CA1041422A - Antibodies tagged with radiant energy emitters - Google Patents

Abstract

Abstract of the Disclosure Dye loading in specific antibody fluorescence is enhanced by grouping dye molecules on a carrier backbone polymer having reactive sites along its length and a reactive end site and bonding the end site to the antibody to be dye tagged. The reactive end site of the carrier molecule is chemically protected while bonding dye molecules along the length of the carrier molecule and the chemical protection is stripped in preparation for antibody tagging.

Description

~ ! :

~ Backqxoun~ o the Invention .
The present invention rela-tes to clinical analysis and more particularly to specific antibody fluorescence processing techniques and materials therefor and generally in immunochemistry.
.
j According to the invention loading of tagging fluorescent . . ^ , tincluding fluorescent and phosPhorescent dye3, or other radiant energy emitters, in such processing is increased to thereby ' lncrease sensitivity of the process without introducing new sources of error.
~D ~1 It is a known clinical laboratory technique to detect and classify a suspect virus by successively exposing fluid contain-;l ~1 ing the sample to highly s~ecific antibodies which are tagyed ¦l with a radiant energy emitter. If the sample carries the antigen of the challenging antibody, the radiant energy emitter will be linked to the virus via the antibody-anti~en reaction and, since ,.,, !! . . - '.
,~ s~vexal antibo~y mo~ecules will be linked to a sinqle viral j, , ' ., molecule, the radiant energ~ emission from the agaregate of emitters thus associated with a viral molecule is, in principle, i!
distinguishable from the backgrouna of tagged antibodies. However, ... . .
01 in the state of the art, sensitivity and ability to pick up small guantities of virus are regarded as lot,r.
t¦ In particular, in specific antibodv immunofluorescence, the ! signal level of fluorochrome dyes associated with viral particles ~-., ;~ , i ".
j vl~ ant~body~intermcdiates is ~too~low F indL idual pal~iclc ~ ~ -2~

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detec-tion. Prior art effort~ to impro~e sensiti~ity ~y increased dye loading result in reduced specifici-ty in a number of ways:
(1) As enou~h dye molecules beco~e attached to the molecule, some of them will be close enough to -the active site to produce partial steric shieldingj~ with profound effects on the ~ -~molecule's specificit~.
-(2) Changes in the overall h~drophilicity and net charge . of the resulting molecule will alter its reactivity and solu-:,bility. ~.
,, ,~. (3) Steric and h~drophilicity stresses on the molecule, as : i, well as possible changes in its vibrat,ional behavior, ma~ distort `
the protein's tertiary ~tructure and consequentl~ its specificity.
(i~) Chem:Lcal interact.ions at the bond site ma~ cause alter-, 15 ' ation of the atomic bonding for some distance away from the link's ., Location, possibly involving the specific binding site.
:, It is therefore an im~ortant object of the presen-t invention ' to provide increased concentration of radiant e.nergy emitter bonded..to -the antibody molecule consistent with maintaining the ~: 20 ~. immunochemistry process s~ecificity essentiall~ unimpaired.
, It is a ~urther ob~ect of the invention to increase dye , loading in speci~ic antibody immuno1uorescence consistent with ' ,the preceding object.
: ,l It is a:further object of the invention to increase sensitiv~
25 ~ ',,ity by:at least two orders of magnitu~e ~powe~ of ten of detection~.
' in parts per microliter] compared with Prior art processes con~
s;stent with~one or both of the preceding objects. ~ ' ' . ~

~ 3- ' j : -'1. ~, ' It is a further object of the invention to provide means for detecting and sizing individual viral particles or other ; detected species consistent with one or more of the preceding .~ objects.
~ Summary of the Invention :: According to one embodiment of this invention, there is .
-.~ provided a radiant energy tag base assembly suitable for use as an analytical reagent, the assembly being reactable with a first reactant to form a radiant energy tag assembly which is capable ~0 of tagging a second reactant by reaction between a specif.ic re-active site on each molecule of the first reactant and the second reactant, the tag base assembly comprising a polymeric backbone molecule having reactive end groups and repeating units having re-; active sites of different chemical reactivity, and radicals derived .1 from molecules capable of emitting radiank energy when suitably .~ activated, which radicals are bondea to the reactive sites of the polymeric backbone molecule, to produce side chains on the polymeric ~I backbone molecule, the end groups of the polymeric backbone molecule -~ being reactive with the first reactant whilst the reactive sites are ..
.. 20 essentially unreactive with ~he first reactant~ the tag base assem-.' bly having substantially the same hydrophilicity and net charge per unit volume as the first :reactant. ~
As a further embodiment there is provided a method of producing .
a tag base assembly suitable for use as an analytical reagent com-prising the steps of reacting polymeric backbone molecules having reactive end groups and repeating units having reactive sites of different chemical reactivity with a compound capable of blocking ~:
the reactive end groups, reacting the polymeric backbone molecule .~ with blocked end groups with molecules capable of emitting radiant energy when suitably activated, the reaction between the polymeric backbone molecule and the molecules capable of emitting xadiant energy taking place at the reactive sites of the polymeric back~

