serological testing |
Country Status (1)
Country | Link |
---|---|
GB (1) | GB1577131A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0079145A1 (en) * | 1981-10-23 | 1983-05-18 | FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. | Reagent for use in diagnosis of syphilis and preparation thereof |
EP0274264A1 (en) * | 1986-12-24 | 1988-07-13 | FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. | Reverse passive particle agglutination reagent |
WO1995002186A1 (en) * | 1993-07-07 | 1995-01-19 | Shield Diagnostics Limited | New diagnostic assay for detection of syphilis |
-
1977
- 1977-03-18 GB GB1106576A patent/GB1577131A/en not_active Expired
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0079145A1 (en) * | 1981-10-23 | 1983-05-18 | FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. | Reagent for use in diagnosis of syphilis and preparation thereof |
US4618588A (en) * | 1981-10-23 | 1986-10-21 | Fujizoki Pharmaceutical Co., Ltd. | Reagent and merchandising kit for use in the diagnosis of syphilis and preparation thereof |
EP0274264A1 (en) * | 1986-12-24 | 1988-07-13 | FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. | Reverse passive particle agglutination reagent |
WO1995002186A1 (en) * | 1993-07-07 | 1995-01-19 | Shield Diagnostics Limited | New diagnostic assay for detection of syphilis |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Cannon et al. | An improved serologic method for the determination of the precipitative titers of antisera | |
DE2203377C3 (en) | Water-insoluble immunological diagnostic reagent intended for an agglutination test | |
EP0224134B1 (en) | Process for the quantitative determination of cell populations or subpopulations, and reagent therefor | |
US4259207A (en) | Suspending medium for immunologic reactions | |
US3565987A (en) | Immunochemical determinations of antigens and antibodies | |
DE3002973C2 (en) | ||
GB1561920A (en) | Direct agglutination reagent for pregnancy testing | |
DE2134928B2 (en) | Immunological reagent and process for its preparation | |
US3562384A (en) | Immunological indicator and test system | |
DE602004012161T2 (en) | REDUCING TIME TO SUBMISSION OF BLOOD BANK DIAGNOSTIC RESULTS | |
GB1577131A (en) | Serological testing | |
EP1386160A2 (en) | Method for detecting blood cell antigens and the antibodies in response to the same | |
US3828103A (en) | Indirect hemagglutination test with simultaneous absorption of heterologous antibodies | |
EP0011716B1 (en) | Reagent for the determination of infectious mononucleosis and process for its preparation | |
US3600494A (en) | Serological test for syphilis | |
EP0079145A1 (en) | Reagent for use in diagnosis of syphilis and preparation thereof | |
EP0554657B1 (en) | Method for the determination of antigens or antibodies in the presence of an immune complex | |
EP0363449B1 (en) | Method for detecting antibodies specific for rheumatic diseases, in particular chronic polyarthritis, and reagent therefor | |
US4224306A (en) | Preparation of an infectious mononucleosis antibody neutralizing antigen and product thereof | |
US4228148A (en) | Heterophil antibody differentiation (HAD) test | |
US4139606A (en) | Preparation of antigen and test for heterophil antibody | |
EP0332021B1 (en) | Agent containing amino oxide for use in immuno assays | |
June et al. | Improved detection of immune complexes in human and mouse serum using a microassay adaptation of the C1q binding test | |
DE2844060A1 (en) | Haemagglutination method using two differently sensitised particles - e.g erythrocytes of different sedimentation speeds, allowing two tests on single sample | |
Hornung et al. | Effect of tannic acid and adsorbed purified blood group substances on human erythrocytes. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PS | Patent sealed | ||
PCNP | Patent ceased through non-payment of renewal fee |