CA1083956A

CA1083956A - Microbial antibodies immunoassay

Info

Publication number: CA1083956A
Application number: CA287,947A
Authority: CA
Inventors: Susan M. Mondabaugh; Magdalena U. Gomez
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 1976-10-12
Filing date: 1977-10-03
Publication date: 1980-08-19
Also published as: FR2368037A1; AU2939277A; NL7711155A; JPS5347521A; AU504706B2; DE2745279A1; IT1154002B

Abstract

Abstract Immunoassay for determining the presence of microbial antibodies in mammalian serum which comprises the steps of:
A. Providing a reaction mixture comprising a buffered aqueous medium; serum to be sampled;
antigen derived from said microbes and anti-antibody to the serum to be tested, said antigen or anti-antibody bearing a detectable label;
B. Incubating said reaction mixture at from about 4 to 45°C. for from about 24 to 2 hours at a pH of from about 6.5 to 8.5 to form an antigen-antibody conjugate when said microbial antibody is present in said serum sample; and C. Determining the level of said label as a measure of the presence of said antigen-antibody conjugate; characterized in that the volume ratio of anti-antibody to serum sample is adjusted to a value sufficient to form an agglutinate but less than required to effectuate precipitation of the antigen-anti-body conjugate.

Description

.

.', , .,~. .
The present invention relates to an impr~ved method for immunoassay for antibodies to microbial organisms. The improve-ment involves the use of diluted anti-human lgG to produce a , ~- non-visible agglutinate of the micro~ial antigen-serum antibody conju~ate. Such method provides for maximum label binding and thus maximum assay sensitivity compared to the previously employed conjugate precipitation method. The present improve-ment is particularly useful in a radioimmunoassay for detecting antibodies to Neisseria gonorrhoeae (N.g.) in sera.

.
10 ,One ofthe techniques for assaying for antibodies to microbial organisms involves forming a conjugate between the antibody and an antigen derived from the microbe such as for example from the ce~ w~l thereof. The conjugate is precipitated from the test solution by the presenceof an anti-antibody derived from a different mammalinn species.
By introducing a`suitable labelinto the conjugate utilizin~ either a labelled I antigen or a labelled anti-~ltibody, itis possibl~ to determine the I concentration ofthe antibody in the sample using a previously generated ~ standard cul~ve.

Hen/1609.1977 1~)83 , Thus, for example., de~ection of gonorrhea antibodies ~ in human serum is described in the U~S.. Patent Specification ; No. 3,974,2~9. The method is based on the use of an .-.~ antigen produced by the Neisseria gonorrhoeae organism whose ~ 5 isolation is described in ~urther detail in the German Patent . .
.. Application No. 2,343,264.

. .............. In a specific embodiment of a radioimmunoassay.; described in U.S. Patent Specification No. 3~974,269 N.g.
~' antlbodies ln serum are detected by a process comprising:
: 10 A. Adding anti-human .IgG to the serum to be tested in a buffer aqueous medium;
B.. Thereater adding a heat labile antigen .~ labelled with a radioactive element;
C. Incubating the resulting.mixture at from about 4 to 45C. for from about 2 to ~4 hours at a pH
of from about 6.5 to 8.5 to fo~m an antigen-. antibody conjugate when antibodies are present in the serum; and D. Determining the level of radioactivity as a measure o~ the pxesence of the antigen- ~.
antibody con~ugate.

:

.

~. . ; , . . .. . . .
:' ;' ,:, , .

~L~83~31SI~

.; ~
., ' , .
The precipit~ted conjugate is separated from the solution by either filtration or centrifugation. In accordance with the prior kno~vledge and practice in the art the amount of anti-human IgG used in relation to the `, amount of serum in step A is adjusted to maximize precipitation. The S assumption has al~ ays been that maximum precipitation is equivalent to maximum radioactivity yield. To t}-is end the ratio of anti-human IgG
antibody to serum sample volumes is adjusted to substantially more than 1/1, usually a ratio of 6/1 is used.