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bone molecule/ and unblocking the reactive end groups of the poly-`1 meric backbone molecule, the unblocked end groups being reactable j c/ \J g _~
~, ,1' ,:: :

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V th a first reactant to form a radiant energy tag assembly which is capable of tagging a second reactant by mea~s of a reaction between a specific reactive site on each molecule of the first re-actant and the second reactant, the hydrophilicity and net charge :
per unit volume of the tag base assembly being selected so as to be substantially the same as those of the first reactant with which it is to be reacted.
Detailed Description of Preferred Embodiments Polyethylene imine with a methylamine chain termination (I):
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(I) 2 [CH2cH2NH]n ~ CH3 ,........................................................................ .

: where n is preferably single valued and at least 4 is reacted with benzaldehyde :

, (II) ~ - CHO
.jj , ' ., in aqueous environment to produce (III):

(III) ~ - CH=N - [CH2CH2NH~n CH3 . ', ..
The reaction product (III) is transferred to an apro-tic solvent and ., . ;.~.,:
treated with lithium to produce (IV):

(IV) ~ - CEI=EI - [CH2CEl2l~ 3n ~ CE13 3` and a fluorochrome dye is then bound to it by a demetallation re- :
action at a ~-bromoethyl side chain on the dye to produce (V):

.. ' .
_ jc/ (j~s . .

'~ '~ , '' ', ' ,~ . .
ZZ ', (V) ~ CH=N - LC~2C~l2~]n C~I3 :~ C~2 . D
which on hydrolysis yields (VI):

... . . .
(VI) NH2 ~ [CH2CH21]n C~13 ~ CH
: . - I 2 ~ . D
: which may be treated with thiophosgene to give the thiocyanate ~. . - .
~ .
(VII) NCS [CH2CH2N] ~ CH3 ,, CH2 , . .
:! . I . ;
CH
. . . D . - : :
. whLch in turn ~a~n bc bound to the pr~Leiii molec~le by standard procedures.
~. /;e~ . . -'d~ In ~ of polyethylene imine, other backbone materials l . . . i ;i may lnclude polyethylene oxide, polyamides ~e.g. nylon-6], lou molecular weight [lOO-lO,OOO] polymeric carboxylic and amino acids ~ including polypeptides. The general formula for such structures I , , . .'.
: is: , ~ . (VIII) Ri_~R2_R31~Rs ~

b : '1 ' . ', . -' - ' ' ' l ' . . . .~ . - .. ..
`~ . . . . . . . .
;~ ~ ` . : . - . - i .
- f i I : ~ ' - ' , j . 1 . -6~

.

.
_ ~ . . . r.

-; ( ~
` ~

~s34~ 2 wherein R is an end radical such a~s C~S, CN, COO~I, NEI2, reactive ith the ir~muno~lohin protein; R~ is a hydro~hilic grou~ such ~ as O!~ NH, N, S~, which is essentiallY unreactive ~7ith either -~ the immunoglobin or the s~ecies to be det~cted or its commensals;
R2 and R- are sig~a bonding inter~ediate units, such as tCH2)m where m is at least 2; and D is the dye radical either containing or an end linkage to R4; R5 is another end grou~ which is essentially inert in present context, e.g. ~CH3 and n is 4 to 1,000. . ' ,, ', ~, ~
i ' The polymers may also be copolymers with alternating struc-tures, some or all of which may fit the above criteria.
In connection with maintaining the h~drophilicity and net charge requ.irements given above, the backbone/dye aggregate must contain one polar grou~, e.g. ~OH, -NH, -SH per molecular weight unit portion in a relation to match hydro~hili grou~s per unit '~
volume of the antibo~y to be ta~ed. TyPically the ratio is 1:130 , ` for antibodies used in antibodv-antigen reactions for virus I detections. The backbone may Provide all of said Polar groups, -but in practice, the dye may ~rovide some.
;) ~ The structure of the dye tagqed antibody may be re~resented , ,! as~

, D D

. . . . ...
.. , , . . !
.