Sevcral problems rcmain before such techniques cnn l)e utilized as 10 I the basis for mass screening tests. Onc major prol~lem is the fact that the ~ositive cut off level is three standnrd devintions above thc me~n of a ! group o negative sel;a even at maximum sensitivily. Thus the test vould pick up positive sera having relatively high antibody titers but I vould not accurately pick up sera having weak or borderline concentrations of antibody which still represent subjects tvith positive infections of the microbe agents in question. I~Ioreover, in order to rench this level of sensiti~rity it is critically necessary to add the reagents in specific order, that is the anti-human IgG is added to the buffered test serum follo~ved by addition of the labeIled anti~en. Use of the norn~al order wherein the antigen is added to the serum follo~ved by the anti-human IgG results in a test which is indicated to be of insufficient sensitivity to distinguish bet~veen positive and negative sera ~vith a useful degree of confidence.

'~, ' . : ,',' ` ' , . ' , ,,; ' ' :. , ' ', . :

The present invention relates to an impr.oved method for immunoassay for antibodies to microbial organisms in mammalian serum. In particular, the present improvement relates to immunoassays for said antibodies wherein a second antibody is added to the immunological reaction mixture in order to assist in the efficient precipita.ion of the antigen-microbial specific antibody conjugate from the medium. This :.
second antibody lS eIici~ed from a mammalian species other than the test species and is specific to the IgG fraction of the test species,:i.e. an anti-IgG antibody.
More particularly the present invention relates to an immunoassay for determining the presence of microbial antibodies in mammalian serum which.comprises the steps o~:
A. Providing a reaction mixture comprising a bufered aqueous medium; serum to be sampled;
antigen derived from said microbes and anti-antibody to the serum to be tested, said antigen or anti-antibody bearing a detectable label, said anti-antibody being an anti-human IgG from a mammalian species other than the test species, and being specific to the IgG
. raction of the test species;

B. Incubating said reaction mixture at from about 4 to 45C. for from about 2 to 24 hours at a pH of rom about 6.5 to 8.5 to form an antigen-antibody conjugate when said microbial antibody is present in said serum sample; and "1"",) .: : ::,.; ~:,.,.: ' '::.; :: : -: . .

3~S6 ;

C. Determining the leveL of said l~bel as a measure of the presence of said antigen-antibody conjugate; characterized in that the voIume ratio of an~i-antibody to serum sample is adjusted to a value sufficient to form a non-visible agglutinate but less than requirsd to effectuate precipitation of the antigen-antibody con~ugate.
As is indicated above, it is conventional in this art to utilize an excess volume ratio of anti-IgG serum com-~- pared to test serum when conduc~ing such assay. This is due to the desire to maximize precipitation which it is believed will be equivalent to maximizing the amount of labelled antigen or antibody recovered. It is the efficiency o~ such recove~y that influences to a great degree the sensitivity o the procedure.
It has now been discovered that, contrary to the established beliefs in the art, maximum yield of label is not obtained ~y maximizing the precipitation of the antibody-antigen conjugage. Rather, quite surprisingly, maximum labelrecovery i8 observed to occur in such method when the volume rakio o~ anti-antibody to serum sample i9 adjusted so as to produce a non-visible agglutination of the conjugate. The , . .
ratio needed to produce such agglutination is substantially below that needed ~or precipitation. While the precise volume ratio ranges utilized to effect agglutination will vary depend-ing on the identity of the specific reagents : ,'' ..

!.' . ~
~....
: ' ` . :,, ' : '~ '' ''; ", :

-, : . ' ., ' ',, ' ' ' ' ' . ' ' .. , ~1~83~s~

employe~ in the assay, they will ordinarily be lower than l/l and generally will be between l/2 and 1/250. Thus, for example in an immunoassay for gonorrhea antibody the volume ratio is preferably from 1/5 to 1/250.
Since volumes of reagents used in immu~oassays are relatively small, for example in radioimmunoassays they may be in the order of about 5 ~ul for the serum sample~ it is more convenient to dilute the anti-antibody and add equal volumes of this diluted reagent to the test serum. Dilutions can be accomplished using either serum from the mammalian species in which the anti-antibody was elicited or else phosphate buffered saline (PBS) as diluent.
The improved method of the instant invention can be used in immunoassays directed towards detection of antibodies present in mammalian blood serum which are directed to microbial organisms. Thus the instant method can be utilized as a diagnostic screen in detecting infection by such microbial organisms. Among the microbial organisms to which the method can be directed include, without limitation, bacteria such as Neisseria gonorrhoeae, Neisseria meningiditis, Treponema pallidum, and Streptococcus pyogenes;
~ungi such as EIistoplasma capsalatum; yeast such as Candida albicans; and parasites such as Trichomonas vaginalis.

,, , : . , . , ............ . ., , . " .: . , : .:

:: :, .; ~ . . ....... ,,: . : , ., , :. ......... : . . .:

. . ' :: , :''' '',.,~:; , ,. . ~ :

~ ~083~

. The ormation of the microbial antibody-antigen-conjugate agglutinate can be detected by a number of methods available in the art. Either the antigen derived from the subject microbe or the anti-antibod~ can be labelled in a manner known per se.
Such labels may include a detectable radioactive element such as 3H, 14C, 125I and the like; an enzyme such as described in U.S0 Patent Specification 3,817,837; an electron spin resonance group such as described in U.S. Patent Specifications 3,453,288;
: ~ 3,481,952; 3,507,876 and 3,690,834; a fluorophore such as fluorescamine, MDPF, fluorescein, rhodamine, auramine and the ~; like; or chromophores.

The nnti-~ntibody employed in the practice of the inventioIl is elicitcd from a mammalinn species diffcrcnt than the species ~qhose scrum is to be ¦ tested for the microbial antibody. Thus, for examplc, if human blood 15 scrum is utilized as the test serum then anti-human IgG derived from sheep or goat serum can be utilized. Such materials arc available as articles of . commerce.

.
In preferred embodiment of the invention detection of the agglutinate is accomplish~d by use of a labelled antigcn, most prererably an 1 l 20 ~ labelled a*ltigen. Such a label can be introduced by utilizing procedures.

known in the art, such as for e~nple as described by Syvanen et al., J. Biol. Chem. 248, 37G2 (1973) or the modified procedure described in V .S . Patent Specification No. 3,974,269.

, -.. : . . .... ,: . :., . , , .~ :.:~ .,, ;

391SI~
-:

g The subject conjugate is formed by a reaction which takes place in an aqueous medium at a pH of ~rom about 6.5 to 8.5 during a period of from about 2 to 24 hours at a temperature of from about 4-45C. Reaction is effected by mixing serum under test with a~ueous buffer, labelled antigen and anti-antibody. The order of addition is not critical.
In a typical procedure for the detection of microbial antibodies by radioimmunoassay a reaction mixture comprising 0.1 ml. of phosphate buffered saline (pH 7.2); 5 ~ul of test 10 serum; 5 ~1 o~ anti-antibody diluted 1/2 to 1/200 with buffer or normal serum; and 50 ul of 125I-labelled antigen (8,000 cpm at 70% counting efficiency) is prepared. The reaction mixture is incubated at 4C. overnight (about 16 hours) and at the end of this period 0.5 ml. of PBS is added. The agglutinated material obtained is filtered through a 2.4 cm Whatman GF/C
filter using a Millipore manifold filter apparatus. Although the agglutinated conjugate is not visible it is retained by the filter in the above procedure. The filter is then washed ; well with distilled water and the label detected by counting the radioactivity.

In order to minimize nonspecific binding of antigen, it is desirable to prewash the filter. The preEerred prewash medium is bovine serum albumin although other reagents such as human serum immunoglobulin, ovalbumin and hemoglobin may also be emplo~ed. The preferred prewash is with 0.5 ml of a solution containing 2~ by weight fraction V BSA together with a chelating agent such as 0.01 M ethylenediamino tetraacetic acid (EDTA).

.. . , ... , "
,,, : ,.. :: ... , .,.,~. .. ,,, ,, , .. ,. ,:,.. ... , :

, . . , . -:, : : :., :.. ~ :: , :. . .

!33956 i. Meth'od In a typical procedure ~or the detection o~ Gc antibodies, the following reagents are added and mixed well: O.l ml of phosphate buffered saline, pH 7.2; 5 ~l of patient serum, 5 ~l of a l/~6 dilution of goat antihuman IgG in normal goat serum and 50 ~l of l25I labellea Gc-antigen ~8000 cpm). The order of addition of reagents is not ~important. After an overnight ncubation at 4~C., 0.~50 ml of PBS are added to provide a ;volume large enough to facilitate handling. The antigen-;~10 antibody a~glutinate is filtered through a 2.4 cm. Whatman GF/C
filter using a Millipore Mani~old (The filters must be pre-soaked with a bovine serum albumin solution). After thoroughly washing the filter, it is dried by gentle vacuum and counted in a gamma scintillation counter.

ii. Comparison o present methodolo~y with that of U.S.
Patent Specification No. 3,974,269.