:
. ~, ( !
', `1-.
4 ~ ~ 2 Z
where ~, B, C is th~ basic Y form molecular stxucture of i~munoglobin, C being the stable leg an~ A an~l B containing re-active grou~s thereo'f T~7hich ~articiPate in sPecific antibody ''' agti~en reactions. D re~resents dye molecules linked to the S folded ~ackbone carrier molecules BC, one or more of which may ' be end linked to the end and to one or more side linkages of ~, the immunoglobin. Several of such tag~ed immunoglohins can be`'~ , attached to the antigen source molecule in the antigen-antibody reaction, the exact numbex depending on the number of reactive , LO sites and size of the antigen source.
, , Accordingly, radiant ener~,~ detection (e.g. photometric analysis by conventional means) can be used to determine whether , , the test antibody-antigen reaction has taken place, i'.e. whether ;~ the suspect antigen source was present in a sample challenged, !5 by admixture of ta~ged antibody there~.7ith and treating to induce ,~ reaction. Unrea'cted antibod,y is either at a lower local concen-~, tration or may be ~7ashed aT~ay.
,' Additionally, individual molecular reaction products of antibod~-antigen reaction can h~ pa.s~ed, through'a capillary flow ''' tub~ and individually exami~ed for radiant energy emissio~. The ' ' emission can be used to determine numbers o~ dye molecules I~, ,;`f ' 'attached to the antibody. If the backhone carrier molecules used `'are essentially slngle valued in numbers of repeating units of ''active sites for~ dye attachment. The n~ er of such sites can be~
multiplied by number of sites o~ the antibodY for attachment of ~ 8~

' '. `',1' ., ~o~z2 backbone molecule and differ~nces in emission readinys from sample '-o sample ~lill be traceable to differences in the number of antibodies attached to the viral particle or other antigen ~: source being detected,. which is in turn correlatable to the ; 5 size of.viral particle. ..~
.~ . As used herein "radiant energy" inc:Ludes radioactive X-ray, microwave, and fluorescent energy emission, ."fluorescent" includes ~:.
,. : , ~-. .phosphorescent and "polymer" includes dimers, trimers and large ~ :
. monomers with repeating units as well as polymerized monomers.
: 10 IDetails of conventional radiant energy detection means ana `. .'emitters therefor, are not set out here as the present invention contemplates the use of conventional apparatus, materials and ' ~

techniques modified only to the extent set out above. Examples , : .
!:
`. ,~of disclosures of such conventional apparatus materials and i`
.:. 15 techniques are found in U.S. patents 3497~90 and 3586859. : ..
It is evident that those skilled ln the art, once given .
. the benefit of the foregoing disclosure, may now make numerous ..~, , - .. - . . . j . . :.
-~. other uses and modifications of, and departures from the specific ::
.
~: embodiments described herein without departing from the inventive ~ 20 concepts. Consequently, the invention is to be construed as l~

!~embracing each and every novel feature and novel combination of ' ;:
I .
eatures present in, or possessed by, the apparatus and techniques .' ' . !! : , , .:.
. .herein disclosed and limited solely by the scope and splrit of the .;
appended cl~in~s, .. ,~.-.. . . ~ . . . . i :
,,s,,,~.~ , ,~. . :. ' I' .`, , ~ . . .. I
:`::. !i - .
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Claims (23)

WHAT WE CLAIM IS:

1. A radiant energy tag base assembly suitable for use as an analytical reagent, the assembly being reactable with a first reactant to form a radiant energy tag assembly which is capable of tagging a second reactant by reaction between a specific reactive site on each molecule of the first reactant and the second reactant, the tag base assembly comprising a poly-meric backbone molecule having reactive end groups and repeating units having reactive sites of different chemical reactivity, and radicals derived from molecules capable of emitting radiant energy when suitably activated, which radicals are bonded to the reactive sites of the polymeric backbone molecule, to produce side chains on the polymeric backbone molecule, the end groups of the polymeric backbone molecule being reactive with the first reactant whilst the reactive sites are essentially unreactive with the first reactant, the tag base assembly having substantially the same hydrophilicity and net charge per unit volume as the first reactant.

2. A tag base assembly as claimed in claim 1 having a folded structure.

3. A radiant energy tag assembly comprising a tag base assembly as claimed in claim 1 when bonded to the first reactant.

4. A radiant tag assembly as claimed in claim 3 wherein the constituents of the tag assembly are so selected as to have substantially the same hydrophilicity and the net unit charge per unit volume as the second reactant to be tagged thereby.