A series of 5 Gc culture negative sera (1-5) and 12 Gc culture positive sera (6-17) were run employing the present methodology and also that o U.S. Patent Specification No.
20 3,974,269. As can be seen in Table I, all of the positive sera are detected by the present assay yet four out of twelve sera or 33% are missed by the methodology of U.S. Patent Speci~ica-tion No. 3,974,269. The sensitivity o the method o the present invention is therefore superior to that of the published -~25 one. It is~also important to notice that the binding of the label is also much greater. The ratio of the counts obtained by the *Trade Mark 3g~

present method to the counts obtained according to U.S. Patent Specification No. 3,974,269 is on the average 3.25 (range 1.15- -5.59)-D ¦ D¦

~0 ~ ~;iC~ ~ L'~ ~D ~ O ~ ~1 ~ L~l L') L") (~ ~) r~ i ~) L~) ~i , ~` 8~ I I I I l++++++++++++

3 ~ ~ co o ~ ~ o ~ co ~ ~ u~ ~ a ~ t~1 C~ ~ O O O t~ 9 0 L') ~
~ ~ O ~ ~ ~ ~ O ~ I~ ~ ~D ~ l~ r~ ~ ~ o u~ ~
E~ ' . ' .~ ~ I I 1 1 1 1 1 1 ++++++++
. ~
. ~

D ~,~, 1 ~ ~ ,~ ~ a~ ~ ,,~ "~ ~ ~ cn ~ u~ ~
:' ~: ~ + + + + + + + + + + + +
,.

.: -1 :.' ., .
, ; . .

. ., . . .... . , ., . .. . ' . . .. . . . .
.. , . . ~.' ,.:,, ... ,, , ;, .. ~ : . . .

Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. Immunoassay for determining the presence of microbial antibodies in mammalian serum which comprises the steps of:
A. Providing a reaction mixture comprising a buffered aqueous medium; serum to be sampled;
antigen derived from said microbes and anti-antibody to the serum to be tested, said antigen or anti-antibody bearing a detectable label, said anti-antibody being an anti-human IgG from a mammalian species other than the test species, and being specific to the IgG fraction of the test species;
B. Incubating said reaction mixture at from about 4 to 45°C. for from about 2 to 24 hours at a pH of from about 6.5 to 8.5 to form an antigen-antibody conjugate when said microbial antibody is present in said serum sample; and C. Determining the level of said label as a measure of the presence of said antigen-antibody conjugate, characterized in that the volume ratio of anti-antibody to serum sample is adjusted to a value sufficient to form a non-visible agglutinate but less than required to effectuate precipitation of the antigen-antibody conjugate.

2. Immunoassay according to claim 1 wherein the volume ratio of anti-antibody to serum sample is in the range of between 1/2 and 1/250.

3. Immunoassay according to claim 1 wherein said label is a radioactive element and said immunoassay is a radio-immunoassay.

4. Immunoassay according to claim 2 wherein said label is a radioactive element and said immunoassay is a radio-immunoassay.

5. Immunoassay according to claim 1, 2 or 3 wherein said microbe is Neisseria gonorrhoeae; said mammalian serum is human serum; said detectable label is antigen from Neisseria gonorrhoeae labelled with 125I; and said volume ratio of anti-antibody to serum sample is in the range of from about 1/5 to about 1/250.

6. Immunoassay according to claim 1, 2 or 3 wherein the incubation is carried out at about 4°C. for about 16 hours at a pH of about 7.2

7. Immunoassay according to claim 1, 2 or 3 wherein equal volumes of anti-antibody and serum sample is used, said desired volume ratio being achieved by dilution of said anti-antibody.

8. Immunoassay according to claim 1, 2 or 3 wherein equal volumes of anti-antibody and serum sample is used, said desired volume ratio being achieved by dilution of said anti-antibody, and wherein said diluent is selected from aqueous buffer or normal mammalian serum.

9. Immunoassay according to claim 1, 2 or 3 wherein said microbe is Neisseria gonorrhoeae; said mammalian serum is human serum; said detectable label is antigen from Neisseria gonorrhoeae labelled with 125I; and said volume ratio of anti-antibody to serum sample is in the range of from about 1/5 to about 1/250, and wherein the agglutinated conjugate is separated from the reaction mixture by filtration and said filter is then washed and counted for radioactivity to determine the amount of conjugate collected which is proportional to the microbial antibody level in the serum sample.