5. A tag base assembly or tag assembly as claimed in any one of claims 1-3 wherein the polymeric backbone molecule is a molecule of polyethyleneimine.

6. A tag base assembly or tag assembly as claimed in any one of claims 1-3 wherein the molecules capable of emitting radiant energy when suitably activated comprise fluorochrome dye molecules which emit fluorescence upon excitation by incident light.

7. A tag base assembly or tag assembly as claimed in any one of claims 1-3 wherein the polymeric backbone molecule comprises repeating sigma bond units of sufficient spacing to prevent steric interference and achieve resonance shielding between the radicals capable of emitting radiant energy bonded to adjacent reactive sites of the polymeric backbone molecule, the backbone molecule and the radiant energy emitting radicals being so selected that the backbone molecule does not quench the radiant energy emitted by the radiant energy emitting radicals.

8. A tag assembly as claimed in claim 3 wherein the first reactant is a protein antibody and the second reactant with which it is capable of reacting is an antigen.

9. A tag assembly as claimed in claim 8 wherein the tag base assembly has the general formula wherein R1 is an end group reactive with a protein antibody R2 -R3 and R3 -R4 are repeating structures or dissimilar alternating units, R3 is a hydrophilic atom or group and R2 and R4 each comprise at least two single bonded carbon atoms, D is a fluorochrome dye radical, R5 is an inert group and n is at least 4.

10. A tag assembly as claimed in claim 9 wherein R1 represents a thiocyanate group, R2 and R4 each represent two methylene groups, R3 represents a nitrogen atom and R5 represents a methyl group .

11. A method of producing a tag base assembly suitable for use as an analytical reagent comprising the steps of reacting polymeric backbone molecules having reactive end groups and repeating units having reactive sites of different chemical reactivity with a compound capable of blocking the reactive end groups, reacting the polymeric backbone molecule with blocked end groups with molecules capable of emitting radiant energy when suitably activated, the reaction between the polymeric backbone molecule and the molecules capable of emitting radiant energy taking place at the reactive sites of the polymeric backbone molecule, and unblocking the reactive end groups of the polymeric backbone molecule, the unblocked end groups being reactable with a first reactant to form a radiant energy tag assembly which is capable of tagging a second reactant by means of a reaction between a specific reactive site on each molecule of the first reactant and the second reactant, the hydrophilicity and net charge per unit volume of the tag base assembly being selected so as to be substantially the same as those of the first reactant with which it is to be reacted.

12. A method as claimed in claim 11 wherein the end groups of the polymeric backbone molecule are blocked by reaction with benzaldehyde and unblocked by hydrolysis.

13. A method as claimed in claim 11 wherein the polymeric backbone molecule is a molecule of polyethyleneimine.

14. A method of preparing a radiant energy tag assembly comprising the steps of preparing a tag base assembly by a method as claimed in claim 11 and thereafter reacting the tag base assembly with a first reactant, the first reactant having a plurality of reactive sites, only one of which is reactable with a second reactant to be tagged, the reaction between the tag base assembly and the first reactant taking place at the end groups of the polymeric backbone molecule and the reaction site on the first reactant being so located as not to impair the specificity of a reaction between the first reactant portion of the tag assembly and the second reactant.

15. A method of tagging a molecule comprising the steps of preparing a tag assembly by a method as claimed in claim 14 and then reacting the tag assembly with the second reactant, the second reactant constituting the molecule to be tagged.

16. A method as claimed in claim 15 wherein the first reactant is a protein antibody and the second reactant is an antigen.

17. A method as claimed in claim 15 wherein the tagging reaction is carried out by mixing the tag assembly with the antigen in such a manner that the tag assembly has the same hydrophilicity and net charge per unit volume as the second reactant.

18. A method as claimed in claim 15 comprising the initial step of reactinq the tag assembly with a sample of the second reactant and separating out and decomposing the reaction products formed to retrieve tagged molecules therefrom.

19. A method as claimed in claim 18 comprising the addi-tional step of mixing the tag assembly with a sample which is suspected to contain molecules capable of being tagged by the tag assembly, which additional step is carried out prior to reacting the tag assembly with the major portion of the sample.

20. A method as claimed in claim 15 wherein the molecules capable of emitting radiant energy when suitably activated are fluorochrome dye molecules, the method comprising the additional step of measuring fluorescent emission from the tag assembly-second reactant mixture.

21. A method as claimed in claim 20 utilized in the detection of viruses.

22. A method as claimed in claim 14 wherein the tag base assembly has the general formula wherein D is a dye radical and n represents 4 to 1000.

23. A method as claimed in claim 22, the tag base assembly being in aqueous solution.